Dataset Information

Effects of growth arrest and DNA damage-inducible protein 34 (GADD34) on inflammation-induced colon cancer in mice.

ABSTRACT:

Background

Growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a) is a family of GADD proteins that are induced by DNA damage. GADD34 protein has been suggested to regulate inflammation or host defense systems. However, the in vivo function of GADD34 in inflammation is still unclear. Long lasting inflammation, such as that seen in Crohn's disease and ulcerative colitis, is associated with a higher incidence of colorectal cancer (CRC).

Methods

Using a colitis-associated cancer model, we analysed GADD34-deficient (KO) mice to study the effect of GADD34 on colitis and colorectal tumorigenesis.

Results

We found a higher incidence of CRC in wild-type (WT) mice than in GADD34KO mice. Moreover, dextran sodium sulfate (DSS)-induced inflammatory responses were downregulated by GADD34 deficiency. The expression of pro-inflammatory mediators such as TNF?, IL-6, and iNOS/NOS2 was higher in the colons of WT mice than GADD34KO mice. IL-6 is known to activate STAT3 signalling in colonic epithelial cells and subsequently induced epithelial proliferation. We found that IL-6-STAT3 signalling and epithelial proliferation were higher in WT mice compared with GADD34KO mice.

Conclusions

These results indicated that GADD34 upregulated pro-inflammatory mediator production leading to a higher tumour burden following azoxymethane (AOM)/DSS treatment.

SUBMITTER: Tanaka Y

PROVIDER: S-EPMC4647681 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Publications

Effects of growth arrest and DNA damage-inducible protein 34 (GADD34) on inflammation-induced colon cancer in mice.

Tanaka Yuriko Y Ito Sachiko S Oshino Reina R Chen Nana N Nishio Naomi N Isobe Ken-ichi K

British journal of cancer 20150721 4

<h4>Background</h4>Growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a) is a family of GADD proteins that are induced by DNA damage. GADD34 protein has been suggested to regulate inflammation or host defense systems. However, the in vivo function of GADD34 in inflammation is still unclear. Long lasting inflammation, such as that seen in Crohn's disease and ulcerative colitis, is associated with a higher incidence of colorectal cancer (CRC).<h4>Methods</h4>Using a colitis-associate ...[more]

PMID: 26196182

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Project description:Growth arrest DNA damage-inducible gene 45 beta (GADD45beta) has been known to regulate cell growth, apoptotic cell death, and cellular response to DNA damage. Down-regulation of GADD45beta has been verified to be specific in hepatocellular cancer (HCC) and consistent with the p53 mutant, and degree of malignancy of HCC. This observation was further confirmed by eight HCC cell lines and paired human normal and HCC tumor tissues by Northern blot and immunohistochemistry. To better understand the transcription regulation, we cloned and characterized the active promoter region of GADD45beta in luciferase-expressing vector. Using the luciferase assay, three nuclear factor-kappaB binding sites, one E2F-1 binding site, and one putative inhibition region were identified in the proximal promoter of GADD45beta from -865/+6. Of interest, no marked putative binding sites could be identified in the inhibition region between -520/-470, which corresponds to CpG-rich region. The demethylating agent 5-Aza-dC was used and demonstrated restoration of the GADD45beta expression in HepG2 in a dose-dependent manner. The methylation status in the promoter was further examined in one normal liver cell, eight HCC cell lines, eight HCC tissues, and five corresponding nonneoplastic liver tissues. Methylation-specific polymerase chain reaction and sequencing of the sodium bisulfite-treated DNA from HCC cell lines and HCC samples revealed a high percentage of hypermethylation of the CpG islands. Comparatively, the five nonneoplastic correspondent liver tissues demonstrated very low levels of methylation. To further understand the functional role of GADD45beta under-expression in HCC the GADD45beta cDNA constructed plasmid was transfected into HepG2 (p53 WT) and Hep3B (p53 null) cells. The transforming growth factor-beta was assayed by enzyme-linked immunosorbent assay, which revealed a decrease to 40% in transfectant of HepG2, but no significant change in Hep3B transfectant. Whereas, Hep3B co-transfected with p53 and GADD45beta demonstrated significantly reduced transforming growth factor-beta. The colony formation was further examined and revealed a decrease in HepG2-GADD45beta transfectant and Hep3B-p53/GADD45beta co-transfectant. These findings suggested that methylation might play a crucial role in the epigenetic regulation of GADD45beta in hepatocyte transformation that may be directed by p53 status. Thus, our results provided a deeper understanding of the molecular mechanism of GADD45beta down-regulation in HCC.

Project description:In this study, we describe the growth arrest DNA damage-inducible gene 45beta (GADD45beta), whose expression was significantly down-regulated in the hepatocellular carcinoma (HCC) microarray study and confirmed by Northern blot analysis. The results suggested that expression of GADD45beta was decreased in human liver cancer cell lines HepG2 and Hep3B, but not in normal human embryonic liver cell line CL-48 or normal liver tissue. Histochemistry study and real-time PCR further confirmed that GADD45beta staining in HCC was significantly decreased when compared to surrounding non-neoplastic liver tissue. In further studies of multiple human cancer tissues, GADD45beta strongly stained tissues such as colon cancer, breast cancer, prostate cancer, squamous cell cancer, lymphoma, and leiomyosarcoma, suggesting that the decreased expression of GADD45beta is specific to HCC. Eighty-five cases of primary HCC were further examined by immunohistochemistry and statistical analyses demonstrated that HCC scored lower than matched non-neoplastic liver tissues consistently and significantly. No staining occurred in 12.94% of HCC cases (score = 0, n = 11); 42.35% had weak staining (score = 1, n = 36); 27.06% had moderate staining (score = 2, n = 23); and 17.65% had staining as strong as normal tissue (score = 3, n = 15). Overall, surrounding non-neoplastic liver tissue was highly positive for GADD45beta compared to adjacent neoplastic liver tissues (P < 0.01). We further observed that down-regulation of GADD45beta expression was strongly correlated with differentiation (P < 0.01) and high nuclear grade (P < 0.01). Moreover, we found that expression of GADD45beta was inversely correlated to the presence of mutant p53 in HCC tissue (P < 0.05). Thus, the results of our study suggest that GADD45beta, which is down-regulated in most cases of HCC, remains an ideal candidate for development as a molecular marker in the diagnosis of HCC and as a potential therapeutic target.

Project description:The stress-induced growth arrest and DNA damage-inducible a (GADD45a) gene is up-regulated by mechanical stress with GADD45a knockout (GADD45a(-/-)) mice demonstrating both increased susceptibility to ventilator-induced lung injury (VILI) and reduced levels of the cell survival and vascular permeability signaling effector (Akt). However, the functional role of GADD45a in the pathogenesis of VILI is unknown.We sought to define the role of GADD45a in the regulation of Akt activation induced by mechanical stress.VILI-challenged GADD45a(-/-) mice were administered a constitutively active Akt1 vector and injury was assessed by bronchoalveolar lavage cell counts and protein levels. Human pulmonary artery endothelial cells (EC) were exposed to 18% cyclic stretch (CS) under conditions of GADD45a silencing and used for immunoprecipitation, Western blotting or immunofluoresence. EC were also transfected with mutant ubiquitin vectors to characterize site-specific Akt ubiquitination. DNA methylation was measured using methylspecific polymerase chain reaction assay.Studies exploring the linkage of GADD45a with mechanical stress and Akt regulation revealed VILI challenged GADD45a(-/-) mice to have significantly reduced lung injury on overexpression of Akt1 transgene. Increased mechanical stress with 18% CS in EC induced Akt phosphorylation via E3 ligase tumor necrosis factor receptor–associated factor 6 (TRAF6)–mediated Akt K63 ubiquitination resulting in Akt trafficking and activation at the membrane. GADD45a is essential to this process because GADD45a silenced endothelial cells and GADD45a(-/-) mice exhibited increased Akt K48 ubiquitination leading to proteasomal degradation. These events involve loss of ubiquitin carboxyl terminal hydrolase 1(UCHL1), a deubiquitinating enzyme that normally removes K48 polyubiquitin chains bound to Akt thus promoting Akt K63 ubiquitination. Loss of GADD45a significantly reduces UCHL1 expression via UCHL1 promoter methylation resulting in increased Akt K48 ubiquitination and reduced Akt levels.These studies highlight a novel role for GADD45a in the regulation of site-specific Akt ubiquitination and activation and implicate a significant functional role for GADD45a in the clinical predisposition to VILI.

Dataset Information

Effects of growth arrest and DNA damage-inducible protein 34 (GADD34) on inflammation-induced colon cancer in mice.

Background

Methods

Results

Conclusions

Publications

Effects of growth arrest and DNA damage-inducible protein 34 (GADD34) on inflammation-induced colon cancer in mice.

Similar Datasets

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