8 ± 5.7% increase in death of Jurkat cells. These

results suggest that the S20-3 peptide derived from the HHV-8 K1 protein selectively induces cell death in malignant hematological cells, but is not toxic to normal human cells. The S20-3 peptide kills cells in the see more absence of the Fas receptor To investigate whether S20-3–induced apoptosis depends on the signaling of the Fas receptor, we tested the S20-3 peptide in Fas-resistant HDAC inhibitor Jurkat cell lines I2.1 and I9.2, which have defective FADD and caspase-8 functions, respectively [18]. The S20-3 peptide induced slightly less cell death in I2.1 cells than in the wild type ACY-738 manufacturer Jurkat cells (21% vs. 24% above control; Figure 3A). The response of caspase-8 function-defective

Jurkat cell line I9.2 to the S20-3 peptide was significantly blunted compared with that of wild-type Jurkat cells (14.4% vs. 24% above control; 60% reduction) but not completely eliminated (Figure 3A). In line with this result, we found that the pan-caspase inhibitor z-VAD also only partially blocked S20-3-induced death in BJAB cells (8.9% vs. 13.3% above control; 67% reduction) (Figure 3B) as well as apoptosis induced by the Fas-agonistic antibody CH-11 (14% vs. 29% above control; 48% reduction) (data not shown). Figure 3 The S20-3 peptide–induced cell death is only partially dependent on caspases and involves necroptosis. (A) Jurkat (wild-type), Jurkat I9.2 GPX6 (caspase-8–deficient), and Jurkat I2.1 (FADD-dominant-negative mutant) cell lines were incubated with 100 μM peptide S20-3. (B) BJAB cells were incubated with 100 μM peptide S20-3 in the presence or absence of 20 μM pan-caspase inhibitor z-VAD-FMK. (C) Daudi cells were incubated with 100 μM peptide S20-3 or buffer in the presence or absence of 20 μM pan-caspase inhibitor z-VAD-FMK. After 1 hour of incubation, cells were washed and incubated in complete medium

for 24 hours before flow cytometry analysis. Data in (A) and (B) are shown as means ± SD of triplicate wells; *P < 0.01. Further examination of the cell death induced by the S20-3 peptide in Daudi cells revealed that the S20-3 peptide induced necrosis (33.7% PI–positive cells) rather than apoptosis (0.3% AnnexinV–positive/PI–negative cells) in Fas-resistant Daudi cells (Figure 3C and Additional file 1: Figure S3A), and z-VAD further enhanced this effect (41.1% PI–positive cells) (Figure 3C). An LDH release assay further confirmed that the S20-3 peptide was causing necrosis as early as 1 hour post treatment (Additional file 1: Figure S3B).