The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression

Cell cycle growth arrest is an important cellular response to genotoxic stress. Gadd45, a p53-regulated stress protein, plays an important role in the cell cycle G(2)-M checkpoint following exposure to certain types of DNA-damaging agents such as UV radiation and methylmethane sulfonate. Recent findings indicate that Gadd45 interacts with Cdc2 protein and inhibits Cdc2 kinase activity. In the present study, a series of Myc-tagged Gadd45 deletion mutants and a Gadd45 overlapping peptide library were used to define the Gadd45 domains that are involved in the interaction of Gadd45 with Cdc2. Both in vitro and in vivo studies indicate that the interaction of Gadd45 with Cdc2 involves a central region of the Gadd45 protein (amino acids 65-84). The Cdc2-binding domain of Gadd45 is also required for Gadd45 inhibition of Cdc2 kinase activity. Sequence analysis of the central Gadd45 region reveals no homology to inhibitory motifs of known cyclin-dependent kinase inhibitors, indicating that the Cdc2-binding and -inhibitory domains on Gadd45 are a novel motif. The peptide containing the Cdc2-binding domain (amino acids 65-84) disrupted the Cdc2-cyclin B1 protein complex, suggesting that dissociation of this complex results from a direct interaction between the Gadd45 and Cdc2 proteins. GADD45-induced cell cycle G(2)-M arrest was abolished when its Cdc2 binding motif was disrupted. Importantly, a short term survival assay demonstrated that GADD45-induced cell cycle G(2)-M arrest correlates with GADD45-mediated growth suppression. These findings indicate that the cell cycle G(2)-M growth arrest mediated by GADD45 is one of the major mechanisms by which GADD45 suppresses cell growth.     

Involvement of the interaction between p21 and proliferating cell nuclear antigen for the maintenance of G2/M arrest after DNA damage

Although a major effect of p21, a cyclin-dependent kinase inhibitor, is considered to be exerted during G(1) phase of the cell cycle, p21 gene knock-out studies suggested its involvement in G(2)/M checkpoint as well. Here we demonstrate evidence that p21 is required for the cell cycle arrest at G(2) upon DNA damage. We found that expression of wild-type p21 (p21(WT)), not mutant p21 (p21(PCNA-)) lacking the interaction with proliferating cell nuclear antigen (PCNA), caused G(2) cell cycle arrest in p53-deficient DLD1 colon cancer cell line after the DNA damage by treatment with cis-diamminedichloroplatinum (II). We also found that p21(WT) was associated with Cdc2/cyclin B1 together with PCNA. Furthermore, coimmunoprecipitation experiments revealed that PCNA interacted with Cdc25C at the G(2)/M transition, and this interaction was abolished when p21(WT) was expressed presumably due to the competition between p21(WT) and Cdc25C in the binding to PCNA. These findings suggest that p21 plays a regulatory role in the maintenance of cell cycle arrest at G(2) by blocking the interaction of Cdc25C with PCNA.     

Opposite effect of NF-kappa B and c-Jun N-terminal kinase on p53-independent GADD45 induction by arsenite

Cell cycle checkpoint, a major genomic surveillance mechanism, is an important step in maintaining genomic stability and integrity in response to environmental stresses. Using cells derived from human bronchial epithelial cells, we demonstrate that NF-kappaB and c-Jun N-terminal kinase (JNK) reciprocally regulate arsenic trioxide (arsenite)-induced, p53-independent expression of GADD45 protein, a cell cycle checkpoint protein that arrests cells at the G(2)/M phase transition. Inhibition of NF-kappaB activation by stable expression of a kinase-mutated form of IkappaB kinase caused increased and prolonged induction of GADD45 by arsenite. In contrast, the induction of GADD45 by arsenite was transient and less potent in cells where the NF-kappaB activation pathway was normal. Analysis of the cell cycle profile by flow cytometry indicated that NF-kappaB inhibition potentiates arsenite-induced G(2)/M cell cycle arrest. Abrogation of JNK activation, on the other hand, decreased GADD45 expression induced by arsenite, suggesting a role for JNK activation in GADD45 induction. These results indicate a molecular mechanism by which NF-kappaB and JNK may differentially contribute to cell cycle regulation in response to arsenite.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G(2)/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3beta. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G(2)/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.     

Oxaline, a fungal alkaloid, arrests the cell cycle in M phase by inhibition of tubulin polymerization

Oxaline and neoxaline, fungal alkaloids, were found to inhibit cell proliferation and to induce cell cycle arrest at the G(2)/M phase in Jurkat cells. CBP501 (a peptide corresponding to amino acids 211-221 of Cdc25C phosphatase), which inhibits the G(2) checkpoint, did not affect the G(2)/M arrest caused by oxaline, suggesting that oxaline causes M phase arrest but not G(2) phase arrest. The Cdc2 phosphorylation level of oxaline-treated cell lysate was lower than that of the control cells, indicating that oxaline arrests the M phase. Oxaline disrupted cytoplasmic microtubule assembly in 3T3 cells. Furthermore, oxaline inhibited polymerization of microtubule protein and purified tubulin dose-dependently in vitro. In a binding competition assay, oxaline inhibited the binding of [(3)H]colchicine to tubulin, but not that of [(3)H]vinblastine. These results indicate that oxaline inhibits tubulin polymerization, resulting in cell cycle arrest at the M phase.     

Human immunodeficiency virus type 1 Vpr binds to the N lobe of the Wee1 kinase domain and enhances kinase activity for CDC2

Human immunodeficiency virus type 1 Vpr is a virion-associated accessory protein that has multiple activities within an infected cell. One of the most dramatic effects of Vpr is the induction of cell cycle arrest at the G(2)/M boundary, followed by apoptosis. This effect has implications for CD4(+) cell loss in AIDS. In normal cell cycle regulation, Wee1, a key regulator for G(2)-M progression, phosphorylates Tyr15 on Cdc2 and thereby blocks the progression of cells into M phase. We demonstrate that Vpr physically interacts with Wee1 at the N lobe of the kinase domain analogous to that present in other kinases. This interaction with Vpr enhances Wee1 kinase activity for Cdc2. Overexpression of Wee1 kinase-deficient mutants competes for Vpr-mediated cell cycle arrest, and deletion of the region of Wee1 that binds Vpr abrogates that competition. However, the Vpr mutants I74P and I81P, which fail to induce G(2) arrest, can bind to and increase the kinase activity of Wee1 to the same extent as wild-type Vpr. Therefore, we conclude that the binding of Vpr to Wee1 is not sufficient for Vpr to activate the G(2) checkpoint, and it may reflect an independent function of Vpr.     

Checkpoint kinase 1 regulates diallyl trisulfide-induced mitotic arrest in human prostate cancer cells

We have shown previously that diallyl trisulfide (DATS), a constituent of processed garlic, inhibits proliferation of PC-3 and DU145 human prostate cancer cells by causing G(2)-M phase cell cycle arrest in association with inhibition of cyclin-dependent kinase 1 activity and hyperphosphorylation of Cdc25C at Ser(216). Here, we report that DATS-treated PC-3 and DU145 cells are also arrested in mitosis as judged by microscopy following staining with anti-alpha-tubulin antibody and 4',6-diamidino-2-phenylindole and flow cytometric analysis of Ser(10) phosphorylation of histone H3. The DATS treatment caused activation of checkpoint kinase 1 and checkpoint kinase 2, which are intermediaries of DNA damage checkpoints and implicated in Ser(216) phosphorylation of Cdc25C. The diallyl trisulfide-induced Ser(216) phosphorylation of Cdc25C as well as mitotic arrest were significantly attenuated by knockdown of check-point kinase 1 protein in both PC-3 and DU145 cells. On the other hand, depletion of checkpoint kinase 2 protein did not have any appreciable effect on G(2) or M phase arrest or Cdc25C phosphorylation caused by diallyl trisulfide. The lack of a role of checkpoint kinase 2 in diallyl trisulfide-induced phosphorylation of Cdc25C or G(2)-M phase cell cycle arrest was confirmed using HCT-15 cells stably transfected with phosphorylation-deficient mutant (T68A mutant) of checkpoint kinase 2. In conclusion, the results of the present study suggest existence of a checkpoint kinase 1-dependent mechanism for diallyl trisulfide-induced mitotic arrest in human prostate cancer cells.     

DNA mismatch repair-dependent activation of c-Abl/p73alpha/GADD45alpha-mediated apoptosis

Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.     

Increased levels of Wee-1 kinase in G(2) are necessary for Vpr- and gamma irradiation-induced G(2) arrest

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle arrest at the G(2)/M transition and subsequently apoptosis. Here we examined the potential involvement of Wee-1 in Vpr-induced G(2) arrest. Wee-1 is a cellular protein kinase that inhibits Cdc2 activity, thereby preventing cells from proceeding through mitosis. We previously showed that the levels of Wee-1 correlate with Vpr-mediated apoptosis. Here, we demonstrate that Vpr-induced G(2) arrest correlated with delayed degradation of Wee-1 at G(2)/M. Experimental depletion of Wee-1 by a small interfering RNA directed to wee-1 mRNA alleviated Vpr-induced G(2) arrest and allowed apparently normal progression through M into G(1). Similar results were observed when cells were arrested at G(2) following gamma irradiation. Thus, Wee-1 is integrally involved as a key cellular regulatory protein in the signal transduction pathway for HIV-1 Vpr-induced cell cycle arrest.     

Role of c-Abl kinase in DNA mismatch repair-dependent G2 cell cycle checkpoint arrest responses

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.     

Hepatitis B virus X protein differentially regulates cell cycle progression in X-transforming versus nontransforming hepatocyte (AML12) cell lines.

Hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronically infected HBV patients. To understand mechanism(s) of pX-mediated cellular transformation, we employed two tetracycline-regulated, pX-expressing cell lines, constructed in AML12 immortalized hepatocytes: one a differentiated (3pX-1) and the other a de-differentiated (4pX-1) hepatocyte cell line. Only 3pX-1 cells undergo pX-mediated transformation, via sustained Ras-Raf-mitogen-activated protein kinase pathway activation. pX-nontransforming 4pX-1 cells display sustained, pX-dependent JNK pathway activation. To understand how pX mediates different growth characteristics in 3pX-1 and 4pX-1 cells, we report, herein, comparative cell cycle analyses. pX-transforming 3pX-1 cells display pX-dependent G(1), S, and G(2)/M progression evidenced by cyclin D(1), A, and B(1) induction, and Cdc2 kinase activation. pX-nontransforming 4pX-1 cells display pX-dependent G(1) and S phase entry, followed by S phase pause and absence of Cdc2 kinase activation. Interestingly, 4pX-1 cells exhibit selective pX-induced expression of cyclin-dependent kinase inhibitor p21(Cip1), tumor suppressor p19(ARF), and proapoptotic genes bax and IGFBP-3. Despite the pX-mediated induction of growth arrest and apoptotic genes and the absence of pX-dependent Cdc2 activation, 4pX-1 cells do not undergo pX-dependent G(2)/M arrest or apoptosis. Nocodazole-treated, G(2)/M-arrested 4pX-1 cells exhibit pX-dependent formation of multinucleated cells, similar to human T-cell lymphotropic virus type I Tax-expressing cells. We propose that in 4pX-1 cells, pX deregulates the G(2)/M checkpoint, thus rescuing cells from pX-mediated apoptosis.     

Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes

Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes.     

GADD45beta/GADD45gamma and MEKK4 comprise a genetic pathway mediating STAT4-independent IFNgamma production in T cells

The stress-inducible molecules GADD45beta and GADD45gamma have been implicated in regulating IFNgamma production in CD4 T cells. However, how GADD45 proteins function has been controversial. MEKK4 is a MAP kinase kinase kinase that interacts with GADD45 in vitro. Here we generated MEKK4-deficient mice to define the function and regulation of this pathway. CD4 T cells from MEKK4-/- mice have reduced p38 activity and defective IFNgamma synthesis. Expression of GADD45beta or GADD45gamma promotes IFNgamma production in MEKK4+/+ T cells, but not in MEKK4-/- cells or in cells treated with a p38 inhibitor. Thus, MEKK4 mediates the action of GADD45beta and GADD45gamma on p38 activation and IFNgamma production. During Th1 differentiation, the GADD45beta/GADD45gamma/MEKK4 pathway appears to integrate upstream signals transduced by both T cell receptor and IL12/STAT4, leading to augmented IFNgamma production in a process independent of STAT4.     

Phenyl hydroquinone, an Ames test-negative carcinogen, induces Hog1-dependent stress response signaling

Recently, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae. We further found that phenyl hydroquinone arrested the cell cycle at G(1) and G(2)/M. In this study, we demonstrate that phenyl hydroquinone can arrest the cell cycle at the G(2)/M transition as a result of stabilization of Swe1 (a Wee1 homolog), probably leading to inactivation of Cdc28 (a Cdk1/Cdc2 homolog). Furthermore, Hog1 (a p38 MAPK homolog) was robustly phosphorylated by phenyl hydroquinone, which can stabilize Swe1. On the other hand, Chk1 and Rad53 were not phosphorylated by phenyl hydroquinone, indicating that the Mec1/Tel1 DNA-damage checkpoint was not functional. Mutations of swe1 and hog1 abolished phenyl hydroquinone-induced arrest at the G(2)/M transition and the cells became resistant to phenyl hydroquinone lethality and aneuploidy development. These data suggest that a phenyl hydroquinone-induced G(2)/M transition checkpoint that is activated by the Hog1-Swe1 pathway plays a role in the development of aneuploidy.     

Interaction of CR6 (GADD45gamma ) with proliferating cell nuclear antigen impedes negative growth control

GADD45, MyD118, and CR6 (also termed GADD45alpha, 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.     

UVB-Induced G2 Arrest of Human Melanocytes Involves Cdc2 Sequestration by Gadd45a in Nuclear Speckles

Exposure to solar UVB radiation is involved in the development of cutaneous melanoma. We previously showed that human melanocytes and melanoma cells respond to UVB radiation via a p53-independent pathway involving GADD45A activation. Here, we determined that UVB-induction of Gadd45a is necessary for G2 arrest and that Gadd45a and its partner p21Waf1 co-localize in nuclear bodies called Nuclear Speckles. We further observed that UVB-induced G2 arrest is associated with Cdc2 accumulation in these Nuclear Speckles. Knock-down of Gadd45a expression by RNA interference prevents both UVB-induced Cdc2 accumulation in Nuclear Speckles and G2 arrest. Our results demonstrate that UVB-induced G2 arrest of melanoma cells is Gadd45a-dependent. Furthermore, we show that Cdc2 sequestration by Gadd45a occurs in Nuclear Speckles, suggesting a new role for these nuclear bodies, so far only linked to RNA maturation.     

The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior to the onset of apoptosis

In contrast to extracellular signals, the mechanisms utilized to transduce nuclear apoptotic signals are not well understood. Characterizing these mechanisms is important for predicting how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death domain-containing protein p84N5 can induce apoptosis that is inhibited by association with Rb. The pattern of caspase and NF-kappaB activation during p84N5-induced apoptosis is similar to p53-independent cellular responses to DNA damage. One hallmark of this response is the activation of a G(2)/M cell cycle checkpoint. In this report, we characterize the effects of p84N5 on the cell cycle. Expression of p84N5 induces changes in cell cycle distribution and kinetics that are consistent with the activation of a G(2)/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine blocks p84N5-induced G(2)/M arrest but not subsequent apoptotic cell death. The p84N5-induced checkpoint is functional in ataxia telangiectasia-mutated kinase-deficient cells. We conclude that p84N5 induces an ataxia telangiectasia-mutated kinase (ATM)-independent, caffeine-sensitive G(2)/M cell cycle arrest prior to the onset of apoptosis. This conclusion is consistent with the hypotheses that p84N5 functions in an Rb-regulated cellular response that is similar to that triggered by DNA damage.