Resistance to Fas-mediated apoptosis is restored by cycloheximide through the downregulation of cellular FLIPL in NK/T-cell lymphoma

Abstract

Extranodal NK/T-cell lymphoma (NKTL), nasal type, is a highly aggressive neoplasm and is strongly associated with Epstein-Barr virus (EBV). In this study, we demonstrate that EBV-positive NKTL cell lines, namely, Hank-1, NK-YS, and NK-L, are resistant to Fas-mediated apoptosis induced by anti-Fas antibodies despite high levels of Fas surface expression and no mutation in the Fas gene. Fas stimulation of Hank-1 and NK-YS cells showed little processing of caspase 8, caspase 3, or bid, although the proximal signaling molecules of the deathinducing signaling complex, namely, Fas, Fas-associated protein with a death domain, caspase 8, and bid were present in these cells. Consistent with previous reports on the hypermethylation of death associated protein (DAP) kinase in NKTLs, the promoter of DAP kinase was methylated and its mRNA not detected in Hank-1 cells. However, the restoration of DAP kinase expression by 5-aza-2 0 -deoxycytidine did not sensitize Hank-1 to Fasmediated apoptosis, indicating that DAP kinase deficiency does not contribute to resistance to Fas-mediated apoptosis. Since etoposide-induced apoptosis involved caspase 3 activation in Hank-1 and NK-YS cells, the caspase 3-dependent apoptotic machinery appears to be intact. Interestingly, cotreatment of Hank-1 with cycloheximide, a protein synthesis inhibitor, markedly sensitized cells to Fas-mediated apoptosis along with caspase 8 activation and c-FLIP L (cellular FLICE inhibitory protein long form) downregulation. Moreover, immunohistochemistry on paraffin-embedded tissue revealed c-FLIP expression in 39% (14 of 36) of NKTL patients. Taken together, these findings indicate that c-FLIP L -mediated resistance to Fas contributes to the development and progression of NKTLs. This study also suggests that agents capable of downregulating c-FLIP L could be used to treat NKTL. Laboratory Investigation (2005) 85, 874-884.

Antigen activation and impaired Fas-induced death-inducing signaling complex formation in T-large-granular lymphocyte leukemia

“…22 Expression of c-FLIP-L has been reported to confer resistance to Fas-mediated apoptosis in several tumor cell lines. [43][44][45] Persistent in vitro exposure to antigen, in a model of long-term activation of normal T cells, contributed to apoptosis resistance. 32 In this model, continuous repetitive antigen stimulation reduced DISC assembly and induced higher basal expression of the c-FLIP-L protein, which contributed to apoptosis resistance.…”

Natural killer-cell neoplasms

“…EBVpositive ENKL cell lines have high levels of Fas surface expression but are resistant to Fas-mediated apoptosis. Treatment with cycloheximide, a protein synthesis inhibitor, sensitized cells to Fas-mediated apoptosis, activated caspase 8, and downregulated c-FLIP(L) (cellular FLICEinhibitory protein long form) [37].…”

STAT3 transcription factor is constitutively activated and is oncogenic in nasal-type NK/T-cell lymphoma

“…T-cell receptor-g and immunoglobulin H (Framework region (Fr)1, Fr2 and Fr3]) loci were in the germinal conformation. Cytogenetical analysis disclosed a near tetraploid pattern with a complex formula with numerous numerical and structural chromosome changes as follows: 50-82, ÀXx4 [13],À3 [12], add(3)(p?24) [3], À4 [12], À5 [12], del(6)(q12) [2], þ del(6)(q12) [11], add(6)(q26) [11], del(7)(p1?2p1?5) [9], add(7)(p1?2) [7], À7 [4], À8, À8 [12], À9 [3], À9, À9 [10], À10, À10 [13], À11 [13], add(11)(p11) [4], þ add(11)(p11) [8], add(11)(q22) [10], þ add(11) (q22) [2], À12 [9], À13 [11], À14 [3], À15 [4], À16, [3], À17 [4], À17, À17 [8], add(18)(p11) Â 2 [13], þ 20 [3], þ mar1 [12], þ 1-7mars [11] cp [13]. As some structural abnormalities are present in duplicate, they occurred before endoreduplication, probably as some numerical one, whereas some others obviously occurred as secondary events (Figure 2b).…”

“…5 Few putative oncogenic mechanisms were reported. These involve mutations of genes regulating apoptosis such as Fas and p53, [6][7][8][9][10] as well as expression of P-glycoproteins. 11 Moreover, a low expression level of the granzyme B protease inhibitor 9 was associated with a worse prognosis and cell dedifferentiation, suggesting a role for this protein in the pathophysiology of these malignancies.…”