In-Youb Chang

Oncogenic H-Ras Up-Regulates Expression of ERCC1 to Protect Cells from Platinum-Based Anticancer Agents

Tumors frequently contain mutations in the ras genes, resulting in the constitutive activation of the Ras-activated signaling pathway. The activation of Ras is involved not only in tumor progression but also in the development of resistance of the tumor cells to platinum-based chemotherapeutic agents. To investigate the potential mechanisms underlying this resistance, we analyzed the effect of activated H-Ras on the expression of the nucleotide excision repair genes. Here we identified ERCC1, which is one of the key enzymes involved in nucleotide excision repair, as being markedly up-regulated by the activated H-Ras. From promoter analysis of ERCC1, an increase in the Ap1 transcriptional activity as a result of the expression of the oncogenic H-Ras was found to be crucial for this induction. In addition, ERCC1 small interfering RNA expression was shown to reduce the oncogenic H-Ras-mediated increase in the DNA repair activity as well as to suppress the oncogenic H-Ras-mediated resistance of the cells to platinum-containing chemotherapeutic agents. These results suggest that the oncogenic H-Ras-induced ERCC1, which activates the DNA repair capacity, may be involved in the protection of the cells against platinum-based anticancer agents.

Bcl-2 expression suppresses mismatch repair activity through inhibition of E2F transcriptional activity.

Bcl-2 stimulates mutagenesis after the exposure of cells to DNA-damaging agents. However, the biological mechanisms of Bcl-2-mediated mutagenesis have remained largely obscure. Here we demonstrate that the Bcl-2-mediated suppression of hMSH2 expression results in a reduced cellular capacity to repair mismatches. The pathway linking Bcl-2 expression to the suppression of mismatch repair (MMR) activity involves the hypophosphorylation of pRb, and then the enhancement of the E2F–pRb complex. This is followed by a decrease in hMSH2 expression. MMR has a key role in protection against deleterious mutation accumulation and in maintaining genomic stability. Therefore, the decreased MMR activity by Bcl-2 may be an underlying mechanism for Bcl-2-promoted oncogenesis.

Mismatch-repair protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair

MSH6, a key component of the MSH2–MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70.

Human 8-oxoguanine DNA glycosylase suppresses the oxidative stress induced apoptosis through a p53-mediated signaling pathway in human fibroblasts.

Human 8-oxoguanine DNA glycosylase (hOGG1) is the main defense enzyme against mutagenic effects of cellular 7,8-dihydro-8-oxoguanine. In this study, we investigated the biological role of hOGG1 in DNA damage–related apoptosis induced by hydrogen peroxide (H2O2)–derived oxidative stress. The down-regulated expression of hOGG1 by its small interfering RNA prominently triggers the H2O2-induced apoptosis in human fibroblasts GM00637 and human lung carcinoma H1299 cells via the p53-mediated apoptotic pathway. However, the apoptotic responses were specifically inhibited by hOGG1 overexpression. The p53–small interfering RNA transfection into the hOGG1-deficient GM00637 markedly inhibited the H2O2-induced activation of p53-downstream target proteins such as p21, Noxa, and caspase-3/7, which eventually resulted in the increased cell viability. Although the cell viability of hOGG1-knockdown H1299 p53 null cells was similar to that of the hOGG1 wild-type H1299, after the overexpression of p53 the hOGG1-knockdown H1299 showed the significantly decreased cell viability compared with that of the hOGG1 wild-type H1299 at the same experimental condition. Moreover, the array comparative genome hybridization analyses revealed that the hOGG1-deficient GM00637 showed more significant changes in the copy number of large regions of their chromosomes in response to H2O2 treatment. Therefore, we suggest that although p53 is a major modulator of apoptosis, hOGG1 also plays a pivotal role in protecting cells against the H2O2-induced apoptosis at the upstream of the p53-dependent pathway to confer a survival advantage to human fibroblasts and human lung carcinomas through maintaining their genomic stability. (Mol Cancer Res 2007;5(10):1083–98)

Deoxycholic acid inhibits pacemaker currents by activating ATP‐dependent K+ channels through prostaglandin E2 in interstitial cells of Cajal from the murine small intestine

1 We investigated the role of deoxycholic acid in pacemaker currents using whole‐cell patch‐clamp techniques at 30°C in cultured interstitial cells of Cajal (ICC) from murine small intestine. 2 The treatment of ICC with deoxycholic acid resulted in a decrease in the frequency and amplitude of pacemaker currents and increases in resting outward currents. Also, under current clamping, deoxycholic acid produced the hyperpolarization of membrane potential and decreased the amplitude of the pacemaker potentials. 3 These observed effects of deoxycholic acid on pacemaker currents and pacemaker potentials were completely suppressed by glibenclamide, an ATP‐sensitive K+ channel blocker. 4 NS‐398, a specific cyclooxygenase‐2 (COX‐2) inhibitor, significantly inhibited the deoxycholic acid‐induced effects. The treatment with prostaglandin E2 (PGE2) led to a decrease in the amplitude and frequency of pacemaker currents and to an increase in resting outward currents, and these observed effects of PGE2 were blocked by glibenclamide. 5 We next examined the role of deoxycholic acid in the production of PGE2 in ICC, and found that deoxycholic acid increased PGE2 production through the induction of COX‐2 enzyme activity and its gene expression. 6 The results suggest that deoxycholic acid inhibits the pacemaker currents of ICC by activating ATP‐sensitive K+ channels through the production of PGE2.

Rac1 prevents cisplatin-induced apoptosis through down-regulation of p38 activation in NIH3T3 cells.

In this study, the role of V12‐Rac1 in the cisplatin‐induced apoptosis was investigated. Cisplatin‐induced apoptosis is associated with cytochrome c release, which can be inhibited by V12‐Rac1 expression. The analysis of mitogen‐activated protein kinase activity indicated that V12‐Rac1 expression led to a decrease in p38 activity after exposure to cisplatin but not c‐jun N‐terminal kinase and extracellular signal‐regulated kinase. Using pharmacological inhibitors, it was found that only p38 is a critical mediator in the cisplatin‐induced apoptosis of NIH3T3 cells. This suggests that V12‐Rac1 can stimulate the anti‐apoptotic signaling pathway in response to cisplatin, and that decreased p38 activity caused by V12‐Rac1 expression in cisplatin‐treated NIH3T3 cells is crucial for V12‐Rac1‐dependent cell survival.

Protein Phosphatase 5 Regulates the Function of 53BP1 after Neocarzinostatin-induced DNA Damage

53BP1 (p53-binding protein 1) is a conserved nuclear protein that is phosphorylated in response to DNA damage and rapidly recruited to the site of DNA double strand breaks, demonstrating its role in the early events to DNA damage and repair of damaged DNA. In this study, we used the yeast two-hybrid system to identify proteins that interact with 53BP1. Identification and characterization of 53BP1 protein interactions may help to further elucidate the function and regulation of 53BP1. We identified protein phosphatase 5 (PP5), a serine/threonine phosphatase that has been implicated in multiple cellular function, as a 53BP1-binding protein. This interaction further confirmed that 53BP1 interacts with PP5 in PP5-overexpressing U2OS cells, after radiomimetic agent neocarzinostatin (NCS) treatment. 53BP1 dephosphorylation at Ser-25 and Ser-1778 was accelerated in PP5-overexpressing U2OS cells following NCS treatment, and its dephosphorylation was correlated with reduced phospho-53BP1 foci formation. In contrast, the overexpression of PP5 had no effect on NCS-activated BRCA1-Ser-1524 phosphorylation. Additionally, PP5 down-regulation inhibited the dephosphorylation of 53BP1 on Ser-1778 and the disappearance of phospho-53BP1 foci following NCS treatment. Moreover, non-homologous end-joining activity was reduced in PP5-overexpressing U2OS cells. These findings indicate that PP5 plays an important role in the regulation of 53BP1 phosphorylation and activity in vivo.

Colon cancer progression is driven by APEX1-mediated upregulation of Jagged

Aberrant expression of apurinic-apyrimidinic endonuclease-1 (APEX1) has been reported in numerous human solid tumors and is positively correlated with cancer progression; however, the role of APEX1 in tumor progression is poorly defined. Here, we show that APEX1 contributes to aggressive colon cancer behavior and functions as an upstream activator in the Jagged1/Notch signaling pathway. APEX1 overexpression or knockdown in human colon cancer cell lines induced profound changes in malignant properties such as cell proliferation, anchorage-independent growth, migration, invasion, and angiogenesis in vitro and in tumor formation and metastasis in mouse xenograft models. These oncogenic effects of APEX1 were mediated by the upregulation of Jagged1, a major Notch ligand. Furthermore, APEX1 expression was associated with Jagged1 in various colon cancer cell lines and in tissues from colon cancer patients. This finding identifies APEX1 as a positive regulator of Jagged1/Notch activity and suggests that it is a potential therapeutic target in colon cancers that exhibit high levels of Jagged1/Notch signaling.

Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor α1 Expression

ABSTRACT Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor α1 (GFRα1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRα1 transcription through enhanced binding of NF-κB complexes to the GFRα1 promoter. GFRα1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRα1 expression and invasion in response to GNDF, while overexpression of GFRα1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from β-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRα1 expression may play important roles in pancreatic cancer progression and neuronal cell survival.

Immunohistochemical detection of StarD6 in the rat nervous system.

Steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain 6 (StarD6) is known to be exclusively expressed in germ cells of testis. As little is known about StarD6 expression in the nervous system, we investigated the distribution of StarD6 in rat neural tissues. Immunoreactivity of StarD6 was detected in the brain, spinal cord and dorsal root ganglia; particularly cerebral cortex (layer V and VI), hippocampus, substantia gelatinosa of the spinal cord. We provided compelling evidence that multiple neuronal and glial populations were immunolabelled with anti-StarD6 antibody throughout the nervous system. We postulate that StarD6 might play an important role in lipid sensing of the nervous system based on its immunolocalization in this study.