In vitro and in vivo assays implicating TRAIL-R2 as a candidate dosage-dependent tumor suppressor in B-cell lymphoma. (A) TRAIL-induced apoptosis response in SU-DHL6 (resistant to TRAIL-induced apoptosis) and VAL (sensitive to TRAIL-induced apoptosis) cell lines. Cell surface expression of the 4 TRAIL receptors was determined by flow cytometry. Fresh SUDHL6 and VAL cells were incubated in the presence of recombinant human TRAIL (500 ng/mL). After 24 hours, cell death was quantified by flow cytometric analysis after staining with FITC-conjugated Annexin V and propidium iodide; the percentage of dead cells is indicated. (B) Namalwa and SUDHL6 cell lines, both carrying 8p21 deletion, were transfected with pcDNA3 Flag-TRAIL-R1 or pcDNA3 Flag-TRAIL-R2. Transfected cells were selected in the presence of G418-containing media, and subsequently maintained with 0.25 mg/mL to 0.5 mg/mL G418. Protein expression TRAIL-R1 and TRAIL-R2 were analyzed by Western blot and flow cytometry. Flow cytometry analysis of cell surface expression of TRAIL-R1 and TRAIL-R2 receptors after cell transfection in SU-DHL6 and Namalwa is shown. Apoptosis was determined before and after 24 hours of treatment with TRAIL. Restoration of expression levels of TRAIL-R1 and TRAIL-R2 by less than 2-fold was associated with increased TRAIL-induced apoptosis, as shown by the percentage of apoptotic cells in the cell lines transfected with the empty vector compared with those transfected with pcDNA-TRAIL-R1 and R2.