Oncogene (2001) 20, 7029 ± 7040 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Induction of a TRAIL mediated suicide program by interferon alpha in primary eusion lymphoma Ngoc L Toomey1,4, Vadim V Deyev2,4, Charles Wood3, Lawrence H Boise2, Duncan Scott1, Lei Hua Liu1, Lisa Cabral1, Eckhard R Podack2, Glen N Barber2 and William J Harrington Jr*,1 1Department of Medicine University of Miami School of Medicine, Miami, Florida, FL 33136, USA; 2Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida, FL 33136, USA; 3School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, NE 68588, USA Gammaherpes viruses are often detected in lymphomas Virus (EBV) or Human Herpes Virus Type 8 (HHV-8) arising in immunocompromised patients. We have found have been isolated from lymphomas found in im- that Azidothymidine (AZT) alone induces apoptosis in munosuppressed organ transplant recipients, children Epstein Barr Virus (EBV) positive Burkitt's lymphoma with hereditary immunode®ciencies and patients with (BL) cells but requires interferon alpha (IFN-a) to induce acquired immunode®ciency (AIDS) (Swinnen, 1999; apoptosis in Human Herpes Virus Type 8 (HHV-8) Goldsby and Carroll, 1998; Knowles, 1999). Many of positive Primary Eusion Lymphomas (PEL). Our these tumors can be categorized into distinct subtypes analysis of a series of AIDS lymphomas revealed that based on a variety of morphologic and molecular IFN-a selectively induced very high levels of the Death criteria. For example, AIDS associated large cell Receptor (DR) tumor necrosis factor-related apoptosis- diuse or immunoblastic lymphomas (DLCL, IBL) inducing ligand (TRAIL) in HHV-8 positive PEL lines are often EBV positive while AIDS associated Burkitt's and primary tumor cells whereas little or no induction lymphomas (BL) less frequently contain EBV (Gaidano was observed in primary EBV+ AIDS lymphomas and et al., 1994). A recently de®ned subtype, AIDS related EBV7Burkitt's lines. AZT and IFN-a mediated HHV-8 associated Primary Eusion Lymphoma (PEL) apoptosis in PEL was blocked by stable overexpression (Nador et al., 1996), usually occurs in the setting of of dominant negative Fas Associated Death Domain severe immunode®ciency of advanced HIV infection. (FADD), decoy receptor 2 (DcR2), soluble TRAIL PELs dier from most AIDS lymphomas by their receptor fusion proteins (DR-4 and DR-5) and thymi- absence of a discernable primary tumor mass, infre- dine. Trimeric TRAIL (in place of IFN-a) similarly quent expression of B lymphocyte dierentiation synergized with AZT to induce apoptosis in HHV-8 antigens, and lack of c-myc gene rearrangement positive PEL cells. This is the ®rst demonstration that (Mullaney et al., 2000; Gaidano et al., 2000; Demario IFN-a induces functional TRAIL in a malignancy that and Liebowitz, 1998). In general, immunode®ciency can be exploited to eect a suicide program. This novel related herpesvirus associated lymphomas are aggres- antiviral approach to Primary Eusion lymphomas is sive and poorly responsive to conventional chemother- targeted and may represent a highly eective and apy (Swinnen, 2000; Levine, 2000). relatively non-toxic therapy. Oncogene (2001) 20, Although AZT was originally developed as an anti- 7029 ± 7040. cancer agent, this thymidine nucleoside analog has demonstrated relatively little activity in solid tumors Keywords: human Herpes Virus Type 8; Epstein Barr (Findenig et al., 1996). Interest in AZT was revived virus; TRAIL; apoptosis; lymphoma; FADD only when it was found to inhibit HIV reverse transcriptase (De Clercq, 1992). To exert this antiviral activity, AZT must be phosphorylated by cellular Introduction thymidine kinase (TK) (Arner et al., 1992). Our initial studies indicated that there were two distinctly dierent Gammaherpes viruses are frequently associated with pro-apoptotic eects of AZT and Interferon alpha lymphoproliferative disease in immunocompromised (IFN-a) in primary lymphoma cell lines derived from individuals (Okano and Gross, 2000). Epstein-Barr AIDS patients. EBV+ BL cells underwent apoptosis in the presence of AZT alone while HHV-8+ PELs required the addition of IFN-a to undergo signi®cant *Correspondence: WJ Harrington Jr, University of Miami School of programmed cell death (Lee et al., 1999). Medicine/Sylvester Comprehensive Cancer Center, Room 3400 (D8- Interferons have multiple activities involved in host 4), 1475 NW 12th Avenue, Miami, Florida, FL 33136, USA; defense including anti-proliferative and antiviral eects. E-mail: [email protected] It is known that the interferons, which are potently 4These authors contributed equally to this work Received 14 May 2001; revised 17 July 2001; accepted 2 August upregulated by viruses and double stranded RNA 2001 (dsRNA), can synthesize eectors of apoptosis such as Interferon induces TRAIL in primary effusion lymphoma NL Toomey et al 7030 2'5' oligoadenylate synthetase (2' ±5'A) which activates These data demonstrate that IFN-a upregulates RNAseL and degrades viral mRNA (Player and TRAIL in Primary Eusion Lymphomas while AZT Torrence, 1998). Interferon also induces synthesis of sensitizes these cells to TRAIL mediated apoptosis dsRNA activated protein kinase (PKR), which phos- resulting in the activation of a suicide program in phorylates the translation initiator eIF-2-a and inhibits this cancer. The unique tumorcidal eect of AZT protein synthesis in the cell (Zamanian-Daryoush et al., and IFN-a may have important applications for 2000). Recently, interferons were also found to induce therapy of herpesvirus associated lymphoproliferative expression of the pro-apoptotic protein tumor necrosis disease. factor-related apoptosis-inducing ligand (TRAIL) in human dendritic cells and T lymphocytes which is capable of inducing apoptosis in TRAIL receptor Results expressing targets (Fanger et al., 1999; Grith et al., 1999; Balachandran et al., 2000). AZT and IFN-a act synergistically to induce apoptosis in IFN also potentiates the adaptive immune response HHV-8+ PEL cells through upregulation of major histocompatibility complexes (MHC) and activation of cytotoxic T cells We have demonstrated that AZT and IFN-a have (CTLs) and natural killer cells (NKs) (Mingari et al., clinical activity against malignant herpesvirus asso- 2000). CTLs can recognize and kill cells that present ciated lymphomas (Lee et al., 1999). To further foreign peptides, in association with MHC, through the investigate the apoptotic eects, we studied the perforin/granzyme pathway (Barry et al., 2000). HHV-8+ PEL lines, BC-3 and BCBL-1, as well as Another important mechanism of immune eector the EBV+ AIDS BL primary lines, BL-7 and BL-5. clearance of virally infected or cancerous cells is by All lines were cultured for 48 h in the presence of transmission of an apoptotic signal through ligand medium, AZT (10 mg/ml) or IFN-a (1000 u/ml) binding to members of the tumor necrosis (TNF) alone, or AZT and IFN-a together. As shown in family of receptors (Peter et al., 1997). The binding of Figure 1a, AZT alone induced marked apoptosis in ligands such as Fas-L or TRAIL to extracellular the EBV+Burkitt's lymphoma lines (BL-7, BL-5) receptor domains (APO-1 (Fas/CD95), DR-5) results while IFN-a did not induce apoptosis nor add to in recruitment of components of the death domain the cytopathic eect of AZT. In contrast, HHV-8+ containing the adaptor molecule Fas associated death PEL cells (BC-3, BCBL-1) only underwent marked domain (FADD) and subsequent activation of FLICE apoptosis in the presence of both AZT and IFN-a (caspase 8) by autoproteolysis. Following recruitment (Figure 1a). Similar experiments revealed that other and cleavage of procaspase 8, downstream caspases are HHV-8+ PEL lines, BC-1 and BC-5, also required activated, including caspase 3, resulting in apoptosis. both AZT and IFN-a to undergo signi®cant TRAIL mediated signaling and apoptosis is blocked apoptosis (data not shown). Therefore, a general upon its binding to decoy receptors (DcR1, DcR2) property of HHV-8+ PEL cells was that AZT and which do not recruit FADD (Walczak and Krammer, IFN-a induced substantial apoptosis together but not 2000; Ashkenazi and Dixit, 1999; Bodmer et al., 2000). separately. This cytotoxic eect was seen only in The role of DR-4 in apoptosis is unclear since its virus infected lymphomas as both agents had no expression in FADD7/7 ®broblasts still results in cell eect on herpes virus negative BL cells (Ramos) death (Yeh et al., 1998). Signaling through other (Figure 1b). Further experiments on another herpes- members of the TNF receptor family also may activate virus negative lymphoma cell line (BJAB) demon- non-apoptotic processes such as in¯ammatory re- strated that AZT, with or without IFN-a, induced sponses and lymphoid organogenesis (Magnusson and little or no cytotoxic activity (data not shown). Vaux, 1999). To further investigate the mechanism of AZT and IFN-a induces TRAIL in PEL cells IFN-a mediated apoptosis, we studied a series of lymphoma cell lines and primary lymphoma cells It has recently been shown that IFN-a mediates the derived from AIDS patients. We report here that expression of several pro-apoptotic genes and can HHV-8+ PEL cell lines and primary tumor cells from induce cell death through a FADD dependent a PEL patient express high levels of TRAIL when mechanism (Balachandran et al., 2000). Since IFN-a cultured with IFN-a. Despite the induction of TRAIL potentiated apoptosis in PEL, we investigated its in PEL cells by IFN-a, apoptosis was only potentiated eect on the regulation of pro-apoptotic factors in upon the addition of AZT. In PEL cells, AZT and IFN-a sensitive and resistant B cell lymphomas. IFN-a mediated apoptosis was blocked by expression Accordingly, BC-3 and BL-7 cells were treated for of dominant negative FADD (FADD DN), decoy 8 h with AZT (10 mg/ml), IFN-a (1000 m/ml) or the receptor 2 (DcR2), soluble TRAIL receptor fusion chemotherapeutic agent etoposide (10 mg/ml).