Cell cycle assay Cells were fixed in 75% ethanol, selleck compound 5��105 cells stained with propidium iodide and the cell cycle assay was carried out using the Guava PCA System. The experiment was performed in triplicate and analyzed using CytoSoft 2. 1. 5 software. Statistical analysis Fishers exact test, the chi square test, Mann Whitneys U test or Kruskal Wallis rank test used for categorical variables, and Students t test was used for continuous variables. Clinicopathological characteristics and follow up data were analyzed in association with 5 year disease specific survival. The follow up time was calcu lated from the date of surgery to death. DSS was calcu lated by Kaplan Meier method, and survival differences were assessed in the log rank test.

Variables suggested to be prognostic factors on univariable analysis were subjected to multivariable analysis using a Cox proportional hazards regression model. Data is expressed as mean standard deviation, and P value 0. 05 was considered to indicate statistical significance. All statistical analyses were conducted with SAS software package. Results Structure of HOPX promoter region Genomic structure of HOPX gene is shown in Figure 1A. HOPX has 3 unique transcript variants with 2 discrete promote regions, and all of the 3 transcripts have the same open reading frame. Among the 3 variants, only HOPX B harbours promoter CpG islands encom passed by the first exon and intron. Expression level of HOPX transcripts and protein in PC cell lines We first examined expression of HOPX core transcripts, in which PCR primers were shared for all the 3 tran scripts.

Among the 8 PC cell lines tested, only KLM 1, PK 8, and PK 45 H cells express slight ex pression of HOPX core transcript, where HOPX core transcript is consistent with HOPX transcripts. On the other hand, HOPX B was not detected at mRNA level in any 8 PC cell lines. WB analysis also revealed that HOPX protein was hardly detected in any PC cell lines. We therefore added experiments of IP/WB, which could detect very weak but consistent expression of HOPX protein in KLM 1, PK 8, and PK 45 H at 10 kDa. As HOPX transfectants and positive control TE15 showed considerable expression, we concluded that HOPX expression showed very little, if any, in PC cells.

Characteristics of promoter B methylation in PC cell lines We initially examined the HOPX B promoter methyla tion in all the 8 PC cell lines by bisulfite treatment fol lowed by direct sequencing, and promoter B of HOPX gene was proved to be completely methylated in cytosine residues of CpG islands in 7 PC cells except PANC 1 and MIA Paca2. In order to demonstrate whether HOPX silencing of PC cells is due to epigenetic abnormalities, demethylat ing agents such as 5 Aza dC and/or trichostatin A were added to PC cells, and reactivation of HOPX transcripts Batimastat was evaluated by RT PCR and Q RT PCR.