Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate

Evidence from live cell bioassays shows that the flat mucosa from patients with colon cancer exhibits resistance to bile salt-induced apoptosis. Three independent cell lines derived from the colonic epithelial cell line HCT-116 were selected for resistance to bile salt-induced apoptosis. These cell lines were developed as tissue culture models of apoptosis resistance. Selection was carried out for resistance to apoptosis induced by sodium deoxycholate (NaDOC), the bile salt found in highest concentrations in human fecal water. Cultures of HCT-116 cells were serially passaged in the presence of increasing concentrations of NaDOC. The resulting apoptosis resistant cells were able to grow at concentrations of NaDOC (0.5 mM) that cause apoptosis in a few hours in unselected HCT-116 cells. These cells were then analyzed for changes in gene expression. Observations from cDNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy, and confocal microscopy of immunofluorescently stained preparations indicated underexpression or overexpression of numerous genes at either the protein or mRNA level. Genes that may play a role in apoptosis and early stage carcinogenesis have been identified as upregulated in these cell lines, including Grp78, Bcl-2, NF-κB(p50), NF-κB(p65), thioredoxin peroxidase (peroxiredoxin) 2, peroxiredoxin 4, maspin, guanylate cyclase activating protein-1, PKCζ, EGFR, Ras family Abbreviations :B H 4, tetrahydrobiopterin; BNIP3, Bcl2/adenovirus EIB 19 kD-interacting protein 3; CAMK2D, calcium/calmodulin-dependent protein kinase II delta; DHAP, dihydroxyacetone phosphate; DOC, deoxycholate; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; GAP, glyceraldehyde 3-phosphate; GC, guanylate cyclase; Grp78, 78 kD-glucoseregulated protein; IAP, inhibitor of apoptosis protein; IEF, isoelectric focusing; IKK-β ,I κB-kinase-β ;I P 3, inositol triphosphate; MALDI-MS, matrix assisted laser desorption ionization mass spectroscopy; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MEKK, MEK kinase; NIK, NF-κBinducing kinase; NLS, nuclear localization signal; NaDOC, sodium deoxycholate; NO, nitric oxide; NOS2, inducible NO synthase; ONOO–, peroxynitrite; PDTC, pyrrolidine dithiocarbamate; PKCζ, protein kinase C-zeta; PKG, cGMP-activated protein kinase; PN-1, protease nexin-1; QDPR, quinoid dihydropteridine reductase; SERPIN, serine protease inhibitor; TEM, transmission electron microscopy; TPI, triose phosphate isomerase; Trx, thioredoxin; TPx, Trx peroxidase (peroxiredoxin); TR, Trx reductase; TRAF, tumor necrosis factor receptor-associated factor; TTFA, thenoyl trifluoroacetone.