Novel genetic and epigenetic alterations in ... - Ous-research.no

Journal of Translational Medicine 2008, 6:13http://www.translational-medicine.com/content/6/1/13normal colon tissues from four persons with no knownhistory of colorectal cancer were obtained separately.Immunohistochemical in situ protein expression analysisFive m thick sections of the TMA blocks were transferredonto glass slides for immunohistochemical analyses. Thesections were deparaffinized in a xylene bath for 10 minutesand rehydrated via a series of graded ethanol baths.Heat-induced epitope retrieval was performed by heatingin a microwave oven at full effect (850 W) for 5 minutesfollowed by 15 minutes at 100 W immersed in 10 mM citratebuffer at pH 6.0 containing 0.05% Tween-20. Aftercooling to room temperature, the immunohistochemicalstaining was performed according to the protocol of theDAKO Envision+ K5007 kit (Dako, Glostrup, Denmark).The primary antibody, mouse clone 6D9 anti-MAL[29], was used at a dilution of 1:5000, which allowed forstaining of kidney tubuli as positive control, while theheart muscle tissue remained unstained as negative control[30]. The slides were counterstained with haematoxylinfor 2 minutes and then dehydrated in increasing gradesof ethanol and finally in xylene. Results from the immunohistochemistrywere obtained by independent scoringby one of the authors and a reference pathologist.StatisticsAll P values were derived from two tailed statistical testsusing the SPSS 13.0 software (SPSS, Chicago, IL, USA).Fisher's exact test was used to analyze 2 × 2 contingencytables. A 2 × 3 table and Chi-square test was used to analyzethe potential association between quantitative geneexpression of MAL and promoter methylation status.Samples were divided into two categories according totheir gene expression levels: low expression included sampleswith gene expression equal to, or lower than, themedian value across all cell lines or all tumours, highexpression included samples with gene expression higherthat the median. The methylation status was divided intothree categories: unmethylated, partial methylation, andhypermethylated.ResultsPromoter methylation status of MAL in tissues and celllinesThe promoter methylation status of MAL was analyzedwith MSP (Figure 1). One of 23 (4%) normal mucosasamples from non-cancerous donors and two of 21 (10%)normal mucosa samples taken in distance from the primarytumour were methylated but displayed only lowintensityband compared with the positive control aftergel electrophoresis. Forty-five of 63 (71%) adenomas and49/61 (80%) carcinomas showed promoter hypermethylation.Nineteen of twenty colon cancer cell lines (95%),and 15/26 (58%) cancer cell lines from various tissues(breast, kidney, ovary, pancreas, prostate, and uterus)Methylation mucosa Figure 1samples status and of colorectal the MAL promoter carcinomasnormal colonMethylation status of the MAL promoter in normalcolon mucosa samples and colorectal carcinomas.Representative results from methylation-specific polymerasechain reaction are shown. A visible PCR product in lanes Uindicates the presence of unmethylated alleles whereas aPCR product in lanes M indicates the presence of methylatedalleles. N, normal mucosa; C, carcinoma; Pos, positive control(unmethylated reaction: DNA from normal blood, methylatedreaction: in vitro methylated DNA); Neg, negativecontrol (containing water as template); U, lane for unmethylatedMSP product; M, lane for methylated MSP product.were hypermethylated (Table 1 lists tissue-specific frequencies).The hypermethylation frequency found in normal sampleswas significantly lower than in adenomas (P