A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.

Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells. Finally, IR induction of the human GADD45 gene, an induction that is also defective in AT cells, was dependent on wild-type p53 function. Wild-type but not mutant p53 bound strongly to a conserved element in the GADD45 gene, and a p53-containing nuclear factor, which bound this element, was detected in extracts from irradiated cells. Thus, we identified three participants (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.     

Insulin receptor signaling activated by penta-<Emphasis Type="Italic">O</Emphasis>-galloyl-α-<Emphasis Type="SmallCaps">d</Emphasis>-glucopyranose induces p53 and apoptosis in cancer cells

p53 is essential for cell cycle arrest and apoptosis induction while insulin receptor (IR) signaling is important for cell metabolism and proliferation and found upregulated in cancers. While IR has recently been found to be involved in apoptosis, p53 induction or apoptosis mediated through IR signaling activation has never been documented. Here, we report that the IR signaling pathway, particularly the IR-MEK pathway, mediates biological and biochemical changes in p53 and apoptosis in tumor cells. Specifically, natural compound penta-O-galloyl-α-d-glucopyranose (α-PGG), a previously characterized IR signaling activator, induced apoptosis in RKO cells without significantly affecting its normal counterpart FHC cells. α-PGG induced apoptosis in RKO cells through p53, Bax and caspase 3. Importantly, α-PGG’s ability to elevate p53 was diminished by IR inhibitor and IR-siRNA, suggesting a non-conventional role of IR as being involved in p53 induction. Further studies revealed that α-PGG activated MEK, a downstream signaling factor of IR. Blocking MEK significantly suppressed α-PGG-induced p53 and Bax elevation. All these results suggested that α-PGG induced p53, Bax, and apoptosis through the IR-MEK signaling pathway. The unique activity of α-PGG, a novel IR phosphorylation and apoptosis inducer, may offer a new therapeutic strategy for eliciting apoptotic signal and inhibiting cancer growth.     

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.     

A polymorphism but no mutations in the GADD45 gene in breast cancers

The p53 gene product is part of a pathway regulating growth arrest at the G₁ checkpoint of the cell cycle. Mutation of other components of this pathway, including the products of the ataxia telangiectasia (AT), GADD45, mdm2, and p21^WAF1/CIP1 genes may have effects comparable to mutations in the p53 gene. The GADD45 gene is induced by ionizing radiation and several DNA-damaging xenobiotics. Induction requires the binding of wild-type p53 to an evoulutionarily highly conserved putative intronic p53 binding site in intron 3 of GADD45. We recently analyzed the entire coding region of the p53 gene in primary breast cancers of Midwestern white women and found 21 mutations among 53 tumors (39,6%). We now have shown by direct sequencing that there are no mutations in the intronic p53 binding site of the GADD45 gene in any of the 53 primary breast cancers and no mutations in the entire coding region of the GADD45 gene in a subset of 26 consecutive tumors (12 with p53 mutation and 14 without p53 mutation). The only sequence variation detected was a common polymorphism in intron 3. The absence of mutations in the GADD45 gene, including the putative p53-binding intronic site, suggests that this gene is not a frequent target of mutations in breast cancer. Although mutations of the p53 gene have been studied in a wide spectrum of human cancers, GADD45 has not been examined in any tumor or cell line to the best of our knowledge. Our results raise the possibility that mutation of the GADD45 gene alone is not functionally equivalent to loss of wild-type p53 activity. Received: 14 September 1995     

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.     

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.     

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.     

G2/M arrest by 1,25-dihydroxyvitamin D3 in ovarian cancer cells mediated through the induction of GADD45 via an exonic enhancer

1,25-Dihydroxyvitamin D3 suppresses the growth of multiple human cancer cell lines by inhibiting cell cycle progression and inducing cell death. The present study showed that 1,25-dihydroxyvitamin D3 causes cell cycle arrest at the G2/M transition through p53-independent induction of GADD45 in ovarian cancer cells. Detailed analyses have established GADD45 as a primary target gene for 1,25-dihydroxyvitamin D3. A DR3-type vitamin D response element was identified in the fourth exon of GADD45 that forms a complex with the vitamin D receptor.retinoid X receptor heterodimer in electrophoresis mobility shift assays and mediates the dose-dependent induction of luciferase activity by 1,25-dihydroxyvitamin D3 in reporter assays. Chromatin immunoprecipitation assays have shown that the vitamin D receptor is recruited in a ligand-dependent manner to the exonic enhancer but not to the GADD45 promoter regions. In ovarian cancer cells expressing GADD45 antisense cDNA or GADD45-null mouse embryo fibroblasts, 1,25-dihydroxyvitamin D3 failed to induce G2/M arrest. Taken together, these results identify GADD45 as an important mediator for the tumor-suppressing activity of 1,25-dihydroxyvitamin D3 in human ovarian cancer cells.     

Co-activation of ERK, NF-kappaB, and GADD45beta in response to ionizing radiation

NF-kappaB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45beta is a primary cell cycle element responsive to NF-kappaB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-kappaB, ERK and GADD45beta are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-kappaB transactivation and GADD45beta expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-kappaB. Transfection of dominant negative mutant IkappaB that blocks NF-kappaB nuclear translocation, inhibited ERK activity and GADD45beta expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45beta strikingly blocked IR-induced NF-kappaB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-kappaB, ERK, and GADD45beta are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.     

Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2)

The tumor suppressor p53 is frequently mutated in human cancers. Upon activation it can induce cell cycle arrest or apoptosis. ASPP2 can specifically stimulate the apoptotic function of p53 but not cell cycle arrest, but the mechanism of enhancing the activation of pro-apoptotic genes over cell cycle arrest genes remains unknown. In this study, we analyzed the binding of 53BP2 (p53-binding protein 2, the C-terminal domain of ASPP2) to p53 core domain and various mutants using biophysical techniques. We found that several p53 core domain mutations (R181E, G245S, R249S, R273H) have different effects on the binding of DNA response elements and 53BP2. Further, we investigated the existence of a ternary complex consisting of 53BP2, p53, and DNA response elements to gain insight into the specific pro-apoptotic activation of p53. We found that binding of 53BP2 and DNA to p53 is mutually exclusive in the case of GADD45, p21, Bax, and PIG3. Both pro-apoptotic and non-apoptotic response elements were competed off p53 by 53BP2 with no indication of a ternary complex.     

ER stress and ASK1-JNK activation contribute to oridonin-induced apoptosis and growth inhibition in cultured human hepatoblastoma HuH-6 cells

Workers who are exposed to extreme heat or work in hot environments may be at risk of heat stress. Exposure to extreme heat can result in occupational illnesses and injuries. On the other hand, local and regional heat therapy has been used for the treatment of some cancers, such as liver cancer, lung cancer, and kidney cancer. Although heat stress has been shown to induce the accumulation of p53 protein, a key regulator of cell cycle, apoptosis, DNA repair, and autophagy, how it regulates p53 protein accumulation and what the p53 targets are remain unclear. Here, we show that, among various genotoxic stresses, including ionizing radiation (IR) and ultraviolet (UV) radiation, heat stress contributes significantly to increase p53 protein levels in normal liver cells and liver cancer cells. Heat stress did not increase p53 mRNA expression as well as p53 promoter activity. However, heat stress enhanced the half-life of p53 protein. Moreover, heat stress increased the expression of puma and light chain 3 (LC-3), which are associated with the apoptotic and autophagic function of p53, respectively, whereas it did not change the expression of the cell cycle regulators p21, 14-3-3δ, and GADD45α, suggesting that heat-triggered alteration of p53 selectively modulates the downstream targets of p53. Our study provides a novel mechanism by which heat shock stimulates p53 protein accumulation, which is different from common DNA damages, such as IR and UV, and also provides new molecular basis for heat injuries or heat therapy.     

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.     

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.