Number of genes induced by 5-aza-dC treatment in individual pancreatic CAFs, control fibroblasts and cell lines.

<p>An average of 123±86 genes were induced 5-fold or more by 5-aza-dC treatment in four pancreatic cancer cell lines, 9±10 genes in ten pancreatic CAFs (<i>P</i> = 0.0009) and 17±14 genes in three control pancreatic fibroblast lines.</p

Immunohistochemical analysis of Adam12 protein expression in tissue microarrays.

<p>(A) Adam12 protein expression is undetectable in the granule cell layer of the brain (negative control tissue). (B) Stromal fibroblasts (arrows) surrounding normal pancreatic duct do not label Adam12. (C) Cancer associated fibroblasts (arrows) in a primary pancreatic adenocarcinoma are strongly positive for Adam12 protein; magnification, 20×.</p

Genes upregulated by an overall fold change of ≥2.0 in ten 5-aza-dC-treated pancreatic CAFs relative to untreated CAFs.

<p>Genes upregulated by an overall fold change of ≥2.0 in ten 5-aza-dC-treated pancreatic CAFs relative to untreated CAFs.</p

Unlike Pancreatic Cancer Cells Pancreatic Cancer Associated Fibroblasts Display Minimal Gene Induction after 5-Aza-2′-Deoxycytidine

<div><h3>Purpose</h3><p>Cancer associated stromal fibroblasts (CAFs) undergo transcriptional and phenotypic changes that contribute to tumor progression, but the mechanisms responsible for these changes are not well understood. Aberrant DNA methylation is an important cause of transcriptional alterations in cancer cells but it is not known how important DNA methylation alterations are to CAF behavior.</p> <h3>Experimental Design</h3><p>We used Affymetrix exon arrays to compare genes induced by the DNA methylation inhibitor 5-aza-dC in cultured pancreatic cancer associated fibroblasts, pancreatic control fibroblasts and pancreatic cancer cell lines.</p> <h3>Results</h3><p>We found that pancreatic CAFs and control pancreatic fibroblasts were less responsive to 5-aza-dC-mediated gene reactivation than pancreatic cancer cells (mean+/−SD of genes induced ≥5-fold was 9±10 genes in 10 pancreatic CAF cultures, 17±14 genes in 3 control pancreatic fibroblast cultures, and 134±85 genes in 4 pancreatic cancer cell lines). We examined differentially expressed genes between CAFs and control fibroblasts for candidate methylated genes and identified the disintegrin and metalloprotease, <em>ADAM12</em> as hypomethylated and overexpressed in pancreatic CAF lines and overexpressed in fibroblasts adjacent to primary pancreatic adenocarcinomas.</p> <h3>Conclusions</h3><p>Compared to pancreatic cancer cells, few genes are reactivated by DNMT1 inhibition in pancreatic CAFs suggesting these cells do not harbor many functionally important alterations in DNA methylation. CAFs may also not be very responsive to therapeutic targeting with DNA methylation inhibitors.</p> </div

Effect of 5-aza-dC treatment on DNMT1 protein levels by Western blot analysis of pancreatic CAF and control fibroblast cultures.

<p>DNMT1 protein is depleted (relative to GAPDH) in 5-aza-dC treated HPNE and CAF12 cells.</p

Analysis of <i>ADAM12</i> mRNA expression in pancreatic CAFs and control fibroblasts.

<p>(A) Affymetrix exon array analysis of <i>ADAM12</i> mRNA expression in pancreatic CAFs and control fibroblasts. (B) Quantitative RT-PCR analysis of <i>ADAM12</i> mRNA expression in pancreatic CAFs and control fibroblasts after normalization to <i>GAPDH</i> levels. Each assay was performed in triplicate. Data are means of three independent experiments; bars are SD values.</p

Bisulfite sequencing analysis of <i>ADAM12</i>.

<p>(A) Top: <i>ADAM12</i> gene structure and distribution of CpG dinucleotides. Short vertical bars represent CpG sites. Arrow points to transcriptional start site. Below: Bisulfite genomic sequencing analysis in pancreatic CAFs and control fibroblasts. Open circles represent unmethylated CpG sites, solid black circles methylated CpG sites, and hatched circles partially methylated CpG sites. (B) Bisulfite sequencing chromatograms of the <i>ADAM12</i> promoter in a pancreatic CAF (CAF19) and control fibroblast line (HPNE). Arrows point to cytosine residues.</p

Effect of 5-aza-dC treatment on <i>SFN</i> and <i>TKTL1</i> mRNA expression in 5-aza-dC treated pancreatic CAFs and control fibroblasts, and bisulfite sequencing analysis of <i>TKTL1</i>.

<p>(A) Affymetrix exon array analysis of <i>SFN</i> mRNA expression in five pancreatic CAFs before (green) and after (red) 5-aza-dC treatment. (B) and (C) Quantitative RT-PCR analysis of <i>SFN</i> and <i>TKTL1</i> mRNA expression relative to <i>GAPDH</i> mRNA in pancreatic fibroblast cultures before (blue bars) and after (red bars) 5-aza-dC treatment. Each assay was performed in triplicate. Data are means of three independent experiments; bars are SD values. (D) Top: <i>TKTL1</i> gene structure and distribution of CpG dinucleotides. Short vertical bars represent CpG sites. Arrow points to transcriptional start site. Below: Bisulfite genomic sequencing analysis in pancreatic CAFs and control fibroblasts. Open circles represent unmethylated CpG sites, solid black circles methylated CpG sites, and hatched circles partially methylated CpG sites. (E) Bisulfite sequencing chromatograms of the <i>TKTL1</i> promoter in a pancreatic CAF (CAF19) and control fibroblast lines (HPNE and SC2). Arrows point to cytosine residues.</p

MOESM1 of Establishment and characterization of 6 novel patient-derived primary pancreatic ductal adenocarcinoma cell lines from Korean pancreatic cancer patients

Additional file 1: Figure S1. TP53 histogram of AMCPAC cell lines