The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth.

A remarkable overlap was observed between the gadd genes, a group of often coordinately expressed genes that are induced by genotoxic stress and certain other growth arrest signals, and the MyD genes, a set of myeloid differentiation primary response genes. The MyD116 gene was found to be the murine homolog of the hamster gadd34 gene, whereas MyD118 and gadd45 were found to represent two separate but closely related genes. Furthermore, gadd34/MyD116, gadd45, MyD118, and gadd153 encode acidic proteins with very similar and unusual charge characteristics; both this property and a similar pattern of induction are shared with mdm2, whic, like gadd45, has been shown previously to be regulated by the tumor suppressor p53. Expression analysis revealed that they are distinguished from other growth arrest genes in that they are DNA damage inducible and suggest a role for these genes in growth arrest and apoptosis either coupled with or uncoupled from terminal differentiation. Evidence is also presented for coordinate induction in vivo by stress. The use of a short-term transfection assay, in which expression vectors for one or a combination of these gadd/MyD genes were transfected with a selectable marker into several different human tumor cell lines, provided direct evidence for the growth-inhibitory functions of the products of these genes and their ability to synergistically suppress growth. Taken together, these observations indicate that these genes define a novel class of mammalian genes encoding acidic proteins involved in the control of cellular growth.     

Genistein affects histone modifications on Dickkopf-related protein 1 (DKK1) gene in SW480 human colon cancer cell line

Genistein (GEN) is a plant-derived isoflavone and can block uncontrolled cell growth in colon cancer by inhibiting the WNT signaling pathway. This study aimed to test the hypothesis that the enhanced gene expression of the WNT signaling pathway antagonist, DKK1 by genistein treatment is associated with epigenetic modifications of the gene in colon cancer cells. Genistein treatment induced a concentration-dependent G2 phase arrest in the human colon cancer cell line SW480 and reduced cell proliferation. Results from several other human colon cancer cell lines confirmed the growth inhibitory effects of genistein. Overexpression of DKK1 confirmed its involvement in growth inhibition. Knockdown of DKK1 expression by siRNA slightly induced cell growth. DKK1 gene expression was increased by genistein in SW480 and HCT15 cells. DNA methylation at the DKK1 promoter was not affected by genistein treatment in all the cell lines tested. On the other hand, genistein induced histone H3 acetylation of the DKK1 promoter region in SW480 and HCT15 cells. This indicates that increased histone acetylation is associated with the genistein-induced DKK1 expression. The association between histone acetylation and DKK1 gene expression is confirmed by the histone deacetylase inhibitor trichostatin A (TSA) treatment. In conclusion, genistein treatment decreases cell growth and proliferation in colon cancer cell lines. The effect is associated with the increased DKK1 expression through the induction of histone acetylation at the DKK1 promoter region.     

C/EBPalpha regulation of the growth-arrest-associated gene gadd45.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is expressed in postmitotic, differentiated adipocytes and is required for adipose conversion of 3T3-L1 cells in culture. Temporal misexpression of C/EBPalpha in undifferentiated adipoblasts leads to mitotic growth arrest. We report here that growth arrest- and DNA damage-inducible gene 45 (gadd45) is preferentially expressed in differentiated 3T3-L1 adipocytes similar to phenotype-associated genes. Furthermore, C/EBPalpha transactivates a reporter plasmid containing 1.5 kb of the gadd45 promoter region. The proto-oncogene myc, which inhibits adipocyte differentiation, abrogates C/EBPalpha activation of gadd45. gadd45 is known to be a target of the tumor suppressor p53 in a G1 checkpoint activated by DNA damage. Immunoprecipitation of radiolabeled proteins with conformation-specific antibodies revealed that wild-type p53 is expressed throughout 3T3-L1 adipocyte development, including the postmitotic period characterized by the accumulation of gadd45 and C/EBPalpha. A stable 3T3-L1 subline was engineered to express a dominant negative p53, human p53(143ala). The p53(143ala) subline differentiated to adipocytes and showed appropriate developmental expression of gadd45. These findings suggest that postmitotic growth arrest is coupled to adipocyte differentiation via C/EBPalpha stimulation of growth arrest-associated and phenotype-associated genes.     

Butyrate as a model for "gene-regulating chemoprevention and chemotherapy."

Recent progress in molecular genetics has facilitated understanding of the mechanisms of carcinogenesis. However, there is not yet any effective therapy or prevention for cancer based on the molecular mechanisms of carcinogenesis. So-called "gene therapy" for cancer is expected to become a new method of treatment, but there are still several serious problems with gene therapy. As a matter of fact, it seems impossible to adopt gene therapy for prevention. We therefore tried to develop a different method of cancer prevention or therapy based on the molecular mechanisms of carcinogenesis. For instance, the tumor-suppressor gene p53 is mutated in about 50% of human malignancies. It is known that p53 stimulates the promoter activities of p21/WAF1, gadd45 and bax genes, resulting in cell cycle arrest, DNA repair and apoptosis, respectively. Therefore, chemical compounds that can stimulate these genes should compensate for the function of p53. As a model of this, we found that histone deacetylase inhibitors such as butyrate or trichostatin A dramatically stimulate the p21/WAF1 gene promoter through the Spl sites, resulting in cell cycle arrest. Interestingly, another group has recently reported that phenylbutyrate, which is also known as a histone deacetylase inhibitor, is very effective for leukemia patients. We therefore consider methods of up-regulating p21/WAF, gadd45 or bax genes should be useful for cancer therapy and termed this method "Gene-regulating chemotherapy". Theoretically, the chemicals up-regulating such genes should be also useful for chemoprevention, and we also termed it as "Gene-regulating chemoprevention". In conclusion, we propose that "Gene-regulating chemotherapy or chemoprevention" may be a promising new method for cancer therapy or prevention and histone deacetylase inhibitor is a good candidate for this method.     

Effects of genistein and daidzein on the cell growth,cell cycle,and differentiation of human and murine melanoma cells(1)

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G(2)/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G(1) phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.     

Implication of mitogen-activated protein kinase in the induction of G1 cell cycle arrest and gadd45 expression by the carotenoid fucoxanthin in human cancer cells

Background

The precise mechanism of the anti-tumor action of fucoxanthin has yet to be elucidated. We previously reported that gadd45a and gadd45b might play a role in the G1 arrest induced by fucoxanthin. In the present study, we show that several MAPKs modulate the induction of gadd45 and G1 arrest.

Methods

HepG2 and DU145 cells were used. The cell cycle was analyzed using flow cytometry. Expression of gadd45 was assayed by Northern blot and/or quantitative RT-PCR analyses. Activation of MAPK was assayed by Western blot analysis.

Results

Inhibition of p38 MAPK enhanced the induction of gadd45a expression and G1 arrest by fucoxanthin in HepG2 cells. Inhibition of ERK enhanced gadd45b expression but had no effect on the induction of G1 arrest by fucoxanthin in HepG2 cells. Inhibition of SAPK/JNK suppressed the induction of gadd45a expression and G1 arrest by fucoxanthin in DU145 cells.These data suggest that gadd45a is closely related with the G1 arrest induced by fucoxanthin, and that the pattern of MAPK involvement in the induction of gadd45a and G1 arrest by fucoxanthin differs depending on the cell type.

General significance

The implication of GADD45 and MAPK involvement in the anti-tumor action of carotenoids is first described.     

Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.     

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.     

Cell cycle-related signaling pathways modulated by peripheral benzodiazepine receptor ligands in colorectal cancer cells

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce both apoptosis and G1/G0 cell cycle arrest in colorectal cancers. The signaling pathways leading to cell cycle arrest are still unknown. Using cDNA array technology, we identified signaling molecules involved in cell cycle arrest induced by the PBR ligands FGIN-1-27 and PK 11195. Differential gene expression was confirmed by semi-quantitative RT-PCR or Western blot analysis of gene products. The PBR ligand-mediated signaling involved the upregulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, cdc16, and the cell cycle inhibitors gadd45 and gadd153, the downregulation of the cyclins D1 and B1, as well as the inactivation of ERK1/2. The p21-deficient colorectal cancer cell line HCT116 p21-/- was significantly less sensitive to PBR ligands than the parental HCT116 wild-type cells, demonstrating the functional involvement of p21WAF1/CIP1 in PBR ligand-mediated G1 arrest. This study thus revealed PBR ligand-triggered signaling pathways leading to cell cycle arrest. Moreover, we showed the functional implication and interaction of differentially expressed gene products and provided a model of signaling pathways involved in PBR ligand-induced G1 arrest. These results form the basis for future PBR ligand-mediated therapeutic approaches.     

Regulation of Gadd45a mRNA expression in vascular smooth muscle under growth and stress conditions

In order to identify differentially expressed genes under growth conditions, quiescent vascular smooth muscle cells (VSMCs) were stimulated with foetal calf serum (FCS) or platelet-derived growth factor-BB (PDGF-BB) for different time periods. Analysing the gene expression by the differential display (DD) method, we identified the cDNA of the growth arrest and DNA damage inducible gene 45a (Gadd45a, also known as gadd45 and gadd45a). Treatment with FCS or PDGF-BB led to a transient down-regulating of Gadd45a expression during the G0/G1 phase and maximal expression when cells had completed division. We found that expression of p53 and BRCA1 mRNA precedes Gadd45a mRNA expression with a maximal induction in the S phase. As in smooth muscle cells, a similar pattern of the Gadd45a mRNA expression was observed in knockout Gadd45a(-/-) cultured mouse embryonic fibroblasts (MEFs). However, no differences between Gadd45a(+/+) and Gadd45a(-/-) cell lines were observed regarding their kinetics of cell division. These experiments suggest a function of Gadd45a when cells exit the cell cycle rather than when regulating the entry into the S phase.     

Hypermethylation of growth Arrest DNA-damage-inducible gene 45 in non-small cell lung cancer and its relationship with clinicopathologic features

The growth arrest DNA-damage-inducible protein 45 (GADD45) can serve as a key coordinator of the stress response by regulating cell cycle progression, genomic stability, DNA repair, and other stress-related responses. Although deregulation of GADD45 expression has been reported in several types of human tumors, its role in lung cancer is still unknown. DNA hypermethylation of promoter CpG islands is known to be a major mechanism for epigenetic inactivation of tumor suppressor genes. We investigated the methylation status of GADD45 family genes (GADD45A, B, and G) in 139 patients with non-small cell lung cancer (NSCLC) using methylation-specific PCR (MSP) and correlated the results with clinicopathologic features of the patients. Methylation frequencies in tumors were 1.4% for GADD45A, 7.2% for GADD45B, and 31.6% for GADD45G. RT-PCR and MSP analysis showed that promoter methylation of the GADD45G gene resulted in downregulation of its mRNA expression. GADD45G methylation was significantly more frequent in female patients than male patients (P = 0.035). This finding suggests that methylation-associated down-regulation of the GADD45G gene may be involved in lung tumorigenesis.