Joobae Park

Aberrant Methylation of APC, MGMT, RASSF2A, and Wif-1 Genes in Plasma as a Biomarker for Early Detection of Colorectal Cancer

Purpose: To identify epigenetic molecular makers in plasma for the early detection of colorectal cancer. Experimental Design: We retrospectively analyzed the methylation status of 10 genes in fresh-frozen tissues and corresponding plasma samples from 243 patients with stage I and II sporadic colorectal cancer, 276 healthy individuals, and plasma from 64 colorectal adenoma patients using methylation-specific PCR. The methylation score (Mscore) was used to find molecular markers with high sensitivity and specificity. Results: Of the 243 colorectal cancer tissues, methylation was detected in 18% for p14, 34% for p16, 27% for APC, 34% for DAPK, 32% for HLTF, 21% for hMLH1, 39% for MGMT, 24% for RARβ2, 58% for RASSF2A, and 74% for Wif-1. Receiver operator characteristic curve analysis in plasma from 243 patients with cancer and 276 healthy individuals showed that the M score of any single gene had a sensitivity of <40% after controlling for age, sex, and tumor location. The specificity of the M score was not different between multigene and single gene analyses, but the sensitivity of the M score was significantly increased by multigene analysis. For all patients, the M score in a model including APC, MGMT, RASSF2A, and Wif-1 genes had a sensitivity of 86.5% and a specificity of 92.1% when 1.6 was used as a cutoff. In this model, the M score had a positive predictive value of 90.6% and a negative predictive value of 88.8%. Conclusion: The present study suggests that tumor-specific methylation of APC, MGMT, RASSF2A, and Wif-1 genes might be a valuable biomarker in plasma for the early detection of colorectal cancer. (Clin Cancer Res 2009;15(19):6185–91)

Tumor-Specific Methylation in Bronchial Lavage for the Early Detection of Non-Small-Cell Lung Cancer

PURPOSE The aim of this study was to identify tumor-specific methylation in bronchial lavage for the early detection of non-small-cell lung cancer (NSCLC) by differentiating the age-related methylation from the tumor-specific methylation in NSCLC. PATIENTS AND METHODS Eighty-five NSCLC patients and 127 cancer-free subjects participated in this study. Aberrant methylation at the promoters of the p16, Ras association domain family 1A (RASSF1A), fragile histidine triad (FHIT), H-cadherin, and retinoic acid receptor beta (RARbeta) genes were evaluated in the resected tumor tissues and bronchial lavage samples of NSCLC patients and in the bronchial lavage samples of cancer-free subjects by methylation-specific polymerase chain reaction. RESULTS Of the 127 cancer-free samples, methylation was detected in 6% for p16, 13% for RARbeta, 3% for H-cadherin, 4% for RASSF1A, and 28% for FHIT. Hypermethylation of the p16, RARbeta, H-cadherin, and RASSF1A genes was not associated with patient age and smoking, whereas hypermethylation of the FHIT promoter occurred more frequently in older patients (P =.02) and was associated with exposure to tobacco smoke (P =.001). A strong correlation between age and smoking was found in patients with hypermethylation of the FHIT gene (r = 0.36; P =.03). A total of 68% of the bronchial lavage samples from the 85 NSCLC patients showed methylation of at least one of p16, RARbeta, H-cadherin, and RASSF1A genes. CONCLUSION Our study suggests that tumor-specific methylation of the p16, RASSF1A, H-cadherin, and RARbeta genes may be a valuable biomarker for the early detection of NSCLC in bronchial lavage, and that the age-related methylation of FHIT gene in the normal bronchial epithelium is related to the exposure to tobacco smoke.

Elevated mRNA levels of DNA methyltransferase‐1 as an independent prognostic factor in primary nonsmall cell lung cancer

Despite many reports about the involvement of DNA methyltransferases (DNMTs) in human cancers, including nonsmall cell lung cancer (NSCLC), the clinicopathologic significance of DNMTs in primary NSCLC remains to be elucidated.

Cohypermethylation of p16 and FHIT Promoters as a Prognostic Factor of Recurrence in Surgically Resected Stage I Non–Small Cell Lung Cancer

Despite advances in the detection and treatment of lung cancer, the prognosis for patients with lung cancer is poor, partly as a result of recurrences. We retrospectively analyzed the relationship between recurrence and survival in patients with non-small cell lung cancers (NSCLC), and the promoter methylation of p16, GSTP1, FHIT, H-cadherin, and RARbeta2 genes to identify a prognostic molecular marker associated with the recurrence of NSCLC. Methylation status from 335 paraffin blocks was determined by methylation-specific PCR. Of the 335 NSCLC samples, promoter methylation was detected in 35% for p16, 39% for RARbeta2, 42% for H-cadherin, 7% for GSTP1, and 21% for FHIT. Recurrence was observed in 39% (132 of 335) of the patients. Recurrence was significantly associated with histology (P = 0.001) and pathologic stage (P = 0.009). Hypermethylation of any single gene was not associated with recurrence in patients. However, cohypermethylation of p16 and FHIT genes in stage I NSCLCs was associated with an increased risk of recurrence [odds ratio, 6.43; 95% confidence interval (CI), 1.04-20.19; P = 0.02] and poor recurrence-free survival after surgery (hazard ratio, 2.03; 95% CI, 1.09-6.23; P = 0.02). In addition, their survival after recurrence was also 4.62 times poorer (95% CI, 1.27-16.48; P = 0.005) than for those without cohypermethylation of both genes. In conclusion, the present study suggests that cohypermethylation of p16 and FHIT genes in patients with stage I NSCLC may be a valuable biomarker for predicting the recurrence-associated prognosis of the disease.

Behavioural pharmacology of polygalasaponins indicates potential antipsychotic efficacy.

Polygalasaponins were extracted from a plant (Polygala tenuifolia Willdenow) that has been prescribed for hundreds of years to treat psychotic illnesses in Korean traditional medicine. Previous in vitro binding studies suggested a potential mechanism for its antipsychotic action, as polygalasaponin was shown to have an affinity for both dopamine and serotonin receptors [Psychopharmacol. Bull. 31 (1995) 139.]. In the present study we have investigated the functional in vivo actions of this material in tests that are predictive of dopamine and serotonin antagonist activities. Polygalasaponin (25-500 mg/kg) was shown to produce a dose-related reduction in the apomorphine-induced climbing behaviour (minimum effective dose [ED(min)] 25 mg/kg ip, 250 mg/kg sc and po), the 5-hydroxytryptamine (5-HTP)-induced serotonin syndrome (ED(min) 50 mg/kg ip) and the MK-801-induced hyperactivity (ED(min) 25 mg/kg ip) in mice. This compound also reduced the cocaine-induced hyperactivity (ED(min) 25 mg/kg ip) in rats. These results demonstrated that polygalasaponin has dopamine and serotonin receptor antagonist properties in vivo. This might suggest its possible utility as an antipsychotic agent.

Association of the SUV39H1 histone methyltransferase with the DNA methyltransferase 1 at mRNA expression level in primary colorectal cancer.

This study was aimed at investigating the involvement of the SUV39H1 histone methyltransferase on the epigenetic change of euchromatic promoter in colorectal cancer. We retrospectively analyzed the mRNA levels of SUV39H1 and the promoter methylation of the p14ARF, p16INK4a and HLTF genes as well as the mRNA levels of DNA methyltransferase 1 (DNMT1) in fresh frozen tissues from 219 colorectal cancer patients. The mRNA levels of the SUV39H1 and DNMT1 were assessed via quantitative real‐time PCR and the methylation profiles of the CpG islands were determined using methylation‐specific PCR. The mRNA levels of SUV39H1 and DNMT1 were elevated in 25% and 42% of 219 colorectal cancers, respectively. The hypermethylation of the p14ARF, p16INK4a and HLTF genes occurred in 36%, 51% and 34% of the patients. The elevated mRNA levels of SUV39H1 were not associated with the hypermethylation of the 3 genes. However, the mRNA levels of DNMT1 were significantly different between patients with elevated mRNA levels of SUV39H1 and those without (1.62 ± 0.84, 0.91 ± 0.81, respectively; p = 0.007). Patients with elevated mRNA levels of SUV39H1 showed a higher prevalence of DNMT1 elevation than those without (61 vs. 35%, p = 0.0008). Patients with an elevated mRNA level of SUV39H1 had a 2.71 (95% CI = 1.09–4.48, p = 0.002) times greater risk of an elevated mRNA level of DNMT1, after controlling for age and gender. In conclusion, the present study suggests that SUV39H1 is significantly associated with DNMT1, but not with euchromatic promoter methylation in colorectal cancer.

CpG island hypermethylation of E-cadherin (CDH1) and integrin α4 is associated with recurrence of early stage esophageal squamous cell carcinoma

The prognosis of esophageal squamous cell carcinoma (ESCC) patients remains very poor, which is partially due to a high rate of recurrence. This study was aimed at identifying a recurrence‐associated epigenetic prognostic marker in patients with ESCC. We retrospectively analyzed the CpG island hypermethylation of the p16, Wif‐1, sFRP1, integrin α4, CDH1, DAP kinase and RARβ2 genes in 251 ESCCs. The methylation status was determined by methylation‐specific PCR. Hypermethylation was detected in 52% for p16, 25% for RARβ2, 43% for CDH1, 21% for integrin α4, 57% for sFRP1, 38% for DAP kinase and 35% for Wif‐1. Recurrence was observed in 131 (52%) of the 251 cases. For stage I cancers, CDH1 methylation was associated with a high risk of recurrence (OR = 5.26, 95% CI = 1.48–18.67; p = 0.01) and a poor recurrence‐free survival after surgery (HR = 3.13, 95% CI = 1.21–8.09; p = 0.02). The hazard of failure after recurrence was about 13.17 (95% CI = 2.46–70.41; p = 0.003) times higher in patients with Wif‐1 methylation than in those without. For stage II cancers, integrin α4 methylation was associated with an increased risk of recurrence (OR = 3.03, 95% CI = 1.09–8.37; p = 0.03) and a poor recurrence‐free survival (HR = 2.12, 95% CI = 1.13–3.98; p = 0.03). In conclusion, the present study suggests that hypermethylation of CDH1 and integrin α4 genes may be used as recurrence‐associated prognostic indicators in stage I and stage II ESCCs, respectively.

Promoter Methylation of Retinoic Acid Receptor Beta 2 and the Development of Second Primary Lung Cancers in Non–Small-Cell Lung Cancer

PURPOSE To investigate whether the promoter hypermethylation of retinoic acid receptor beta 2 (RARbeta2) is associated with the development of second primary lung cancers (SPLCs) differentially according to smoking status in primary non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS We retrospectively analyzed the relationship between RARbeta2 methylation and the SPLC development in a total of 342 NSCLCs. The methylation status of RARbeta2 was determined by using methylation-specific polymerase chain reaction. The difference in the time to SPLC development was analyzed by using the log-rank test and the Cox proportional hazards model. The median follow-up was 4.1 years. RESULTS SPLCs developed in 19 (5.6%) of the 342 NSCLCs, and overall incidence rate of SPLC development was 1.54 per 100 patient-years. SPLCs did not occur in 39 patients who had not smoked. After controlling for possible confounding factors, the hazard of failure for former smokers with RARbeta2 hypermethylation was about 2.87 (95% CI, 0.92 to 13.64; P =.08) times higher compared to those without RARbeta2 methylation. However, for current smokers, hypermethylation of the RARbeta2 was found to have a protective effect against the SPLC development (hazard ratio = 0.23; 95% CI, 0.11 to 0.87; P =.03). CONCLUSION Hypermethylation of RARbeta2 promoter had a differential effect on the development of SPLCs in NSCLC, and this was dependent on smoking status. Our study suggests that a combination of retinoids and/or a demethylating agent may be effective in the prevention of SPLCs in never-smokers and former smokers with NSCLC.

Activation of protein kinase B (Akt) signaling after electroconvulsive shock in the rat hippocampus.

Akt (protein kinase B, PKB) is one of the major downstream pathways of neurotrophin signaling and plays important roles in the cell survival and synaptic plasticity of the central nervous system. Electroconvulsive shock (ECS) has neurotrophic effect and it affects the synaptic plasticity. It can activate another major pathway of neurotrophin signaling, i.e., Ras-Raf-MEK-Erk cascade. In this paper, the authors investigated whether ECS can activate Akt signaling in the rat hippocampus. After a single ECS, the phosphorylation of Akt was increased, as were the signals detected by phospho-PDK1 substrate antibody, which suggests the activation of PDK1, an upstream molecule of Akt. The phosphorylation of downstream molecules of Akt, forkhead transcription factors (FKHR), endothelial nitric oxide synthase (eNOS), and glycogen synthase kinase-3beta (GSK-3beta) was also increased. The increased phosphorylation of Akt appeared within 5 min of ECS and its time frame paralleled that of the phosphorylation of Erks. Taken together, these results suggest that ECS activates Akt signaling over a similar time scale to that of Erks in the rat hippocampus.

Functional importance of the anaphase-promoting complex-Cdh1-mediated degradation of TMAP/CKAP2 in regulation of spindle function and cytokinesis.

ABSTRACT Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein (TMAP), is a novel microtubule-associated protein that is frequently upregulated in various malignances. However, its cellular functions remain unknown. A previous study has shown that its protein level begins to increase during G1/S and peaks at G2/M, after which it decreases abruptly. Ectopic overexpression of TMAP/CKAP2 induced microtubule bundling related to increased microtubule stability. TMAP/CKAP2 overexpression also resulted in cell cycle arrest during mitosis due to a defect in centrosome separation and subsequent formation of a monopolar spindle. We also show that degradation of TMAP/CKAP2 during mitotic exit is mediated by the anaphase-promoting complex bound to Cdh1 and that the KEN box motif near the N terminus is necessary for its destruction. Compared to the wild type, expression of a nondegradable mutant of TMAP/CKAP2 significantly increased the occurrence of spindle defects and cytokinesis failure. These results suggest that TMAP/CKAP2 plays a role in the assembly and maintenance of mitotic spindles, presumably by regulating microtubule dynamics, and its destruction during mitotic exit serves an important role in the completion of cytokinesis and in the maintenance of spindle bipolarity in the next mitosis.