Constitutive expression of c-FLIP in Hodgkin and Reed-Sternberg cells

Abstract

Crosslinking of the transmembrane receptor CD95/Fas leads to activation of a signaling cascade resulting in apoptosis. c-FLIP is a recently described protein that potently inhibits Fas-mediated apoptosis and has been shown to be a key factor in germinal center B cell survival. Because Hodgkin and Reed-Sternberg cells in classical Hodgkin's disease (cHD) are also resistant to Fas-mediated apoptosis we studied the role of c-FLIP in classical HD. High levels of c-FLIP protein were identified in two Fas-resistant Hodgkin-derived cell lines. In contrast to other tumor cells, inhibition of protein synthesis by cycloheximide did not lead to down-regulation of c-FLIP protein in these HD cell lines. Furthermore, Fas-mediated apoptosis was only partially restored suggesting that normal regulation of c-FLIP was disrupted. The in vivo relevance of these findings was supported by demonstration of significant c-FLIP expression by immunohistochemistry in 18 of 19 evaluable cases of primary HD. Taken together, c-FLIP is constitutively expressed in HD and may therefore be a major mechanism responsible for Fas-resistance in HD.

Figure 1.

HD cell lines L428 and L1236 are resistant to Fas-mediated apoptosis. Jurkat, L1236, and L428 cells (5 × 10⁵) were incubated for 24 hours with 100 ng/ml of agonistic anti-Fas mAb CH11 or the IgM-isotype control mAb. Apoptosis was assessed after 24 hours of incubation as described in Materials and Methods. Results are shown as percentages of apoptotic cells (MV of three independent experiments; bars, SD).

Figure 2.

c-FLIP mRNA is expressed in Fas-resistant HRS cell lines L1236 and 428, and in their physiological counterpart, the GC B cells. cDNAs from L1236, L428, and freshly isolated GC B cells were submitted to 35 cycles of RT-PCR amplification using oligonucleotides specific for the long isoform of c-FLIP. Products were analyzed by agarose gel electrophoresis and ethidium bromide staining. cDNA from K562 was co-amplified as a positive control. Equal amounts of cDNAs were verified by amplifying GAPDH transcripts.

Figure 3.

c-FLIP protein is not down-regulated in cultured HRS cells after treatment with CHX. Cells were incubated with 1 or 10 μg/ml of CHX. At time points of 0 hours, 12 hours, and 24 hours, 1 × 10 cells were harvested, lysed, and cell lysates were submitted to Western blotting using polyclonal c-FLIP_l antibody. Time points are indicated. A: Western blots from BJAB, L428, and L1236 cells at various time points of treatment with 1 μg/ml of CHX. The 55-kd band represents c-FLIP_l, the 42-kd band represents β-actin. B: Western blots from BJAB, L428, and L1236 cells at various time points of treatment with 10 μg/ml of CHX.

Figure 4.

Treatment with 10 μg/ml of the protein synthesis inhibitor CHX leads to a moderate sensitization to Fas-mediated apoptosis in L428 cells. Jurkat, L1236 and L428 cells (5 ×10⁵) were incubated for 24 hours with varying concentrations of CHX (1 μg/ml for Jurkat cells, 10 μg/ml for both L1236 and L428 cells), 100 ng/ml of agonistic anti-Fas mAb CH11 or the IgM-isotype control mAb. Apoptosis was assessed at indicated time points as described in Materials and Methods. Results are shown as percentages of apoptotic cells (MV of three independent experiments; bars, SD). □, Jurkat, 1 μg/ml CHX, 100 ng/ml isotype; ▪, Jurkat, 1 μg/ml CHX, 100 ng/ml CH11; ▵, L428, 10 μg/ml CHX, 100 ng/ml isotype; ▴, L428, 10 μg/ml CHX, 100 ng/ml CH11; ○, L1236, 10 μg/ml CHX, 100 μg/ml isotype; •, L1236, 10 μg/ml CHX, 100 ng/ml CH11.

Figure 5.

FACS analyses of Jurkat, L428, and L1236 cells after 24 hours of combined treatment with CHX and Fas-agonistic mAb CH11. Jurkat, L1236, and L428 cells (5 × 10⁵) were incubated for 24 hours with varying concentrations of CHX (1 μg/ml for Jurkat cells, 10 μg/ml for both L1236 and L428 cells), 100 ng/ml agonistic anti-Fas mAb CH11 or the IgM-isotype control mAb. The cells were incubated with phycoerythrin-labeled Annexin V stained with propidium iodide and submitted to FACS analyses. 10,000 events were recorded. Twenty percent of dots are shown. Percentages of apoptotic cells are indicated. A: Jurkat cells after a 24-hour treatment with 1 μg/ml of CHX and an isotype mAb. B: Jurkat cells after a 24-hour treatment with 1 μg/ml of CHX and 100 ng/ml of CH11. C: L428 cells after a 24-hour treatment with 10 μg/ml of CHX and an isotype mAb. D: L428 cells after a 24-hour treatment with 10 μg/ml of CHX and 100 ng/ml of CH11. E: L1236 cells after a 24-hour treatment with 10 μg/ml of CHX and an isotype-matched control mAb. F: L1236 cells after a 24-hour treatment with 10 μg/ml of CHX and 100 ng/ml of CH11.

Figure 6.

c-FLIP protein is expressed in HRS cells in HD-involved tissue. Serial sections from HD-involved tissues were stained either with a polyclonal anti-c-FLIP antibody or an anti-CD30 mAb to show HRS cells and then counterstained with hemalaun. The number of c-FLIP⁺ cells was estimated and compared to the number of CD30⁺ cells. Cases with 75 to 100% of HRS cells being positive for c-FLIP were categorized as ++, whereas cases with 25 to 75% of c-FLIP⁺ HRS cells were classified as +. Striated muscle tissue and GCs served as positive controls. A: Primary HD case stained with c-FLIP Ab showing one HRS cell. B: Primary HD case stained with a polyclonal c-FLIP Ab showing two HRS cells surrounded by lymphoid cells. C: Specimen of striated muscle tissue infiltrated by a B cell non-Hodgkin’s lymphoma stained with polyclonal c-FLIP Ab. D: Reactive lymph node with three GCs and vascular endothelial cells stained with polyclonal c-FLIP Ab. Original magnifications: ×200 (A); ×400 (B); ×100 (C and D).