DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

PURPOSE: Impotence is one of the major complications occurring in prostate cancer patients after radical prostectomy (RP). Self-repair of the injured nerve has been observed in animal models and in patients after RP. However, the downstream signalling is not well documented. Here, we found that the DAPK/CIP2A complex is involved in GAS6/AXL-related Schwann cell proliferation. MATERIALS AND METHODS: The 3 groups were a sham group, a 14-day post-bilateral cavernous nerve injury (BCNI) group and a 28-day post-BCNI group. Erectile function was assessed and immunohistochemistry was performed. The rat Schwann cell RSC96 line was chosen for gene knockdown, cell viability, western blot, immunofluorescence and co-immunoprecipitation assays. RESULTS: The intracavernosal pressure was low on the 14th day after BCNI and partially increased by the 28th day. GAS6 and p-AXL expression gradually increased in the cavernous nerve after BCNI. RSC96 cells incubated with a GAS6 ligand showed increased levels of p-ERK1/2 and p-AKT. Moreover, DAPK and CIP2A.p-AXL and p-DAPK and CIP2A complexes were identified by both immunoblotting and co-immunoprecipitation. CONCLUSION: The DAPK/CIP2A complex is involved in GAS6/AXL-related Schwann cell proliferation. CIP2A inhibits PP2A activity, which results in p-DAPK(S308) maintenance and promotes Schwann cell proliferation. CIP2A is a potential target for the treatment of nerve injury after RP.

Metformin-treated cancer cells modulate macrophage polarization through AMPK-NF-κB signaling.

Accumulating evidence is indicating metformin to possess the potential ability in preventing tumor development and suppressing cancer growth. However, the exact mechanism of its antitumorigenic effects is still not clear. We found that metformin suppressed the ability of cancer to skew macrophage toward M2 phenotype. Metformin treated cancer cells increased macrophage expression of M1-related cytokines IL-12 and TNF-α and attenuated M2-related cytokines IL-8, IL-10, and TGF-ß expression. Furthermore, metformin treated cancer cells displayed inhibited secretion of IL-4, IL-10 and IL-13; cytokines important for inducing M2 macrophages. Conversely, M1 inducing cytokine IFN-γ was upper-regulated in cancer cells. Additionally, through increasing AMPK and p65 phosphorylation, metformin treatment activated AMPK-NF-κB signaling of cancer cells that participate in regulating M1 and M2 inducing cytokines expression. Moreover, Compound C, an AMPK inhibitor, significantly increased IL-4, IL-10, and IL-13 expression while BAY-117082, an NF-κB inhibitor, decreased expression. In metformin-treated tumor tissue, the percentage of M2-like macrophages decreased while M1-like macrophages increased. These findings suggest that metformin activates cancer AMPK-NF-κB signaling, a pathway involved in regulating M1/M2 expression and inducing genes for macrophage polarization to anti-tumor phenotype.

CIP2A is a poor prognostic factor and can be a diagnostic marker in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma. CIP2A has recently been described as a prognostic marker in many cancers. In this study, we assessed the value of this novel prognostic marker in PTC. A total of 178 surgical specimens of both benign and malignant thyroid tumors were collected. Immunohistochemical staining for CIP2A, HBME-1, galectin-3, and CK19 was performed. Western blotting for CIP2A was also performed. CIP2A was expressed in 85.3% of malignant tumors and 12.1% of benign tumors. ROC analysis showed that the AUC for CIP2A was higher than those for other tumor markers. Western blotting showed that CIP2A expression was higher in PTC than in other tumors. Poor progression-free survival was observed in the high-CIP2A expression group. High CIP2A expression is a poor prognostic factor and can be a diagnostic marker in PTC. The presence of any two of the three indicated makers (CIP2A, galectin-3, and HBME-1) is strongly correlated with the diagnosis of PTC.

Breast cancer therapy and age difference in cardiovascular disease risks: A population-based cohort study in Taiwan.

OBJECTIVE: Cardiovascular events induced in breast cancer patients receiving radiotherapy (RT), chemotherapy (CT), or a combination of both (CRT) can increase the risk of death. This nationwide population-based study aims to estimate the risk of cardiovascular complications with a follow-up period of 5 years. MATERIALS AND METHODS: The study cohorts consisted of all patients hospitalized with a principal diagnosis of breast cancer who underwent treatment in 2002. The Cox proportional hazard model and Kaplan-Meier plot were analyzed to compare the cardiovascular event-free survival rate among breast cancer patients treated with different modalities. RESULTS: Of the 5514 breast cancer patients identified, 289 patients had cardiovascular disease (CVD): 110 (5.7%) from the surgery-alone group, 24 (4.1%) from the RT group, 79 (4.6) from the CT group, and 76 (5.8%) from the CRT group. Breast cancer patients who undergo CT and CRT at an age less than 55 years had a higher risk of CVD when compared with the surgery-alone group (for both groups, p < 0.001). By contrast, breast cancer patients aged over 55 years had no increased risk of CVD among the different treatment modalities. CONCLUSION: Breast cancer patients receiving CT and/or CRT have a higher risk of CVD, especially younger patients (aged < 55 years). Therefore, regular examinations of cardiac functions and electrocardiogram should be considered in cases of young breast cancer patients who are receiving CT and/or CRT.

SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A.

SET is known as a potent PP2A inhibitor, however, its oncogenic role including its tumorigenic potential and involvement in the development of chemoresistance in non-small cell lung cancer (NSCLC) has not yet been fully discussed. In present study, we investigated the oncogenic role of SET by SET-knockdown and showed that SET silencing impaired cell growth rate, colony formation and tumor sphere formation in A549 cells. Notably, silencing SET enhanced the pro-apoptotic effects of paclitaxel, while ectopic expression of SET diminished the sensitivity of NSCLC cells to paclitaxel. Since the SET protein was shown to affect chemosensitivity, we next examined whether combining a novel SET antagonist, EMQA, sensitized NSCLC cells to paclitaxel. Both the in vitro and in vivo experiments suggested that EMQA and paclitaxel combination treatment was synergistic. Importantly, we found that downregulating p-Akt by inhibiting SET-mediated protein phosphatase 2A (PP2A) inactivation determined the pro-apoptotic effects of EMQA and paclitaxel combination treatment. To dissect the critical site for EMQA functioning, we generated several truncated SET proteins. By analysis of the effects of EMQA on the binding affinities of different truncated SET proteins to PP2A-catalytic subunits, we revealed that the 227-277 amino-acid sequence is critical for EMQA-induced SET inhibition. Our findings demonstrate the critical role of SET in NSCLC, particularly in the development of chemoresistance. The synergistic effects of paclitaxel and the SET antagonist shown in current study encourage further validation of the clinical potential of this combination.

Hypoxia Induces a Metabolic Shift and Enhances the Stemness and Expansion of Cochlear Spiral Ganglion Stem/Progenitor Cells.

Previously, we demonstrated that hypoxia (1% O2) enhances stemness markers and expands the cell numbers of cochlear stem/progenitor cells (SPCs). In this study, we further investigated the long-term effect of hypoxia on stemness and the bioenergetic status of cochlear spiral ganglion SPCs cultured at low oxygen tensions. Spiral ganglion SPCs were obtained from postnatal day 1 CBA/CaJ mouse pups. The measurement of oxygen consumption rate, extracellular acidification rate (ECAR), and intracellular adenosine triphosphate levels corresponding to 20% and 5% oxygen concentrations was determined using a Seahorse XF extracellular flux analyzer. After low oxygen tension cultivation for 21 days, the mean size of the hypoxia-expanded neurospheres was significantly increased at 5% O2; this correlated with high-level expression of hypoxia-inducible factor-1 alpha (Hif-1α), proliferating cell nuclear antigen (PCNA), cyclin D1, Abcg2, nestin, and Nanog proteins but downregulated expression of p27 compared to that in a normoxic condition. Low oxygen tension cultivation tended to increase the side population fraction, with a significant difference found at 5% O2 compared to that at 20% O2. In addition, hypoxia induced a metabolic energy shift of SPCs toward higher basal ECARs and higher maximum mitochondrial respiratory capacity but lower proton leak than under normoxia, where the SPC metabolism was switched toward glycolysis in long-term hypoxic cultivation.

VEGF-D as a marker in the aid of malignant metastatic pleural effusion diagnosis.

BACKGROUND: The sensitivity in cytology diagnosis of malignant metastatic pleural effusion (MMPE) is insufficient nowadays due to the similarity of the reactive mesothelial cells and malignant cells. Vascular endothelial growth factor (VEGF) is one of the key factors in tumor lymphangiogenesis and metastasis. Therefore, the aim of this study was to evaluate the value of VEGF and its homologs in the aid of MMPE diagnosis. METHODS: A total of 217 pleural effusions samples were eligible for analysis. Among them, 81 malignant and 22 benign cases were made into the cell blocks for the immunocytochemical (ICC) staining of VEGF-A, VEGF-C, VEGF-D, VEGFR-2, and VEGFR-3 expression. Another 114 samples (41 malignant and 73 benign cases) were subjected to the ELISA test for the protein level of VEGF-D. RESULTS: In a total of 156 MMPE, only VEGF-D expression by ICC stain was significantly different between malignant (92.6%) and benign cases (9.1%) with P<0.001 in either nuclear or cytoplasmic staining. Only 6 malignant cases showed negative stain results. In addition, 3 of the 4 lung small cell carcinoma were immunoreactive for VEGF-D. However, some lymphocytes also showed nuclear staining pattern of VEGF-D. In contrast, the ELISA test for the VEGF-D protein levels failed to demonstrate the difference between malignant and benign pleural effusions. CONCLUSIONS: Among VEGF homologs, MMPE from various kinds of tumor origin, VEGF-D showed 92.6% rate of positive expression. ICC stain of VEGF-D is a useful marker in the aid of MMPE diagnosis.

Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

Afatinib has anti-tumor effect in non-small cell lung carcinoma (NSCLC) with epidermal growth factor receptor (EGFR) mutation. We found afatinib can also induce apoptosis in NSCLC cells without EGFR mutation through CIP2A pathway. Four NSCLC cell lines (H358 H441 H460 and A549) were treated with afatinib to determine their sensitivity to afatinib-induced cell death and apoptosis. The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively. Reduction of Elk-1 binding to the CIP2A promoter and suppression of CIP2A transcription were analyzed. In vivo efficacy of afatinib against H358 and H460 xenografts tumors were also determined in nude mice. Afatinib induced significant cell death and apoptosis in H358 and H441 cells, but not in H460 or A549 cells. The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation. Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1. Clinical samples showed that higher CIP2A expression predicted a poor prognosis and Elk-1 and CIP2A expressions were highly correlated. In conclusion, afatinib induces apoptosis in NSCLC without EGFR mutations through Elk-1/CIP2A/PP2A/AKT pathway.

Tamoxifen induces apoptosis through cancerous inhibitor of protein phosphatase 2A-dependent phospho-Akt inactivation in estrogen receptor-negative human breast cancer cells.

INTRODUCTION: Tamoxifen, a selective estrogen receptor (ER) modulator, may affect cancer cell survival through mechanisms other than ER antagonism. In the present study, we tested the efficacy of tamoxifen in a panel of ER-negative breast cancer cell lines and examined the drug mechanism. METHODS: In total, five ER-negative breast cancer cell lines (HCC-1937, MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3) were used for in vitro studies. Cellular apoptosis was examined by flow cytometry and Western blot analysis. Signal transduction pathways in cells were assessed by Western blot analysis. The in vivo efficacy of tamoxifen was tested in xenograft nude mice. RESULTS: Tamoxifen induced significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3 cells, but not in HCC-1937 cells. Tamoxifen-induced apoptosis was associated with inhibition of cancerous inhibitor of protein phosphatase 2A (CIP2A) and phospho-Akt (p-Akt) in a dose-dependent manner. Ectopic expression of either CIP2A or Akt protected MDA-MB-231 cells from tamoxifen-induced apoptosis. In addition, tamoxifen increased protein phosphatase 2A (PP2A) activity, and tamoxifen-induced apoptosis was attenuated by the PP2A antagonist okadaic acid in the sensitive cell lines, but not in resistant HCC-1937 cells. Moreover, silencing CIP2A by small interfering RNA sensitized HCC-1937 cells to tamoxifen-induced apoptosis. Furthermore, tamoxifen regulated CIP2A protein expression by downregulating CIP2A mRNA. Importantly, tamoxifen inhibited the in vivo growth of MDA-MB-468 xenograft tumors in association with CIP2A downregulation, whereas tamoxifen had no significant effect on CIP2A expression and anti-tumor growth in HCC-1937 tumors. CONCLUSIONS: Inhibition of CIP2A determines the effects of tamoxifen-induced apoptosis in ER-negative breast cancer cells. Our data suggest a novel "off-target" mechanism of tamoxifen and suggest that CIP2A/PP2A/p-Akt signaling may be a feasible anti-cancer pathway.

TD-19, an erlotinib derivative, induces epidermal growth factor receptor wild-type nonsmall-cell lung cancer apoptosis through CIP2A-mediated pathway.

Some patients with nonsmall-cell lung cancer (NSCLC) without epidermal growth factor receptor (EGFR) mutations still respond to gefitinib and erlotinib, suggesting that there may be a mechanism(s) other than the EGFR pathway that mediates the tumoricidal effects. In the current study, we tested the efficacy of TD-19, a novel compound chemically modified from erlotinib, which has more potent apoptotic effects than erlotinib in EGFR wild-type NSCLC cell lines. TD-19 induced significant cell death and apoptosis in H358, H441, H460, and A549 cells, as evidenced by increased caspase-3 activity and cleavage of procaspase-9 and poly (ADP-ribose) polymerase. The apoptotic effect of TD-19 in H460 cells, which were resistant to erlotinib, was associated with downregulation of cancerous inhibitor of protein phosphatase 2A (CIP2A), increased protein phosphatase 2A (PP2A) activity, and decreased AKT phosphorylation, but minimal effects on EGFR phosphorylation. Overexpression of CIP2A partially protected the H460 cells from TD-19-induced apoptosis. Okadaic acid, a known PP2A inhibitor, significantly reduced TD-19-induced apoptosis, while forskolin, which increased PP2A activity, increased the apoptotic effect of TD-19. TD-19 inhibited the growth of H460 xenograft tumors by ∼80%. We conclude that TD-19 exerted tumoricidal effects on NSCLC cells. TD-19 provides proof that the CIP2A pathway may be a novel target for the treatment of EGFR wild-type NSCLC.

CIP2A mediates erlotinib-induced apoptosis in non-small cell lung cancer cells without EGFR mutation.

BACKGROUND: Epidermal growth factor receptor (EGFR) inhibitors show favorable clinical response in some patients with non-small cell lung cancer (NSCLC) who have no EGFR mutation, indicating alternative mechanisms for their tumoricidal effects. We previously showed erlotinib, a selective EGFR antagonist, inhibited the growth of sensitive hepatocellular carcinoma cells by inhibiting the cancerous inhibitor of protein phosphatase 2A (CIP2A) pathway. The aim of this study was to determine if erlotinib can also inhibit the growth of NSCLC cells by inactivating the CIP2A-dependent signaling pathway. METHODS: Four NSCLC cell lines (H358 H441 H460 and A549) were treated with erlotinib to determine their sensitivity to erlotinib-induced cell death and apoptosis. Expression of CIP2A and the downstream AKT were analyzed. The effects of CIP2A on erlotinib-induced apoptosis were confirmed by overexpression of CIP2A and knockdown of CIP2A gene expression in the sensitive cells and resistant cells, respectively. In vivo efficacy of erlotinib against H358 xenograft tumor was also determined in nude mice. RESULTS: Erlotinib induced significant cell death and apoptosis in H358 and H441 cells, as evidenced by increased caspase 3 activity and cleavage of pro-caspase 9 and PARP, but not in H460 or A549 cells. The apoptotic effect of erlotinib in the sensitive H358 cells was associated with downregulation of CIP2A, increase in PP2A activity and decrease in AKT phosphorylation. Overexpression of CIP2A and AKT protected the sensitive H358 cells from erlotinib-induced apoptosis. Knockdown of CIP2A gene expression by siRNA enhanced the erlotinib-induced apoptotic in the resistant H460 cells that resembled the sensitive H358 cells. Erlotinib also inhibited the growth of H358 tumors in nude mice. CONCLUSIONS: The CIP2A-dependent pathway mediates the tumoricidal effects of erlotinib on NSCLC cells without EGFR mutations in vitro and in vivo. CIP2A may be a novel molecular target against NSCLC for future drug development.

Signal transducer and activator of transcription 3 as molecular therapy for non-small-cell lung cancer.

INTRODUCTION: Targeting signal transducer and activator of transcription 3 (STAT3), a transcription factor that modulates survival-directed transcription, is often persistently activated in epidermal growth factor receptor (EGFR) wild-type non-small-cell lung cancer (NSCLC). The aim of this study was to determine whether sorafenib and its derivative can inhibit EGFR wild-type NSCLC via STAT3 inactivation. METHODS: EGFR wild-type NSCLC cell lines (A549 H292 H322 H358 and H460) were treated with sorafenib or SC-1, a sorafenib derivative that closely resembled sorafenib structurally but was devoid of kinase inhibitory activity. Apoptosis and signal transduction were analyzed. In vivo efficacy was determined in nude mice with H460 and A549 xenograft. RESULTS: SC-1 had better effects than sorafenib on growth inhibition and apoptosis in all tested EGFR wild-type NSCLC lines. SC-1 reduced STAT3 phosphorylation at tyrosine 705 in all tested EGFR wild-type NSCLC cells. The expression of STAT3-driven genes, including cylcin D1 and survivin, was also repressed by SC-1. Ectopic expression of STAT3 in H460 cells abolished apoptosis in SC-1-treated cells. Sorafenib and SC-1 enhanced Src homology-2 containing protein tyrosine phosphatase-1 (SHP-1) activity, whereas knockdown of SHP-1, but not SHP-2 or protein-tyrosine phosphatase 1B (PTP-1B), by small interference RNA reduced SC-1-induced apoptosis. SC-1 significantly reduced H460 and A549 tumor growth in vivo through SHP-1/STAT3 pathway. CONCLUSIONS: SC-1 provides proof that targeting STAT3 signaling pathway may be a novel approach for the treatment of EGFR wild-type NSCLC.

Risk of colorectal cancer in type 2 diabetic patients: a population-based cohort study.

OBJECTIVE: Most of the existing findings on the association between diabetes mellitus and colorectal cancer were generated from studies in Western societies. However, significant differences in cancer incidence and cancer-prone lifestyles are apparent between Asian and Western countries. This study aims to estimate the risks of colorectal cancer in the diabetic population in Taiwan by conducting a large-scale, controlled cohort study. METHODS: From Taiwan's Longitudinal Health Insurance Database 2005 (LHID2005), a total of 37 001 diabetic patients were identified. We also obtained data for four controls per patient, matched for sex, age and year of first entry into the LHID2005. All patients were followed up from the date of entry into the LHID2005 until they developed colorectal cancer or to the end of 2006, whichever was earlier. We used Cox's regression models to assess the risk of developing colorectal cancer, with adjustment for sex, age, comorbid disorders, and socioeconomic characteristics. RESULTS: We identified 37 001 diabetic patients and 148 004 controls. The adjusted hazard ratio for colorectal cancer in diabetes mellitus patients was 2.1 (95% confidence interval, 1.82-2.42) compared with controls. The risk was significant to both men and women. The adjusted hazard ratios for colorectal cancer were 2.03 (95% confidence interval, 1.68-2.47) in male diabetes mellitus patients and 2.17 (95% confidence interval, 1.77-2.67) in female diabetes mellitus patients. CONCLUSIONS: Our findings based on a large population-based cohort study provide evidence that diabetes mellitus may increase the risk of colorectal cancer in Asians.

Idiopathic pulmonary fibrosis in Taiwan - a population-based study.

BACKGROUND: This study took advantage of a large population-based database of the Taiwan National Health Insurance (NHI) to investigate the epidemiology of idiopathic pulmonary fibrosis (IPF) in Taiwan. METHODS: This is a retrospective cohort study based on secondary analysis of prospectively collected data in the NHI system and governmental data on death registry in Taiwan during 1997-2007. By using the broad and narrow definitions for IPF, we estimated incidence and prevalence rates of IPF, and its associated clinical outcomes. RESULTS: The estimates of annual IPF incidence rates became more stable after 2000, ranging between 0.9 and 1.6 cases per 100,000 persons. The prevalence rates became more than twofold from 2000 to 2007 (from 2.8 to 6.4 cases per 100,000 persons for the broad definition, and from 2.0 to 4.9 cases per 100,000 persons for the narrow definition). Men of age older than 75 years had markedly higher incidence and prevalence rates than other groups. Around 40% of all incidences and about 30% of prevalent cases occurred in this population group. The median survival time after IPF diagnosis was 0.9 year (interquartile range (IQR), 0.2-2.5 years) and 0.7 year (IQR, 0.1-2.3 years) for the broad and narrow definitions, respectively. Progression of IPF was the leading cause of death, followed by cancer. CONCLUSIONS: In Taiwan, elderly men were the major group suffering from IPF. Survival time was short after IPF diagnosis, and the poor survival was largely attributable to quick IPF progression after diagnosis.

Preoperative proteinuria is associated with long-term progression to chronic dialysis and mortality after coronary artery bypass grafting surgery.

AIMS: Preoperative proteinuria is associated with post-operative acute kidney injury (AKI), but whether it is also associated with increased long-term mortality and end-stage renal disease (ESRD) is unknown. METHODS AND RESULTS: We studied 925 consecutive patients undergoing CABG. Demographic and clinical data were collected prospectively, and patients were followed for a median of 4.71 years after surgery. Proteinuria, according to dipstick tests, was defined as mild (trace to 1+) or heavy (2+ to 4+) according to the results of the dipstick test. A total of 276 (29.8%) patients had mild proteinuria before surgery and 119 (12.9%) patients had heavy proteinuria. During the follow-up, the Cox proportional hazards model demonstrated that heavy proteinuria (hazard ratio [HR], 27.17) was an independent predictor of long-term ESRD. There was a progressive increased risk for mild proteinuria ([HR], 1.88) and heavy proteinuria ([HR], 2.28) to predict all-cause mortality compared to no proteinuria. Mild ([HR], 2.57) and heavy proteinuria ([HR], 2.70) exhibited a stepwise increased ratio compared to patients without proteinuria for long-term composite catastrophic outcomes (mortality and ESRD), which were independent of the baseline GFR and postoperative acute kidney injury (AKI). CONCLUSION: Our study demonstrated that proteinuria is a powerful independent risk factor of long-term all-cause mortality and ESRD after CABG in addition to preoperative GFR and postoperative AKI. Our study demonstrated that proteinuria should be integrated into clinical risk prediction models for long-term outcomes after CABG. These results provide a high priority for future renal protective strategies and methods for post-operative CABG patients.

SUMO modification modulates the transrepression activity of PLZF.

Small ubiquitin-like modifier (SUMO) modification has recently been shown to associate with transcriptional regulation and nuclear body formation. Here, we show that transcription factor PLZF can be SUMO modified at lysine residue 242, 387 and 396. Converting these three SUMO acceptor Lys to Arg 3KR does not significantly affect PLZF nuclear body formation, which is distinct from the scenario of PML sumoylation in PML nuclear body formation. Furthermore, PLZF-3KR markedly reduced the transcriptional repression activity, correlating with a loss of PLZF-mediated growth suppression. These results reveal an important role of SUMO modification in PLZF-mediated transcriptional repression.

Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors.

Small ubiquitin-like modifier (SUMO) modification has emerged as an important posttranslational control of protein functions. Daxx, a transcriptional corepressor, was reported to repress the transcriptional potential of several transcription factors and target to PML oncogenic domains (PODs) via SUMO-dependent interactions. The mechanism by which Daxx binds to sumoylated factors mediating transcriptional and subnuclear compartmental regulation remains unclear. Here, we define a SUMO-interacting motif (SIM) within Daxx and show it to be crucial for targeting Daxx to PODs and for transrepression of several sumoylated transcription factors, including glucocorticoid receptor (GR). In addition, the capability of Daxx SIM to bind SUMO also controls Daxx sumoylation. We further demonstrate that arsenic trioxide-induced sumoylation of PML correlates with a change of endogenous Daxx partitioning from GR-regulated gene promoter to PODs and a relief of Daxx repression on GR target gene expression. Our results provide mechanistic insights into Daxx in SUMO-dependent transcriptional control and subnuclear compartmentalization.

Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen.

In this study, a serotype-specific monoclonal antibody (mAb), D(2) 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His-Arg/Lys-Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.