Cholangiocarcinoma (CCA) is the most common primary liver cancer in Northeastern Thailand where liver fluke infection is highly endemic. Although aberrant DNA methylation in CCA has been reported by several investigators, little is known regarding the associations between them. In the present study, the results obtained from our previously published methylation array were analyzed and 10 candidate genes involved in DNA repair [protein phosphatase 4 catalytic subunit (PPP4C)], apoptosis [runt related transcription factor 3 (RUNX3), interferon regulatory factor 4 (IRF4), ubiquitin C‑terminal hydrolase L1 (UCHL1) and tumor protein p53 inducible protein 3 (TP53I3)], cell proliferation [cyclin D2 (CCND2) and Ras association domain family member 1 (RASSF1)], drug metabolism [aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and solute carrier family 29 member 1 (SLC29A1)] and angiogenesis [human immunodeficiency virus‑1 tat interactive protein 2 (HTATIP2)] were selected for quantification of their methylation levels in 54 CCA and 19 adjacent normal tissues using methylation‑sensitive high‑resolution melting. The associations between the methylation status of the individual genes and clinical parameters were statistically analyzed. High methylation levels were observed in UCHL1, IRF4, CCND2, HTATIP2 and TP53I3. The median methylation level of UCHL1 was 57.3% (range, 3.15 to 88.7%) and HTATIP2 was 13.6% (range, 7.5 to 36.7%). By contrast, low methylation of HTATIP2 and UCHL1 was identified in adjacent normal tissues. The methylation status of HTATIP2 and UCHL1 was associated with patients' overall survival. CCA patients with high methylation of HTATIP2 and low methylation of UCHL1 exhibited longer overall survival. In addition, multivariate Cox regression analysis demonstrated that UCHL1 methylation was an independent factor for CCA with hazard ratio of 1.81 (95% confidence interval, 1.01‑3.25) in high methylation group. The combination of HTATIP2 and UCHL1 methylation status strongly supported their potential predictive biomarker in which patients with CCA who had high methylation of HTATIP2 and low methylation of UCHL1 showed longer overall survival than those with low HTATIP2 methylation and high UCHL1 methylation. In conclusion, the present study revealed the value of aberrant DNA methylation of HTATIP2 and UCHL1, which may serve as a potential predictive biomarker for CCA.

To assess the presence and absence of mycoplasma contamination in cell culture, Fourier transform infrared (FTIR) microspectroscopy, coupled with multivariate analysis, was deployed to determine the biomolecular changes in hepatocellular carcinoma cells, HepG2, before and after mycoplasma contamination. The contaminated HepG2 cells were treated with antibiotic BM-Cyclin to decontaminate the mycoplasma, and polymerase chain reaction (PCR) was then performed to confirm the presence or the absence of mycoplasma contamination. The contaminated and decontaminated HepG2 cells were analyzed by FTIR microspectroscopy with principal component analysis (PCA) and peak integral area analysis. The results showed that the FTIR spectra of contaminated HepG2 cells demonstrated the alteration in the IR spectra corresponding to the lipid, protein, and nucleic acid regions. PCA analysis distinguished the spectral differences between the groups of mycoplasma-contaminated and -decontaminated cells. The PCA loading plots suggest that lipid and protein are the main contributed molecules for the difference between these two cell groups. Peak integral area analysis illustrated the increase of lipid and nucleic acid and the decrease of protein contents in the contaminated HepG2 cells. FTIR microspectroscopy is, therefore, proven to be a potential tool for assessing mycoplasma removal by monitoring biomolecular alterations in cell culture.

Cholangiocarcinoma (CCA) is a malignancy of bile duct epithelial cell lining. In the past decade, the incidence and mortality rates of CCA have been increasing worldwide. The effective treatment modality for CCA is surgical resection. Adjuvant chemotherapy and/or radiotherapy are optional therapy for unresectable and postoperative CCA. Unfortunately, the responsiveness of CCA patients to chemotherapy was relatively low and 5-year survival rate was poor. We have analyzed data which were obtained from our previous study on methylation microarray based on chemotherapeutic drug resistance and found aberrant DNA methylation of genes related to chemotherapy. This present study aimed to quantitate DNA methylation levels of 10 candidate genes related to chemotherapy in 54 CCA and 19 normal adjacent tissues using methylation-sensitive high resolution melting (MS-HRM) analysis. The methylation level of each gene was statistically analyzed with clinical parameters. Genes with high methylation level were UCH-L1 and IRF4, and genes with moderate methylation were CCND2, HTATIP2 and TP53I3. We found significant difference in methylation levels of HTATIP2 (P=0.036) and UCH-L1 (P=0.012) between short and long survival groups. Patients with high methylation level of HTATIP2 and low methylation of UCH-L1 showed longer overall survival (P=0.03 and P=0.044, respectively). Interestingly, chemotherapeutic responders showed higher methylation level of HTATIP2 than non-responders (P=0.020), whereas no significant difference in UCH-L1 methylation level was observed between these two groups. The methylation levels of HTATIP2 and UCH-L1 were low in normal adjacent tissues. In patients who received chemotherapy, patients who had high methylation of HTATIP2 and low methylation of UCH-L1 showed longer overall survival (P<0.001 and P=0.041, respectively). Moreover, HTATIP2 methylation was associated with tumor progression in which lower methylation level was found in CCA patients with positive lymph node metastasis (P=0.029). In conclusion, our finding indicates the great value of DNA methylation levels of HTATIP2 and UCH-L1 as prognostic and predictive markers for CCA patients. Citation Format: Temduang Limpaiboon, Chaiyachet Nanok, Siriporn Proungvitaya, Patcharee Jearanaikoon, Ake Pugkhem, Anucha Puapairoj. Aberrant DNA methylation of HTATIP2 and UCH-L1 as prognostic and predictive biomarkers for cholangiocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A37.

Background: Major histocompatibility complex class I chain related A (MICA/PERB11.1) molecules are expressed as transmembrane proteins. They are ligands recognized by the natural killer cell receptor group (NKG)2D, an activating receptor on NK cells, gamma delta T cells and activated CD8 T cells. Up-regulated MICA expression was reported in many tumors. Shedding of MICA from tumor cells as soluble MICA (sMICA) is a mechanism for immune evasion. MICA proteins could be presented in various sizes from 43 to 75 kDa depending on degree of glycosylation. Eight potential N-glycosylation sites in a MICA molecule were reported. This study aimed to investigate the size of sMICA in sera of cervical cancer patients and women blood donors by western blot technique. Methods: The sMICA*009 plasmid DNA was transfected to COS7 cells for recombinant sMICA protein production in culture supernatant. The recombinant sMICA protein was harvested and validated with mouse monoclonal anti-MICA antibody (WW6B7) by indirect ELISA and immunobloting. Molecular weight of deglycosylated recombinant sMICA protein treated with N-glycosidase F enzyme and sMICA in cervical cancer and healthy sera was determined. Results: The recombinant sMICA and serum sMICA showed 62 kDa and 45 kDa in molecular weight, respectively. The molecular weight of recombinant sMICA treated with N-glycosidase F enzyme was reduced to 45 kDa which equal to serum sMICA indicating that sMICA in sera of patients and blood donor controls was in a deglycosylated form. Conclusion: Recombinant sMICA produced in COS7 had a molecular weight of 62 kDa. In contrast, sMICA in sera was in a deglycosylated form and had a molecular weight of 45 kDa equal to in vitro deglycosylation with N-glycosidase F enzyme of produced recombinant sMICA. The mechanism and kinetic of deglycosylation of sMICA in sera await further investigation. It would be interesting to see whether this form of sMICA would affect sMICA detection in sera. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2344.