FLIPL mediates Fas-resistance in EBV-positive Burkitt’s lymphoma cell lines. (A) FLIPL transcripts were detected by RT-PCR analysis of total RNA samples. The amplification product corresponding to the mature 1,440-bp coding sequence is indicated. Higher-molecular-weight products representing unprocessed transcripts were also detected and can be seen in the Mutu-BL III and Jijoye lanes. FLICE/caspase-8 transcript levels were determined with primers specific for the caspase-homology domain (792-bp). (B) Caspase-8/FLIPL competitive PCR analysis. FLIPLand FLICE/caspase-8 transcript levels were directly compared in the same sample by performing RT-PCR reactions containing primer pairs for both genes under conditions of limiting dNTPs. Gene-specific products and their sizes are indicated on the right and left, respectively. (C) The FLICE/FLIPL ratios were calculated from arbitrary values obtained by scanning densitometry of the gel from (B) and are graphically represented. Data were obtained from scans using 3 different exposures. Results are expressed as mean ± standard deviation and are representative of at least 3 separate experiments. (D) Direct comparison of FLICE/caspase-8 and FLIPL protein expression. Immunoblot analysis was performed on a panel of EBV-negative and EBV-positive BL cell lysates. The same membranes were used for analysis of both FLICE/caspase-8 and FLIPL by sequential antibody hybridizations. The 55-kD forms of both proteins are indicated, as well as the FLIPL p12 cleavage product predominant in the Ramos cell line.
