Comparative phenotypic studies of duct epithelial cell lines derived from normal human pancreas and pancreatic carcinoma

Abstract

We have investigated the mRNA/protein expression of several tyrosine kinase receptors, growth factors, and p16INK4A cyclin inhibitor in cell lines derived from normal human pancreatic duct epithelium (HPDE) and compared them with those of five pancreatic ductal carcinoma cell lines. Cultured HPDE cells express low levels of epidermal growth factor receptor (EGFR), erbB2, transforming growth factor (TGF)-alpha, Met/hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF). They also expressed high levels of amphiregulin but did not express EGF and cripto. The expression levels were similar in primary normal HPDE cells and those expressing transfected E6E7 genes of human papilloma virus-16, but their immortalization appeared to enhance the expression of EGFR and Met/HGFR. In comparison, pancreatic carcinoma cell lines commonly demonstrated overexpression of EGFR, erbB2, TGF-alpha, Met/HGFR, VEGF, and KGF, but they consistently showed marked down-regulation of amphiregulin mRNA expression. In contrast to all carcinoma cell lines that showed deletions of the p16 gene, HPDE cells consistently demonstrated normal p16 genotype and its mRNA expression. This is the first report that compares the phenotypic expression of cultured pancreatic ductal carcinoma cells with epithelial cell lines derived from normal human pancreatic ducts. The findings confirm that malignant transformation of human pancreatic duct cells commonly results in a deregulation of expression of various growth factors and receptors.

Figure 1.

The mRNA expression of epidermal growth factor receptor family proteins and ligands in duct epithelial cell lines derived from normal human pancreas (HPDE) and pancreatic carcinomas (PKs). HPDE6(P6), primary culture of normal pancreatic duct epithelium; HPDE1-E6E7(P9), HPDE5-E6E7(P6), pre-crisis LXSN16E6E7 (retroviral expression vector carrying the E6E7 genes of HPV16) infected cells from two separate individuals; HPDE4-E6E7(P18) and HPDE6-E6E7(P18), immortalized LXSN16E6E7 infected HPDE cells from two other individuals; PK1, PK8, PK9, PK45, and PK59, pancreatic ductal carcinoma cell lines. Hybridization with 18 S ribosomal RNA probe showed the relative loading amount of RNA samples. All samples were hybridized on a single membrane.

Figure 2.

The expression of met (hepatocyte growth factor receptor), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF) in HPDE and pancreatic carcinoma cell lines. (See Figure 1 ▶ for details). All samples were hybridized on a single membrane.

Figure 3.

The p16^INK4A genotype and its expression in pancreatic ductal cell lines. A Southern blot analysis for p16 showed retention of normal p16 gene in HPDE cell lines and its homozygous deletion in four of five PK lines. The PK-59 cell line demonstrated 50% loss of hybridization signal, indicating heterozygous deletion. In contrast to p16, the hybridization signals for c-myc, c-met (hepatocyte growth factor receptor), and epidermal growth factor receptor (EGFR) genes among HPDE and adenocarcinoma (PK) cell lines were similar among these cell lines. Variability could be attributed to unequal amounts of DNA samples. B Northern blot analysis showed expression of p16 mRNA in all HPDE cell lines and in PK-59 but its absence in the other four PK cell lines with homozygous deletion of the gene.

Figure 4.

Immunoblot analyses for levels of EGFR, erbB2, and Met/HGFR proteins in immortalized HPDE16-E6E6 (passage 23) and PK-9 cell lines. Studies were performed on cells in preconfluent (PC) and confluent (C) cultures. The levels of EGFR and Met/HGFR were not influenced by the culture condition, but erbB2 levels were higher in confluent cells. Note also the higher levels in PK-9 as compared with the HPDE6-E6E7 cells.