Gadd45a在抑制细胞转化和肿瘤恶性进展中的作用

Gadd45a,一个受p53和BRCA1调节的生长阻滞和DNA损伤基因,在抑制细胞转化和肿瘤恶性进展中扮演重要的角色.Gadd45a可以通过抑制细胞生长以及促进DNA损伤修复等间接或者直接方式维持基因组稳定性,从而抑制细胞转化和肿瘤的恶性进展.此外,Gadd45a还可通过对一些信号传导通路的调节,参与肿瘤发生发展的抑制.     

HDAC inhibitors induce apoptosis but not cellular senescence in Gadd45α-deficient E1A+Ras cells

HDAC inhibitors (HDIs) induce irreversible cell cycle arrest and senescence in E1A+Ras expressing cells. Furthermore, HDIs activate Gadd45α/NF-κB signaling pathway to suppress apoptosis thereby promoting the cell survival. Here, to clarify the role of Gadd45α in realization of the antiapoptotic program, we compared wild-type E1A+Ras cells and the cells with knockout of gadd45α gene (Gadd45α−/− cells). As in Gadd45α-expressing E1A+Ras cells, HDIs induce irreversible cell cycle arrest in Gadd45α−/− cells, but the arrested cells do not senesce and eventually die due to activation of the apoptotic death program. These data suggest that the expression of Gadd45α is involved in maintaining the balance of pro- and anti-apoptotic stimuli, while lack or loss of Gadd45 directs the cells to apoptosis after HDIs treatment. Appropriately Gadd45α-deficient cells demonstrate a higher level of pro-apoptotic signals, whereas the anti-apoptotic program is suppressed. The elevated apoptotic background of Gadd45α−/− cells is accompanied by higher levels of Ser15-phosphorylated p53 and p21/Waf1 proteins that additionally commit the cells to HDIs-induced apoptosis. Additionally, loss of Gadd45α protein activates the DDR signaling pathway as demonstrated by nuclear pATM staining, accumulation of γH2AX foci and an increase of single-strand DNA breaks. Thus, in wild-type E1A+Ras cells the p53-dependent expression of Gadd45α is necessary not only for DNA repair and HDI-induced cellular senescence, but also to withstand to apoptosis after DNA damage and stress. Therefore the use of HDIs in combination with agents that block Gadd45α function may have promise for cancer therapy.     

Oligomerization of human Gadd45a protein

Gadd45a is an 18-kDa acidic protein that is induced by genotoxic and certain other cellular stresses. The exact function of this protein is not known. However, there is evidence for its involvement in growth control, maintenance of genomic stability, DNA repair, cell cycle control, and apoptosis. Consistently, Gadd45a has previously been shown to interact in vitro and/or in vivo with a number of proteins playing central roles in these cellular processes: proliferating cell nuclear antigen, p21(Cip1/Waf1), Cdc2-CyclinB complex, MTK1, and histones. Adding to this complexity, we have found that Gadd45a self-associates in solution, both in vitro and when expressed in the cell. Moreover, Gadd45a can complex with the two other members of the Gadd45 family of stress-induced proteins, human Gadd45b (MyD118) and Gadd45g (CR6). Gel-exclusion chromatography, native gel electrophoretic analysis, enzyme-linked immunosorbent assay, and chemical cross-linking showed that recombinant Gadd45a forms dimeric, trimeric, and tetrameric species in vitro, the dimers being the predominant form. Deletion mutant and peptide scanning analyses suggest that Gadd45a has two self-association sites: within N-terminal amino acids 33-61 and within 40 C-terminal amino acids. Despite the low abundance of Gadd45a in the cell, oligomer-forming concentrations can probably be achieved in the foci-like nuclear structures formed by the protein upon overexpression. Evidence for a potential role of Gadd45a self-association in altering DNA accessibility on damaged nucleosomes is presented.     

Gadd45a expression induces Bim dissociation from the cytoskeleton and translocation to mitochondria

Gadd45a, a p53- and BRCA1-regulated stress protein, has been implicated in the maintenance of genomic fidelity, probably through its roles in the control of cell cycle checkpoint and apoptosis. However, the mechanism(s) by which Gadd45a is involved in the induction of apoptosis remains unclear. We show here that inducible expression of Gadd45a protein causes dissociation of Bim, a Bcl2 family member, from microtubule-associated components and translocation to mitochondria. The Bim accumulation in mitochondria enhances interaction of Bim with Bcl-2, relieves Bax from Bcl-2-bound complexes, and subsequently results in release of cytochrome c into the cytoplasm. Suppression of endogenous Bim greatly inhibits Gadd45a induction of apoptosis. Interestingly, Gadd45a interacts with elongation factor 1alpha (EF-1alpha), a microtubule-severing protein that plays an important role in maintaining cytoskeletal stability, and inhibits EF-1alpha-mediated microtubule bundling, indicating that the interaction of Gadd45a with EF-1alpha disrupts cytoskeletal stability. A mutant form of Gadd45a harboring a deletion of EF-1alpha-binding domain fails to inhibit microtubule stability and to induce Bim translocation to mitochondria. Furthermore, coexpression of EF-1alpha antagonizes Gadd45a's property of suppressing cell growth and inducing apoptosis. These findings identify a novel link that connects stress protein Gadd45a to the apoptotic machinery and address the importance of cytoskeletal stability in apoptotic response to DNA damage.     

Gadd45 mutations are uncommon in human tumour cell lines

GADD45 is an evolutionarily conserved gene that encodes a small acidic, nuclear protein and is an example of a p53 responsive gene. Gadd45 protein has been shown to interact with PCNA and also p21^waf1. It has been implicated in growth arrest, DNA repair, chromatin structure and signal transduction. The confusing biochemical data has been clarified by the demonstration that Gadd45 null mice have a phenotype strikingly similar to that of p53 null mice, being tumour prone and showing marked genomic instability. We have tested the hypothesis that mutations in the GADD45 coding region might substitute for p53 abnormalities in tumour cell lines where p53 is wild type. After generating cDNA from mRNA in a panel of 24 cell lines we sequenced the GADD45 cDNA and have demonstrated that no mutations can be observed, even in the p53 wild type cell lines. Such data suggest that Gadd45 mutations are uncommon in human cancer. From this we postulate that, despite the phenotype of the GADD45 null mouse, GADD45 is unlikely to be the key mechanistic determinant of the tumour suppressor activity of the p53 pathway.
Note on nomenclature: We have employed GADD45 to designate the gene and Gadd45 to designate the encoded protein. This gene has also be denoted GADD45 α elsewhere in the literature.     

Gadd45a interacts with aurora-A and inhibits its kinase activity

Centrosome stability is required for successful mitosis in mammalian cells. Amplification of the centrosome leads to chromosomal missegregation and generation of aneuploidy, which are closely associated with cell transformation and tumorigenesis (Doxsey, S. J. (2001) Nat. Cell Biol. 3, E105-E108; Hinchcliffe, E. H., and Sluder, G. (2001) Genes Dev. 15, 1167-1181; Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., and Doxsey, S. J. (2001) Cancer Res. 61, 2212-2219). However, there are currently limited insights into mechanism(s) for this critical biological event. Here we show that Gadd45a, a DNA damage-inducible protein that is regulated by tumor suppressors p53 and BRCA1, participates in the maintenance of centrosome stability. Mouse embryonic fibroblasts derived from gadd45a knock-out mice exhibit centrosome amplification (designated as increased centrosome numbers). Introduction of exogenous Gadd45a into mouse embryonic fibroblasts isolated from gadd45a-null mice substantially restored the normal centrosome profile. In contrast to p21(waf1/cip1), which ensures coordinated initiation of centrosome, Gadd45a had no significant effect on centrosome duplication in S phase. Interestingly Gadd45a was found to physically associate with Aurora-A protein kinase, whose deregulated expression results in centrosome abnormality. Furthermore Gadd45a was demonstrated to strongly inhibit Aurora-A kinase activity and to antagonize Aurora-A-induced centrosome amplification. These findings identify a novel mechanism for Gadd45a in the maintenance of centrosome stability and broaden understandings of p53- and BRCA1-regulated signaling pathways in maintaining genomic fidelity.     

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.     

Crystal structure of human Gadd45 reveals an active dimer

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45, revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45 identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.     

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.     

Gadd45a deletion aggravates hematopoietic stem cell dysfunction in ATM-deficient mice

Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM^-/-) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM^-/- HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM^-/- mice. Instead, ATM and Gadd45a double knockout (ATM^-/- Gadd45a^-/-) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM^-/- HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM^-/- Gadd45a^-/- HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM^-/- Gadd45a^-/- mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM^-/- mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM^-/- HSCs.     

Genomic Instability in Gadd45a-/- Cells is Coupled with S-Phase Checkpoint Defects

Gadd45a is a p53-regulated gene whose protein product, like p53, is involved in maintenanceof genome stability. Specifically, deletion of Gadd45a leads to extensive aneuploidy as aconsequence of centrosome amplification and subsequent abnormal segregation of chromosomesduring mitosis. S-phase checkpoints were investigated in Gadd45a-/- cells to determine possibledefects contributing to the uncoupling of centrosome duplication and DNA replication. In thepresence of hydroxyurea, Gadd45a-/- mouse embryo fibroblasts show increased centrosomeamplification coupled with loss of a sustained S-phase checkpoint. Gadd45a deletion allowsanother form of genomic instability, gene amplification, when p21 (Cdkn1a gene product) isdeleted also. Gene amplification in Gadd45a-/-p21-/- cells correlated with loss of both G1 andS-phase checkpoints. Multiple conditions of nutrient deprivation failed to prevent DNAsynthesis in Gadd45a-/- cells. Gadd45a is therefore required for proper S-phase control andcheckpoints under multiple conditions of nutrient deprivation. It is proposed that loss of S-phasecontrol may account for both the uncoupling of DNA replication and centrosome duplication,and conferring gene amplification proficiency in cells lacking Gadd45a-/-. This is of particularimportance for solid tumors, which may lack sufficient nutrients yet are unable to elicitcheckpoints preventing genomic instability under these conditions.     

BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention

BRCA1 is involved in maintaining genomic integrity and, as a regulator of the G2/M checkpoint, contributes to DNA repair and cell survival. The overexpression of BRCA1 elicits diverse cellular responses including apoptosis due to the stimulation of specific signaling pathways. BRCA1 is normally regulated by protein turnover, but is stabilized by BARD1 which can recruit BRCA1 to the nucleus to form a ubiquitin E3 ligase complex involved in DNA repair or cell survival. Here, we identify BARD1 as a regulator of BRCA1-dependent apoptosis. Using transfected MCF-7 breast cancer cells, we found that BRCA1-induced apoptosis was independent of p53 and was stimulated by BRCA1 nuclear export. Conversely, BARD1 reduced BRCA1-dependent apoptosis by a mechanism involving nuclear sequestration. Regulation of apoptosis by BARD1 was reduced by BRCA1 cancer mutations that disrupt Ub ligase function. Transfection of BRCA1 N-terminal peptides that disrupted the cellular BRCA1-BARD1 interaction caused a loss of nuclear BRCA1 that correlated with increased apoptosis in single cell assays, but did not alter localization or expression of endogenous BARD1. Reducing BARD1 levels by siRNA caused a small increase in apoptosis. Our findings identify a novel apoptosis inhibitory function of BARD1 and suggest that nuclear retention of BRCA1-BARD1 complexes contributes to both DNA repair and cell survival.     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.