Journal Article

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

Cara L. Crowley-Weber, Claire M. Payne, Mary Gleason-Guzman, George S. Watts, Bernard Futscher, Caroline N. Waltmire, Cheray Crowley, Katerina Dvorakova, Carol Bernstein, Mary Craven, Harinder Garewal and Harris Bernstein

in Carcinogenesis

Volume 23, issue 12, pages 2063-2080
Published in print December 2002 | ISSN: 0143-3334
Published online December 2002 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/23.12.2063
Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate

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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 members, PKA, PI(4,5)K, TRAF2 and BIRC1 (IAP protein). Under-expressed mRNAs included BNIP3, caspase-6, caspase-3 and serine protease 11. NF-κB was constitutively activated in all three resistant cell lines, and was responsible, in part, for the observed apoptosis resistance, determined using antisense oligonucleotide strategies. Molecular and cellular analyses of these resistant cell lines has suggested potential mechanisms by which apoptosis resistance may develop in the colonic epithelium in response to high concentrations of hydrophobic bile acids that are associated with a Western-style diet. These analyses provide the rationale for the development of hypothesis-driven intermediate biomarkers to assess colon cancer risk on an individual basis.

Keywords: BH4, 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-glucose-regulated protein; IAP, inhibitor of apoptosis protein; IEF, isoelectric focusing; IKK-β, IκB-kinase-β; IP3, inositol triphosphate; MALDI-MS, matrix assisted laser desorption ionization mass spectroscopy; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MEKK, MEK kinase; NIK, NF-κB-inducing 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.

Journal Article.  14566 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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