GADD153 is an independent prognostic factor in melanoma: immunohistochemical and molecular genetic analysis

The main role of growth arrest and DNA damage-inducible (GADD) genes is to block proliferation at G1 and G2 checkpoints in response to DNA damage. The goal of this study was to examine the expression of GADD genes in primary melanomas with respect to prognosis. GADD34 was found in 73% of the primary melanomas investigated. GADD45 and GADD153 were positive in 60% and 80% of primary melanomas, respectively. Cox regression demonstrated that only GADD153 had any independent prognostic impact. We therefore decided to establish a PCR assay for detection of GADD153 in paraffin-embedded tissue. GADD153 deletion was found in 3/26 melanomas. None of the 3 cases with GADD153 deletion showed any expression of GADD153. Sequencing analysis detected polymorphism T-C at amino acid position 10 in 6/23 melanomas. In 6 cases with GADD153 polymorphism, GADD153 expression was found in 2 melanomas with a maximum GADD153 index of 10%. We postulate that the GADD gene family plays an important role in melanoma progression.     

RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly

DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.     

Hydrogen peroxide induces GADD153 in Jurkat cells through the protein kinase C-dependent pathway

Growth arrest and DNA damage-inducible gene 153 (GADD153) is a CCAAT/enhancer binding protein (C/EBP) related gene and is induced in response to various stimuli including DNA damaging agents, UV irradiation, and serum starvation. In this study, we investigated which intracellular signals contribute to the expression of GADD153 mRNA in Jurkat cells in response to oxidative stress using several kinds of kinase inhibitors. GADD153 mRNA expression was immediately enhanced following hydrogen peroxide exposure and was significantly inhibited by treatment with H-7, staurosporin, and Ro-31-8220. In particular, rottlerin, a PKCdelta specific inhibitor, markedly attenuated hydrogen peroxide-induced GADD153 mRNA expression even at 1 microM. Treatment with a potent PKC activator, phorbol-12-myristate-13-acetate (PMA), augmented GADD153 mRNA in Jurkat cells in the presence of hydrogen peroxide, although PMA alone induced GADD153 mRNA marginally. Hydrogen peroxide significantly enhanced the AP-1 binding activity of the nuclear extract from Jurkat cells to the GADD153 AP-1 binding site. AP-1 binding activity was suppressed by rottlerin treatment. These findings indicate that PKC, especially PKCdelta, plays an important role in the induction of GADD153 mRNA following oxidative stress.     

Impact of α-Targeted Radiation Therapy on Gene Expression in a Pre-Clinical Model for Disseminated Peritoneal Disease when Combined with Paclitaxel

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.     

Interaction with BRCA2 suggests a role for filamin-1 (hsFLNa) in DNA damage response

The BRCA2 tumor suppressor plays significant roles in DNA damage response. The human actin binding protein filamin-1 (hsFLNa, also known as ABP-280) participates in orthogonal actin network, cellular stress responses, signal transduction, and cell migration. Through a yeast two-hybrid system, an in vitro binding assay, and in vivo co-immunoprecipitations, we identified an interaction between BRCA2 and hsFLNa. The hsFLNa binding domain of BRCA2 was mapped to an internal conserved region, and the BRCA2-interacting domain of hsFLNa was mapped to its C terminus. Although hsFLNa is known for its cytoplasmic functions in cell migration and signal transduction, some hsFLNa resides in the nucleus, raising the possibility that it participates in DNA damage response through a nuclear interaction with BRCA2. Lack of hsFLNa renders a human melanoma cell line (M2) more sensitive to several genotoxic agents including gamma irradiation, bleomycin, and ultraviolet-c light. These results suggest that BRCA2/hsFLNa interaction may serve to connect cytoskeletal signal transduction to DNA damage response pathways.     

Regulation of DNA repair throughout the cell cycle

The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.     

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.