FAS (CD95) mutations are rare in gastric MALT lymphoma but occur more frequently in primary gastric diffuse large B-cell lymphoma - PubMed (original) (raw)

Figure 2

Biological activity of CD 95 mutants isolated from three cases of gastric DLBCL (cases 1, 2, and 3 in Table 2). A: T47D breast carcinoma and Jurkat T cells were cotransfected with either FAS wild-type, FAS mutant 1, 2, or 3 (corresponding to cases 1, 2, and 3 in Table 2), or vector control, together with the luciferase reporter vector, pGL3. After transfection cells were cultured with or without sFASL (T47D 30 ng/ml; Jurkat cells 10 ng/ml) for 24 hours. Luciferase expression was assayed thereafter, and percent viability was calculated by the following formula: (luciferase activity with sFASL/luciferase activity without sFASL) × 100. The data shown are the mean results (± SEM) of three independent experiments, each performed in triplicate. The P values for the differences between FAS wild-type and mutant 1, 2, and 3 transfected cells in comparison to vector-only transfectants were: T47D cells; wild-type, P < 0.08, mutant 1, P < 0.0004, mutant 2, P < 0.00004, mutant 3, P < 0.6. Jurkat cells: wild-type, P < 0.0008, mutant 1, P < 0.00001, mutant 2, P < 0.00001, mutant 3, P < 0.0005 (Student’s _t_-test). B: Detection of transfected FAS wild-type and mutant 1, 2, and 3 mRNA expression by RT-PCR. RT+ = 1. strand cDNA reaction with reverse transcriptase; RT− = negative control without reverse transcriptase in 1. strand cDNA reaction; water control = negative control without addition of cDNA reaction product to the PCR. C: Detection of transfected FAS wild-type and mutant 1, 2, and 3 protein by Western blotting with an antibody against a V5 sequence tag located at the 3′ end of the transfected genes. In the lane “mut 2” no protein band was detected, because this mutant harbors a stop codon in the intracellular death domain, located 5′ to the V5 sequence tag, which is therefore not expressed.