Fas expression prevents cholangiocarcinoma tumor growth

Cholangiocarcinoma continues to have a dismal prognosis with an overall sur vival rate of less than 10%. An increased understanding of the molecular on cogenesis of this tumor is needed. Fas/APO-1 (CD95) receptor and Fas ligand have been implicated as key factors in apoptosis. In this study we have ex amined the role of the Fas receptor in the growth of cholangiocarcinoma. Th e purpose of this study was to evaluate the role of the Fas receptor in the induction of apoptosis in cholangiocarcinoma and to assess the role of the Fas receptor in cholangiocarcinoma tumorigenesis. Human cholangiocarcinoma cells, SK-ChA-1, were evaluated for Fas receptor expression using fluoresc ence-activated cell sorting (FACS). Distinct cell populations (Fas-positive and Fas-negative) were isolated by FAGS and cloned from single cell diluti ons. Fas expression was assessed by FACS and reverse transcriptase-polymera se chain reaction (RT-PCR). Cell populations were further characterized by their sensitivity to anti-Fas monoclonal antibody at 72 hours. Cell viabili ty and apoptotic index were evaluated by trypan blue cell count and termina l deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end- labeling (TUNEL) assay, respectively. Distinct cell populations were evalua ted for their ability to form tumors in BALB/c nude mice (2.5 x 10(6) cells per subcutaneous injection). After 4 weeks, tumors were evaluated for tumo r area by caliper measurement and Fas expression by RT-PCR. Maintenance of biliary phenotype was assured by means of AE-1 (cytokeratin) immunohistoche mistry. populations of Fas-positive and Fas-negative cells mere identified, isolated, and confirmed by FACS and RT-PCR. Treatment of Fas-positive cell s with anti-Fas monoclonal antibody produced an 80% reduction in cell viabi lity compared to no decrease in viability in Fas-negative cells by trypan b lue cell count. TUNEL staining showed an apoptotic index of 75% fdr Fas-pos itive cells incubated with anti-Fas monoclonal antibody and no significant evidence of apoptosis in the Fas-negative cells, When cholangiocarcinoma ce lls were subcutaneously injected into nude mice, only Fas-negative cells fo rmed tumor nodules; Fas-positive cells failed to form tumor nodules. The an alyzed tumors lacked Fas messenger RNA by RT-PCR but maintained the biliary cytokeratin AE-1 by immunohistochemistry. Fas receptor expression is an im portant mediator of apoptosis in cultured human cholangiocarcinoma cells an d appears to be a critical determinant of cholangiocarcinoma tumor growth i n nude mice.