Both deregulated growth and blocks in differentiation cooperate in the multistage process of leukemogenesis. Thus, understanding functional interactions between genes that regulate normal blood cell development, including cell growth and differentiation, and how their altered expression contributes to leukemia, is important for rational drug design. Previously, we have shown that the zinc finger transcription factor Egr-1 plays a role in monocytic differentiation. Ectopic expression of Egr-1 in M1 myeloblastic leukemia cells was observed to activate the macrophage differentiation program in the absence of the differentiation inducer interleukin 6 (IL-6) and to promote terminal differentiation in its presence. In addition, we have shown that deregulated expression of the proto-oncogene c-myc blocks the myeloid terminal differentiation program. Here we show that restoring expression of Egr-1 in M1 cells that express deregulated c-Myc abrogates the c-Myc block in terminal differentiation, resulting in cells that undergo functional macrophage maturation. However, there is an absence of both growth arrest and cell adhesion. In addition, Egr-1 expression diminished M1myc leukemogenicity in vivo. These findings indicate that Egr-1 can act as a tumor suppressor gene and suggest that Egr-1 or Egr-1 targets may provide important tools for differentiation therapy in certain leukemic phenotypes.

Previously, we have shown that Fos/Jun transcription factor complexes function as positive modulators of myeloid differentiation. Fos, which is stably induced during normal myeloid differentiation, is not induced upon differentiation of M1 myeloblastic leukemia cells. Establishing M1 cells that express a beta-estradiol-conditional FosER chimera, we show that in the absence of the differentiation inducer interleukin-6 (IL-6), Fos expression in M1 myeloblasts promoted apoptotic cell death, entailing cytochrome c release and caspase-9 activation. In contrast, in the presence of IL-6, Fos-mediated apoptosis was abrogated, and Fos promoted terminal differentiation, increasing the sensitivity of M1 cells to be induced for differentiation by IL-6. Fos-mediated apoptosis was accelerated by deregulated c-Myc. Furthermore, restoring Fos expression in M1 partially abrogated the block imparted by deregulated c-Myc on the myeloid differentiation program, increased the sensitivity of the cells to be induced for differentiation, and curtailed their leukemic phenotype. These data provide evidence that Fos/Jun transcription factor complexes play a role in modulating both myeloid cell survival and differentiation and suggest that genetic lesions that alter Fos expression may cooperate with deregulated c-Myc in leukemogenesis.

Tightly regulated c-Myc expression is crucial for normal hematopoiesis, and alterations in the level of c-Myc expression or protein structure are associated with many hematological malignancies. Deregulated Myc expression has been shown to block myeloid terminal differentiation as well as concomitantly induce p53 independent apoptosis through the Fas/CD-95 pathway. This observed apoptosis was not completely penetrant, however it impacted on the neoplastic potential of the blocked myeloid cells. It has been previously shown that deregulated c-Myc, in combination with the deregulation of an apoptotic suppressor such as Bcl-2, can effectively transform hematopoietic cells. Since acute myeloid leukemia (AML) results from a clonal expansion of myeloid cells that are blocked from differentiating, there is significance to the mechanisms behind the apoptosis seen during the Myc mediated block in myeloid differentiation. To facilitate the study of Myc, conditional alleles were used that employed fusing the estrogen homone receptor binding (ER) domain onto the carboxy terminus of the Myc protein. This MycER mutant was originally determined to have no impact on the various proliferative or apoptotic functions of Myc. Consequently, this conditional ER chimeric Myc has been widely used in a variety of studies as a means of titrating Myc activity. Here, we show that the presence of the ER binding domain on the c-terminus of Myc alters its ability to induce apoptosis during the Myc mediated block in myeloid differentiation by affecting the Fas/CD-95 ligand receptor pathway. Our data showed that M1 myeloid leukemic cells stably transfected with MycER (M1MycER), and stimulated to differentiate with interleukin-6 (IL-6) were blocked for terminal differentiation but lacked the apoptotic phenotype normally seen with M1 cells expressing the wild type Myc transgene (M1Myc). Further study of M1MycER cells showed low expression of the death receptor signaling protein RIP when compared to M1Myc cells. RIP has been shown to be an important component of the Fas/CD-95 death receptor pathway. Furthermore, during IL-6 treatment, M1MycER cells showed down-regulation of GADD45 alpha, as well as increased levels of activated Akt, both indicative of NFkB activation. Others have shown that NFkB can be activated through the Fas/CD-95 pathway during the inhibition of Fas/CD-95 mediated apoptosis. The presence of activated Akt in the stimulated M1MycER cells coincides with an increase in the transcriptional level of MCL-1. MCL-1 is an anti-apoptotic member of the Bcl-2 family induced by Akt. In M1MycER cells, increased transcriptional levels of DAD-1 and Bax, both binding partners of MCL-1, suggest possible mechanisms for affecting cytochrome-c release in the mitochondria and cell survival. Taken as a whole, these results offer new insights into how mutations in cMyc as well as the various components of the NF-kB branch of the Fas/CD-95 pathway can impact on the aggressiveness of leukemias.

Gadd45 expression, which is stress inducible, has been associated with growth arrest, but the exact role of gadd45 family genes in apoptosis still remains unclear. We have found that myeloid progenitor cells from gadd45a and gadd45b-deficient mice are more sensitive to ultra-violet radiation, VP-16 or daunorubicin induced apoptosis. indicating that gadd45a or gadd45b protect haematopoetic cells from DNA damaging agents. To determine, how gadd45a or gadd45b proteins exert their anti-apoptotic function, bone marrow cells from wild-type and gadd45a or gadd45b deficient mice were exposed to ultraviolet radiation (UV) and analyzed for expression of stress responsive kinases, including JNK and p38. It was observed that P38 and JNK were activated in wt bone marrow cells in response to UV but not in bone marrow cells defecient in gadd45a. Also, the transcription factor NF-kB was activated in wt bone marrow cells, but not in gadd45a−/− cells. The pharmacological inhibitor SB203580 specific for p38, increased apoptosis in reponse to UV, indicating that p38 is implicated in signaling myeloid cell survival. SB203580 was observed also to inhibit the expression of certain NF-kB target genes, including cIAP-1, c-IAP-2, bcl-2 and bcl-xl, in gadd45a+/+ cells but not in gadd45a deficient bone marrow cells. Taken together this data provides first evidence for the role gadd45a plays in the control of hematopoietic cell survival in response to UV, via modulation of P38 MAPK and NF-kB signaling pathways. Unlike in gadd45a−/− bone marrow cells, p38 activation appeared not to be impaired in gadd45b−/− cells, indicating that gadd45b is not involved in p38 activation in myeloid cells. However, UV induced JNK activation was sustained in gadd45b−/− myeloid cells compared to wt cells, indicating that gadd45b is a negative modulator of UV induced JNK signaling in myeloid cells. UV induced activation of MKK4 an upstream regulator of JNK also was impaired in gadd45b−/−. NF-kB was also found activated in wt cells, but not in gadd45b−/− cells. This data indicates that in bone marrow cells exposed to UV, NF-kB induced expression of Gadd45b plays a protective role against UV induced apoptosis via inhibition of MKK4 kinase which in turn results in suppression of JNK activity. Taken together this data provides evidence that Gadd45a and Gadd45b protect haematopoetic cells from genotoxic-stress induced apoptosis via distinct signaling pathways.

The proliferation and differentiation of hematopoietic cells are tightly regulated to maintain cellular homeostasis. Understanding the mechanism of this regulation may identify potential therapeutic targets against leukemia. Previous works have shown that several negative regulators of differentiation, namely oncogenes c-Myb, E2F-1 and c-Myc were capable of blocking the IL6-mediated myeloid terminal differentiation program of M1 myeloblastic leukemia cells. On the other hand, a positive regulator of differentiation, Egr-1 has been shown to activate the macrophage differentiation program of M1 cells in the absence of IL-6. Interestingly, recent work showed that Egr-1 could override the block of differentiation imparted by deregulated c-Myc in the presence of IL-6, and could reverse the leukemic phenotype associated with deregulated c-Myc. From such observations, we asked whether exogenous expression of Egr-1 in M1 cells could override the earlier block of differentiation imparted by the oncogenes c-Myb or E2F-1. The established M1Myb-Egr cells and M1E2F-Egr cells were analyzed. The M1E2F-Egr cells underwent growth arrest followed by macrophage differentiation and subsequently apoptosis. In addition, exogenous Egr-1 only partially abrogated c-Myb block of differentiation. The M1Myb-Egr cells failed to undergo growth arrest, however, were able to enter intermediate-late stage macrophage differentiation with concomitant phagocytic functionality. These data demonstrate that E2F and c-Myb each block myeloid differentiation via different mechanisms. Egr-1 can completely override the E2F block but cannot abrogate the c-Myb block to allow M1 cells to terminally differentiate or growth arrest. Furthermore, Egr-1 appears to behave as a tumor suppressor, and therefore may serve as a possible target against various forms of leukemia.

Myeloid leukemic cells (M1) proliferate continuously in culture unless induced by Interleukin-6 (IL6) to undergo a terminal differentiation program into macrophages, followed by apoptosis. M1 cells lack the tumor suppressor p53, which is a critical determinant of the cellular decision to either growth arrest and repair DNA damage or to undergo apoptosis. Activation of a temperature sensitive p53 protein (p53 val) at the permissive temperature in M1 cells results in rapid apoptosis. IL6 treatment blocks this p53-mediated apoptosis. Towards understanding the basis for this p53-mediated apoptosis and its abrogation by IL6, we have shown that at the permissive temperature p53 activates the pro-apoptotic Fas/CD95 pathway by up regulating the Fas/CD95 receptor and cleaving antiapoptotic c-FLIP. On the other hand, antagonistic Fas antibody protects against apoptosis. IL6 decreases Fas/CD95 ligand expression and prevents cleavage of FLIP. Treatment of M1p53ts cells with the specific Akt inhibitor Ly294002 abrogated IL6 protection and resulted in Flip cleavage, suggesting that IL-6 blocks apoptosis by phosphorylating and activating P13kinase/Akt, which in turn promotes FLIP stability. Inhibition of the ERK pro-survival pathway did not abrogate IL6 protection against p53 apoptosis like Akt inhibition caused. Nevertheless M1p53ts-Flip cell lines fail to completely protect against p53 mediated apoptosis, suggesting that FLIP is itself insufficient to protect against apoptosis. Therefore we examined what other proteins may synergize with FLIP to protect from apoptosis, and found that that the pro-survival bcl2 protein family member MCL-1 is strongly up regulated by IL6 in our M1p53 cell line. Generation of M1p53-FLIP-MCL1, M1p53-MCL1, and M1p53 -uncleavable FLIP cell lines is underway to determine whether there is synergy between MCL-1 and FLIP in escape from p53 mediated apoptosis of M1p53 cells. Thus far taken together these data support a model for leukemic progression where cells that acquire the ability to produce an autocrine survival factor, such as IL6, can bypass normal p53 surveillance function by targeting downstream inhibitors of apoptosis such Akt, c-Flip and MCL-1.

Growth arrest and DNA damage, Gadd45 gene family members are rapidly induced by genotoxic agents as well as by apoptosis and differentiation inducing cytokines. Their role in hemetopoiesis, wherein proliferation, differentiation and apoptosis integrate to maintain cellular homeostasis, is not clear. Using bone marrow cells from gadd45a or gadd45b deficient and wild type littermate mice we have investigated the role of Gadd45 proteins in cytokine induced myeloid cell differentiation in vitro. Bone marrow cells obtained from either gadd45a or gadd45b deficient mice displayed compromised cytokines (IL3, GM-CSF, M-CSF or G-CSF) induced myelopoiesis, resulting in a quantitatively decreased population of mature myeloid cells. Immuno-phenotyping with antibodies to cell surface molecules associated with myeloid cell maturation confirmed impaired myeloid cell maturation in Gadd45a or b deficient bone marrow cells treated with the above cytokines. Analysis of apoptosis by annexin-V and PI staining followed by FACS analysis showed a substantially higher apoptosis in Gadd45a−/− as well as gadd45b−/− cells compared to wild type cells after treatment with M-CSF or G-CSF. Gadd45a−/− as well as gadd45b−/− bone marrow cells were found to be less clonogenic in methylcellulose medium. Morphologically compact and round colonies consisting of immature myeloid cells prevailed over dispersed- colonies consisting of mature myeloid cells in gadd45- deficient cells cultured in methyl cellulose containing IL-3. Furthermore, colony re-plating assay showed better self-renewal abilities in gadd45a−/− as well as gadd45b−/− progenitors, compared to wild type progenitor cells. Altered myelopoiesis in gadd45 a or b deficient mice was further confirmed in vivo by intra-peritoneal administration of sodium casienate - a known inducer of inflammatory response and myelopoiesis in mice bone marrow. Sodium casienate failed to enhance myelopoiesis in gadd45a or gadd45b deficient mice bone marrow, while wild type littermate mice showed a rapid induction of myelopoiesis. Simultaneously peritoneal exudates collected from gadd45 deficient mice consisted of 2–3 fold less myeloid cells compared to age matched wild type control mice after sodium casienate treatment. Gadd45a−/− or gadd45b−/− mice showed a slow recovery after myelo-suppressive effect of antimetabolite 5-Fluorouracil, which further confirmed that gadd45 deficiency leads to delayed myelopoiesis in mouse. Mechanistic aspects of Gadd45 deficiency, which results in impaired myelopoiesis are under investigation.

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.

Transforming growth factor-beta(TGF-beta)-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. In this report, we identify GADD45b as an effector of TGF-beta-induced apoptosis. GADD45b has been shown to be a positive mediator of apoptosis induced by certain cytokines and oncogenes. We show that Gadd45b is an immediate-early response gene for TGF-beta and that the proximal Gadd45b promoter is activated by TGF-beta through the action of Smad2, Smad3, and Smad4. We show that ectopic expression of GADD45b in AML12 murine hepatocytes is sufficient to activate p38 and to trigger apoptotic cell death, whereas antisense inhibition of Gadd45b expression blocks TGF-beta-dependent p38 activation and apoptosis. Furthermore, we also show that TGF-beta can activate p38 and induce apoptosis in mouse primary hepatocytes from wild-type mice, but not from Gadd45b(-/-) mice. All of these findings suggest that GADD45b participates in TGF-beta-induced apoptosis by acting upstream of p38 activation.

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. © 2002 Wiley‐Liss, Inc.

Jak3, a member of the Janus kinase family of cytoplasmic tyrosine kinases, is expressed at low levels in immature hematopoietic cells and its expression is dramatically up-regulated during the terminal differentiation of these cells. To better understand the role of Jak3 in myeloid cell development, we have investigated the role of Jak3 in myeloid cell differentiation using the 32Dcl3 cell system. Our studies show that Jak3 is a primary response gene for granulocyte colony-stimulating factor (G-CSF) and the accumulation of tyrosine phosphorylated Jak3 correlated with cell growth inhibition and terminal granulocytic differentiation in response to G-CSF. Ectopic overexpression of Jak3 in 32Dcl3 cells resulted in an acceleration of the G-CSF-induced differentiation program that was preceded by G(1) cell cycle arrest, which was associated with the up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) and down-regulation of Cdk2, Cdk4, Cdk6, and Cyclin E. In addition, ectopic overexpression of Jak3 appears to result in the inactivation of PKB/Akt and Stat3-mediated proliferative pathways in the presence of G-CSF. Similarly, overexpression of Jak3 in primary bone marrow cells resulted in an acceleration of granulocytic differentiation in the presence of granulocyte-macrophage colony-stimulating factor, which was associated with their growth arrest in the G(1) phase of the cell cycle. Taken together, these results indicate that Jak3-mediated signals play an important role in myeloid cell differentiation.

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 (M yD88, Gadd45α, MyD118/Gadd45β, GADD45γ, MyD116/ Gadd34 ) genes, that have been cloned by virtue of being co-ordinately induced upon the onset of terminal myeloid differentiation and following exposure of cells to stress stimuli. In recent years it has become evident that MyD/Gadd play a role 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. MyD/Gadd are also involved in inflammatory responses to invading micro-organisms, and response to environmental stress and physiological stress, such as hypoxia, which results in ischemic tissue damage. An intricate network of interactions among MyD/GADD genes and gene products appears to control their diverse functions. Deregulated growth, increased cell survival, compromised differentiation and deficiencies in DNA repair are hallmarks of malignancy and its progression. Thus, the role MyD/Gadd play in negative growth control, including cell cycle arrest and apoptosis, and in DNA repair, make them attractive molecular targets for tumor suppression. The role MyD/Gadd play in innate immunity and host response to hypoxia also make these genes and gene products attractive molecular targets to treat immunity and inflammation disorders, such as septic shock and ischemic tissue damage.

GADD45, MyDII8, and CR6 (also termed GADD45 alpha, beta, and gamma) comprise a family of genes that encode for related proteins playing important roles in negative growth control, including growth suppression. Data accumulated suggest that MyD118/GADD45/CR6 serve similar but not identical functions along different apoptotic and growth suppressive pathways. It is also apparent that individual members of the MyD118/GADD45/CR6 family are differentially induced by a variety of genetic and environmental stress agents. The MyD118, CR6, and GADD45 proteins were shown to predominantly localize within the cell nucleus. Recently, we have shown that both MyD118 and GADD45 interact with proliferating cell nuclear antigen (PCNA), a protein that plays a central role in DNA replication, DNA repair, and cell cycle progression, as well as with the universal cyclin-dependent kinase inhibitor p21, In this work we show that also CR6 interacts with PCNA and p21, Moreover, it is shown that CR6 interacts with PCNA via a domain that also mediates interaction of both GADD45 and MyD118 with PCNA. Importantly, evidence has been obtained that interaction of CR6 with PCNA impedes the function of this protein in negative growth control, similar to observations reported for MyD118 and GADD45 (1).

Using a variety of differentiation-inducible myeloid cell lines, we previously showed that the zinc-finger transcription factor early growth response gene 1 (Egr-1) is a positive modulator of macrophage differentiation and negatively regulates granulocytic differentiation. In this study, high-efficiency retroviral transduction was used to ectopically express Egr-1 in myeloid-enriched or stem cell-enriched bone marrow cultures to explore its effect on the development of hematopoietic progenitors in vitro and in lethally irradiated mice. It was found that ectopic Egr-1 expression in normal hematopoietic progenitors stimulates development along the macrophage lineage at the expense of development along the granulocyte or erythroid lineages, regardless of the cytokine used. Moreover, Egr-1 accelerated macrophage development by suppressing the proliferative phase of the growth-to-macrophage developmental program. The remarkable ability of Egr-1 to dictate macrophage development at the expense of development along other lineages resulted in failure of Egr-1-infected hematopoietic progenitors to repopulate the bone marrow and spleen, and thereby prevent death, in lethally irradiated mice. These observations further highlight the role Egr-1 plays in monocytic differentiation and growth suppression.