Bcl-2 is overexpressed and alters the threshold for apoptosis in a cholangiocarcinoma cell line.
Cholangiocarcinoma is a malignant neoplasm originating from cholangiocytes. The mechanisms responsible for oncogenesis of cholangiocytes are unknown. Resistance to apoptosis, especially by altered expression of B-cell lymphoma/leukemia 2 (Bcl-2) family members, has been implicated as a mechanism contributing to malignant transformation. Thus, our aim was to test the hypothesis that altered expression of Bcl-2 family members by cholangiocarcinoma cells renders them resistant to apoptosis. We compared the apoptotic threshold and expression of the Bcl-2 protein family members, Bcl-2, Bcl-XL, and Bax, in two human cell lines: 1) nonmalignant human cholangiocytes immortalized by transfection with the simian virus 40 (SV 40) large T antigen; and 2) a malignant human cholangiocarcinoma cell line. Apoptosis was induced pharmacologically using beauvericin. Bcl-2, Bcl-x long, and Bax protein expression were evaluated by immunoblot analysis, and Bcl-2 expression was modulated using antisense technology. The cholangiocyte and malignant/nonmaligant phenotype of both cell lines was verified using both in vitro and in vivo approaches. Beauvericin induced apoptosis of nonmalignant cholangiocytes in a concentration- (0 to 25 micromol/L) and time- (0 to 6 hours) dependent manner. In contrast, malignant cholangiocytes were resistant to apoptosis. Although expression of Bcl-x long and Bax protein were similiar in the two cell lines, Bcl-2 protein expression was 15-fold greater in malignant than in nonmalignant cholangiocytes. An 18 mer bcl-2 antisense oligonucleotide reduced expression of Bcl-2 protein by 50% and increased the rate of beauvericin-induced apoptosis more than threefold in the malignant cells. Our results support the hypothesis that resistance to apoptosis by overexpression of Bcl-2 may be a feature of cholangiocarcinoma.
Harnois
D M
DM
Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Internal Medicine, Mayo Medical School, Clinic and Foundation, Rochester, MN 55905, USA.
Que
F G
FG
Celli
A
A
LaRusso
N F
NF
Gores
G J
GJ
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis; D001650:Bile Duct Neoplasms / Q000737:chemistry / Q000473:pathology*; D001653:Bile Ducts, Intrahepatic; D018281:Cholangiocarcinoma / Q000737:chemistry / Q000473:pathology*; D047630:Depsipeptides; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis*; D014407:Tumor Cells, Cultured
D000900:Anti-Bacterial Agents; D047630:Depsipeptides; D016376:Oligonucleotides, Antisense; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; C004456:beauvericin