Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions.
OBJECTIVE
The objective of this study was to establish a flow cytometry assay for measuring c-FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis.


METHODS
Two c-FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c-FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal-to-noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c-FLIP expression was analysed for association with clinicopathological parameters and survival.


RESULTS
Rabbit polyclonal c-FLIP by Abcam and the IntraStain kit by Dako performed best. c-FLIP expression was detected in tumour cells in all 69 effusions (expression range 21-100%, median = 80%). No association was found between c-FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c-FLIP levels and expression of the previously studied apoptosis marker cleaved caspase-3 (P = 0.029).


CONCLUSIONS
An assay for measuring c-FLIP in cytology specimens is presented. c-FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.
Division of Pathology, Oslo University Hospital, Norwegian Radium Hospital, Oslo, Norway.
H P
HP
Dong
A K
AK
Ree Rosnes
A J
AJ
Bock
A
A
Holth
V A
VA
Flørenes
C G
CG
Trope'
B
B
Risberg
B
B
Davidson
D000230:Adenocarcinoma / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D005122:Exudates and Transudates / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008722:Methods; D008875:Middle Aged; D010051:Ovarian Neoplasms / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D010996:Pleural Effusion / Q000378:metabolism / Q000473:pathology*; D016019:Survival Analysis; D014407:Tumor Cells, Cultured
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053148:Caspase 3