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

Correspondence to: W J Harrington Jr, University of Miami School of Medicine/Sylvester Comprehensive Cancer Center, Room 3400 (D8-4), 1475 NW 12th Avenue, Miami, Florida, FL 33136, USA; E-mail: wharring@med.miami.edu

^aN L Toomey and VV Deyev contributed equally to this work

Gammaherpes viruses are often detected in lymphomas arising in immunocompromised patients. We have found that Azidothymidine (AZT) alone induces apoptosis in Epstein Barr Virus (EBV) positive Burkitt's lymphoma (BL) cells but requires interferon alpha (IFN-) to induce apoptosis in Human Herpes Virus Type 8 (HHV-8) positive Primary Effusion Lymphomas (PEL). Our analysis of a series of AIDS lymphomas revealed that IFN- selectively induced very high levels of the Death Receptor (DR) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HHV-8 positive PEL lines and primary tumor cells whereas little or no induction was observed in primary EBV+ AIDS lymphomas and EBV-Burkitt's lines. AZT and IFN- mediated apoptosis in PEL was blocked by stable overexpression of dominant negative Fas Associated Death Domain (FADD), decoy receptor 2 (DcR2), soluble TRAIL receptor fusion proteins (DR-4 and DR-5) and thymidine. Trimeric TRAIL (in place of IFN-) similarly synergized with AZT to induce apoptosis in HHV-8 positive PEL cells. This is the first demonstration that IFN- induces functional TRAIL in a malignancy that can be exploited to effect a suicide program. This novel antiviral approach to Primary Effusion lymphomas is targeted and may represent a highly effective and relatively non-toxic therapy. Oncogene (2001) 20, 7029-7040.

human Herpes Virus Type 8; Epstein Barr virus; TRAIL; apoptosis; lymphoma; FADD

Introduction

Gammaherpes viruses are frequently associated with lymphoproliferative disease in immunocompromised individuals (Okano and Gross, 2000). Epstein-Barr Virus (EBV) or Human Herpes Virus Type 8 (HHV-8) have been isolated from lymphomas found in immunosuppressed organ transplant recipients, children with hereditary immunodeficiencies and patients with acquired immunodeficiency (AIDS) (Swinnen, 1999; Goldsby and Carroll, 1998; Knowles, 1999). Many of these tumors can be categorized into distinct subtypes based on a variety of morphologic and molecular criteria. For example, AIDS associated large cell diffuse or immunoblastic lymphomas (DLCL, IBL) are often EBV positive while AIDS associated Burkitt's lymphomas (BL) less frequently contain EBV (Gaidano et al., 1994). A recently defined subtype, AIDS related HHV-8 associated Primary Effusion Lymphoma (PEL) (Nador et al., 1996), usually occurs in the setting of severe immunodeficiency of advanced HIV infection. PELs differ from most AIDS lymphomas by their absence of a discernable primary tumor mass, infrequent expression of B lymphocyte differentiation antigens, and lack of c-myc gene rearrangement (Mullaney et al., 2000; Gaidano et al., 2000; Demario and Liebowitz, 1998). In general, immunodeficiency related herpesvirus associated lymphomas are aggressive and poorly responsive to conventional chemotherapy (Swinnen, 2000; Levine, 2000).

Although AZT was originally developed as an anti-cancer agent, this thymidine nucleoside analog has demonstrated relatively little activity in solid tumors (Findenig et al., 1996). Interest in AZT was revived only when it was found to inhibit HIV reverse transcriptase (De Clercq, 1992). To exert this antiviral activity, AZT must be phosphorylated by cellular thymidine kinase (TK) (Arner et al., 1992). Our initial studies indicated that there were two distinctly different pro-apoptotic effects of AZT and Interferon alpha (IFN-) in primary lymphoma cell lines derived from AIDS patients. EBV+ BL cells underwent apoptosis in the presence of AZT alone while HHV-8+ PELs required the addition of IFN- to undergo significant programmed cell death (Lee et al., 1999).

Interferons have multiple activities involved in host defense including anti-proliferative and antiviral effects. It is known that the interferons, which are potently upregulated by viruses and double stranded RNA (dsRNA), can synthesize effectors of apoptosis such as 2'5' oligoadenylate synthetase (2'-5'A) which activates RNAseL and degrades viral mRNA (Player and Torrence, 1998). Interferon also induces synthesis of dsRNA activated protein kinase (PKR), which phosphorylates the translation initiator eIF-2- and inhibits protein synthesis in the cell (Zamanian-Daryoush et al., 2000). Recently, interferons were also found to induce expression of the pro-apoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human dendritic cells and T lymphocytes which is capable of inducing apoptosis in TRAIL receptor expressing targets (Fanger et al., 1999; Griffith et al., 1999; Balachandran et al., 2000).

IFN also potentiates the adaptive immune response through upregulation of major histocompatibility complexes (MHC) and activation of cytotoxic T cells (CTLs) and natural killer cells (NKs) (Mingari et al., 2000). CTLs can recognize and kill cells that present foreign peptides, in association with MHC, through the perforin/granzyme pathway (Barry et al., 2000). Another important mechanism of immune effector clearance of virally infected or cancerous cells is by transmission of an apoptotic signal through ligand binding to members of the tumor necrosis (TNF) family of receptors (Peter et al., 1997). The binding of ligands such as Fas-L or TRAIL to extracellular receptor domains (APO-1 (Fas/CD95), DR-5) results in recruitment of components of the death domain containing the adaptor molecule Fas associated death domain (FADD) and subsequent activation of FLICE (caspase 8) by autoproteolysis. Following recruitment and cleavage of procaspase 8, downstream caspases are activated, including caspase 3, resulting in apoptosis. TRAIL mediated signaling and apoptosis is blocked upon its binding to decoy receptors (DcR1, DcR2) which do not recruit FADD (Walczak and Krammer, 2000; Ashkenazi and Dixit, 1999; Bodmer et al., 2000). The role of DR-4 in apoptosis is unclear since its expression in FADD-/- fibroblasts still results in cell death (Yeh et al., 1998). Signaling through other members of the TNF receptor family also may activate non-apoptotic processes such as inflammatory responses and lymphoid organogenesis (Magnusson and Vaux, 1999).

To further investigate the mechanism of AZT and IFN- mediated apoptosis, we studied a series of lymphoma cell lines and primary lymphoma cells derived from AIDS patients. We report here that HHV-8+ PEL cell lines and primary tumor cells from a PEL patient express high levels of TRAIL when cultured with IFN-. Despite the induction of TRAIL in PEL cells by IFN-, apoptosis was only potentiated upon the addition of AZT. In PEL cells, AZT and IFN- mediated apoptosis was blocked by expression of dominant negative FADD (FADD DN), decoy receptor 2 (DcR2), soluble TRAIL receptor fusion proteins and by the addition of thymidine. In contrast to HHV-8+ PEL, EBV+ AIDS BL lines and EBV- BL lines did not express significant amounts of TRAIL, nor undergo increased apoptosis in response to IFN-.

These data demonstrate that IFN- upregulates TRAIL in Primary Effusion Lymphomas while AZT sensitizes these cells to TRAIL mediated apoptosis resulting in the activation of a suicide program in this cancer. The unique tumorcidal effect of AZT and IFN- may have important applications for therapy of herpesvirus associated lymphoproliferative disease.

Results

AZT and IFN- act synergistically to induce apoptosis in HHV-8+ PEL cells

We have demonstrated that AZT and IFN- have clinical activity against malignant herpesvirus associated lymphomas (Lee et al., 1999). To further investigate the apoptotic effects, we studied the HHV-8+ PEL lines, BC-3 and BCBL-1, as well as the EBV+ AIDS BL primary lines, BL-7 and BL-5. All lines were cultured for 48 h in the presence of medium, AZT (10 g/ml) or IFN- (1000 u/ml) alone, or AZT and IFN- together. As shown in Figure 1a, AZT alone induced marked apoptosis in the EBV+Burkitt's lymphoma lines (BL-7, BL-5) while IFN- did not induce apoptosis nor add to the cytopathic effect of AZT. In contrast, HHV-8+ PEL cells (BC-3, BCBL-1) only underwent marked apoptosis in the presence of both AZT and IFN- (Figure 1a). Similar experiments revealed that other HHV-8+ PEL lines, BC-1 and BC-5, also required both AZT and IFN- to undergo significant apoptosis (data not shown). Therefore, a general property of HHV-8+ PEL cells was that AZT and IFN- induced substantial apoptosis together but not separately. This cytotoxic effect was seen only in virus infected lymphomas as both agents had no effect on herpes virus negative BL cells (Ramos) (Figure 1b). Further experiments on another herpesvirus negative lymphoma cell line (BJAB) demonstrated that AZT, with or without IFN-, induced little or no cytotoxic activity (data not shown).

IFN- induces TRAIL in PEL cells

It has recently been shown that IFN- mediates the expression of several pro-apoptotic genes and can induce cell death through a FADD dependent mechanism (Balachandran et al., 2000). Since IFN- potentiated apoptosis in PEL, we investigated its effect on the regulation of pro-apoptotic factors in IFN- sensitive and resistant B cell lymphomas. Accordingly, BC-3 and BL-7 cells were treated for 8 h with AZT (10 g/ml), IFN- (1000 /ml) or the chemotherapeutic agent etoposide (10 g/ml). Total RNA was extracted and analysed for pro-apoptotic gene expression by ribonuclease protection assay (RPA). RPA analysis revealed that IFN- induced markedly higher levels of TRAIL mRNA in the IFN- sensitive PEL cells (BC-3) compared to the resistant EBV+ BL cells (BL-5) (Figure 2a, lanes 2 and 4 compared to lanes 7 and 9). In contrast, AZT and etoposide, at doses sufficient to kill these cells, had no effect on TRAIL expression in either type of lymphoma (Figure 2a, lanes 3 and 5 and lanes 8 and 10). We then investigated the effect of IFN- on a larger panel of lymphoma lines including HHV-8+ PELs, EBV+ BLs (derived from HIV positive patients) and EBV- BL lines. We found that IFN- significantly induced TRAIL mRNA only in the HHV-8+ PEL lines and primary tumor cells (BCBL-LN) derived from a patient with PEL, while little or no induction was noted either in EBV positive or negative BL lines. IFN- also had no effect on the expression of two death receptors, DR-4 and DR-5, which are known to bind to TRAIL (Figure 2b). Further RPA experiments demonstrated that neither AZT nor IFN- affected mRNA or protein surface expression levels of DR-4 or DR-5 in PEL cells (data not shown). FACS analysis demonstrated that IFN- induced TRAIL protein surface expression in PEL cells but not in EBV+ BL cells (Figure 2c, panels 3 and 4 versus panels 1 and 2). Therefore, the ability of IFN- to potentiate AZT mediated apoptosis in PEL correlated with its ability to induce the pro-apoptotic ligand TRAIL in these tumors.

AZT and IFN- mediated apoptosis in PEL is FADD dependent

TRAIL has been reported to induce apoptosis by binding its cognate receptors. Furthermore, TRAIL mediated apoptosis has recently been shown to be dependent upon signaling through the death adaptor protein FADD (Bodmer et al., 2000). To investigate the mechanism of IFN- induced apoptosis we first confirmed that HHV-8+ PEL cells (BCBL-1) did express TRAIL receptors on their surface (DR-4 was more highly expressed than DR-5) (Figure 3a). BC-3 cells also expressed similar levels of DR-4 and DR-5 (data not shown). To study whether apoptosis induced by AZT and IFN- in PEL involved a FADD dependent mechanism, we transfected the BCBL-1 line with a dominant negative construct of FADD that lacks a death effector domain (Balachandran et al., 2000). After G418 selection, we derived several stably transfected subclones which expressed a dominant negative mutation of the FADD protein (BCBL FADD DN) (Figure 3b). BCBL FADD DN cells were completely resistant to AZT and IFN- mediated apoptosis but retained their sensitivity to etoposide (Figure 3c). To determine that resistant cells were not generated by the selection process, clones were also characterized for expression of anti-apoptotic proteins, Bcl-2 and Bcl-x and found to express similar low levels (compared to the parental BCBL-1 cells) of each protein (data not shown). BCBL-1 FADD DN cells also expressed similar levels of DR-4 and DR-5 and TRAIL mRNA when treated with IFN- (Figure 3d). Therefore, our data indicates that apoptosis induced by AZT and IFN- in HHV-8+ PEL is a FADD dependent process.

AZT and IFN- induces cleavage of caspases 3 and 8 in PEL which is inhibited by soluble DR-4, DR-5 or DcR2

Apoptosis mediated through death receptor ligand pathways involves the recruitment of proteins forming the death inducing signaling complex (DISC) resulting in cleavage (activation) of procaspase 8 (FLICE) to its active form and subsequent activation of downstream pro-caspases (Kischkel et al., 1995; Medema et al., 1997). We therefore treated HHV-8+ PEL cells (BCBL-1 and BC-3) and BCBL-DN FADD transfectants (BCBL-1A2) with AZT, IFN-, or AZT plus IFN- and assayed for procaspase 8 cleavage. Treatment with either agent alone resulted in no cleavage of procaspase 8. However in BC-3 and BCBL-1 cells, AZT and IFN- together activated procaspase 8 and procaspase 3 but did not in BCBL-1A2 (FADD DN) cells (Figure 4a, lane 4). We did note some cleavage of procaspase 3 in AZT treated BCBL-1 and BC-3 PEL cells which indicates that AZT alone does cause some degree of apoptosis in PEL cells (lane 3). Treatment with the caspase inhibitor DEVD abrogated AZT and IFN- mediated cleavage of both procaspases in PEL cells (lane 5). Caspase inhibition of AZT and IFN- mediated apoptosis with IETD, a specific caspase 8 inhibitor, was not possible since the inhibitor itself was toxic to PEL cells (data not shown). We then investigated whether inhibition of TRAIL with soluble TRAIL receptors DR-4 and DR-5 could block AZT and IFN- mediated apoptosis. These experiments were also performed with ZB4, a blocking anti-Fas antibody. As demonstrated in Figure 4b, soluble DR-4 and DR-5 together inhibited AZT and IFN- mediated apoptosis, albeit incompletely, while the addition of ZB4 had little effect on AZT and IFN- apoptosis. In contrast, soluble receptors DR-4, DR-5 and ZB4 had no inhibitory effect on AZT mediated apoptosis in EBV+ BL cells (data not shown). Control experiments demonstrated that soluble DR-4 and DR-5 almost completely inhibited TRAIL mediated cell death (at a highly cytotoxic concentration of TRAIL [100 ng/ml]). To further confirm that AZT and IFN- mediated apoptosis occurs principally through TRAIL signaling, we transfected BCBL-1 cells with the decoy receptor for TRAIL (DcR2). Overexpression of DcR2 markedly abrogated the IFN- component of AZT and IFN- mediated apoptosis (Figure 4c).

AZT enhances TRAIL mediated apoptosis in PEL

Although IFN- induced TRAIL expression in PEL, apoptosis was potentiated by the addition of AZT. To determine whether TRAIL could substitute for IFN- and similarly synergize with AZT to induce apoptosis in PEL cells, we first performed dose response experiments and found that titrating soluble trimeric TRAIL, to concentrations of 10-30 ng/ml for 48 h, induced little apoptosis above the baseline seen in untreated PEL cells (BC-3, BCBL-1) (data not shown). We then substituted soluble trimeric TRAIL for IFN- to determine whether it also induced apoptosis when combined with AZT in PEL cells. We found that in PEL cells, TRAIL and AZT caused a synergistic pro-apoptotic effect similar to that caused by IFN- and AZT (Figure 5a, panel E compared to panel C). Similar to AZT and IFN-, AZT and TRAIL were not cytotoxic to FADD DN BCBL-1 cells (Figure 5b). These data indicate that AZT enhances TRAIL mediated apoptosis in HHV-8+ PEL cells.

AZT and IFN- mediated apoptosis does not occur with other antiviral nucleosides and is inhibited by thymidine

HHV-8 and EBV encode viral thymidine kinases (TKS) which have been shown to be capable of phosphorylating AZT (a thymidine nucleoside analog) (Gustafson et al., 2000). Since only herpesvirus associated lymphomas were sensitive to AZT, we investigated whether this cytotoxic effect occurred with other antiviral nucleoside analogues. When combined with IFN-, a cytidine analogue, ddC, and a thymidine analog, d4T, were completely inactive in AZT and IFN- sensitive PEL cells; BCBL-1 and BC-3 (Figure 6a). We therefore reasoned that in herpesvirus lymphomas, if phosphorylation of AZT was necessary for apoptosis then thymidine, which competes with AZT as a substrate for thymidine kinase, should abrogate the apoptotic effect. As expected, thymidine in a dose dependent fashion, blocked apoptosis in both EBV+ BL cells treated with AZT and in HHV-8+ PEL cells treated with AZT and IFN- (Figure 6b). These data suggest that the anti-tumor effect of AZT in both EBV+ BL and PEL may require phosphorylation by a herpesvirus encoded thymidine kinase with substrate specificity for AZT. These results were further supported by the fact that herpesvirus negative cells BJAB and Ramos were unaffected by AZT (data not shown). These data may also explain why AZT and IFN- are inactive in herpesvirus negative lymphomas.

Discussion

Herpesvirus associated lymphomas in immunocompromised patients are usually difficult to treat, as conventional chemotherapy is poorly tolerated and causes further immunosuppression. Biologic or immunomodulatory cancer therapies such as interferons initially held great promise. However, clear-cut clinical efficacy has been restricted to a limited number of diseases, most notably viral associated chronic active hepatitis (CAH) (Zavaglia et al., 2000). Recent data indicate that interferons appear to sensitize cells to FADD dependent apoptosis. Evidence for this comes from work which demonstrates that inhibitors of caspase 8, dominant negative mutation of FADD, or the absence of FADD, block IFN mediated apoptosis (Balachandran, et al., 1998). Recently, understanding of these pro-apoptotic mechanisms has been furthered by the finding that IFN- induces TRAIL expression in monocytes and dendritic cells (Fanger et al., 1999; Griffith et al., 1999). Therefore, an important biologic property of interferons may be to directly or indirectly activate DR ligands such as TRAIL. Our study demonstrates that IFN- mediated induction of TRAIL in malignancies (HHV-8+ PEL) can be exploited to activate a suicidal or fratricidal tumor cell death.

Recent enthusiasm for the use of death receptor ligands such as TRAIL as a cancer therapy has been tempered by evidence of their toxicity in human hepatocytes (Jo et al., 2000). However, the therapeutic index of TRAIL might be improved if specific killing of virally infected tumor cells were enhanced by the addition of antiviral nucleosides. Alternatively, IFN- may induce tolerable, clinically active levels of TRAIL when administered systemically. This effect might be restricted to certain virus associated tumors or disease processes since our data demonstrates that TRAIL is markedly induced by IFN- specifically in HHV-8 infected lymphomas. Whether this effect is unique to HHV-8+lymphomas will require analysis of a greater number of tumors. This marked pro-apoptotic effect between the thymidine analog AZT and IFN- was not reproducible by substituting a cytidine analog, 3TC, or another thymidine analog, d4T. A likely reason for this is that the HHV-8 encoded thymidine kinase specifically phosphorylates AZT. HHV-8 and EBV encoded TKs have been shown to be capable of phosphorylating AZT (Gustafson et al., 2000), however, these (TKs) are expressed during the viral lytic cycle, although low level expression has been reported in HHV-8+ PEL lines (Cannon et al., 1999). Whether AZT itself may induce lytic herpesvirus genes or is phosphorylated by low level expression of viral TK is presently unknown.

AZT and IFN- mediated apoptosis in PEL was blocked in cells expressing dominant negative FADD or DcR2 and soluble TRAIL receptors inhibited AZT and IFN- mediated cytotoxicity in PEL cells. This demonstrates that AZT and IFN- mediated apoptosis in PEL involves signaling through FADD and TRAIL/DR interaction. In contrast to what has been reported in etoposide treated tumors (Gibson et al., 2000), we found no evidence that AZT, at the doses used, induced TRAIL receptors DR-4 and DR-5 (data not shown). We also found that the effect of IFN- in PEL cells can be reproduced with soluble TRAIL and apoptosis potentiated by AZT. This indicates that in PEL, AZT may promote signaling through death receptors rather than by enhancing their (DR-4 and DR-5) expression. A similar phenomenon has been reported in cells transfected with Herpes Simplex Virus (HSV) TK. Apoptosis in these cells was shown to be associated with recruitment of components of the DISC mediated by phosphorylation of the pro-drug GCV (Beltinger et al., 1999). In our study, the viral kinase was present in each tumor cell, therefore transfection was unnecessary. Phosphorylated AZT may enhance death receptor mediated apoptosis via this mechanism which is accentuated by IFN- mediated induction of TRAIL. Another possibility is that AZT may block NF-kappaB which has been shown to be activated by death receptors (Chaudhary et al., 1997; Yang et al., 2000). This may result in an unfettered apoptotic effect. It is possible that AZT may induce mitochondrial damage in HHV-8+ PEL which alone could cause a mild apoptotic effect and recruitment of FADD and procaspase 8. Upon addition of IFN- (or soluble TRAIL) apoptosis is accentuated through activation of a DR/Ligand signal. This would explain the detection of caspase 3 cleavage in PEL cells treated only with AZT. It is also possible that AZT alone may activate a Type II apoptotic pathway while AZT and IFN- together activate a Type I pathway response. The co-existence of Type I and II pathways has recently been demonstrated in Jurkat cells treated with tumor necrosis factor alpha (Johnson et al., 2000). HHV-8 also encodes a putative oncogene, vIRF, which inhibits IFN signaling as well as a viral inhibitor of FADD dependent apoptosis, vFLIP (Gao et al., 1997; Sarid et al., 1999). It is unknown whether the combination of AZT and IFN- affect expression of these virally encoded anti-apoptotic proteins.

EBV+ BL, although quite sensitive to AZT alone, did not express significant amounts of TRAIL in response to IFN-. IFN- also did not increase the apoptotic effect of AZT in EBV+ BL. There are several potential reasons for the disparate effects of IFN- in PEL and EBV+ BL. IFN- signaling that activates DR ligands or other components of IFN signaling may be defective in these EBV+ BL lines. Alternatively, other pro-apoptotic effects of AZT such as mitochondrial damage may be much more pronounced and predominant in EBV+ BL cells. It is possible that AZT alone might activate other DR ligand pathways in EBV+ BL. It is interesting to note that like PEL cells, EBV+ cells were also killed by a thymidine analog (AZT) and not by other antiviral nucleosides and that this effect was also inhibited by thymidine. We have recently noted that high dose AZT has marked clinical activity in AIDS related EBV+ primary central nervous system lymphoma (Raez et al., 1999). We recently observed a marked clinical improvement in a PEL patient with lymphomatous meningitis treated with parenteral AZT and IFN- after having failed conventional chemotherapy.

The ability to specifically induce TRAIL mediated apoptosis in PEL may prove quite clinically relevant. Laboratory and clinical studies should further define the anti-tumor mechanism of these agents and their therapeutic potential.

Materials and methods

Cell lines

PEL cell lines, BCBL-1, BC-2, BC-3, and BL-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). BC-5 was donated by Dr Ethel Cesarman of Cornell University. BCBL-1 and BC-3 are infected with only HHV-8, while BC-1, BC-2 and BC-5 are co-infected with HHV-8 and EBV. The primary PEL isolate BCBL-LN was obtained from a diagnostic paracentesis performed on an AIDS patient with lymphomatous ascites. The lymphoma did not express B cell surface antigens (CD19, CD20) but its lineage was B cell as defined by gene rearrangement studies. Analysis of tumor DNA by PCR was strongly positive for HHV-8 and EBV (Arvanitakis et al., 1996). BL-5, BL-7, BL-8, and SM-1 are primary cell lines derived from AIDS patients with EBV+ AIDS related BL and carry the typical t8 : 14 c-myc translocation. P3HR-1, BJAB and Ramos are established BL lines and were obtained from the ATCC. P3HR-1 is EBV+ and BJAB and Ramos are EBV-.

Apoptosis analyses

Apoptosis was determined by annexin V-FITC/propidium iodide (P.I.) flow analysis. 5.0´10⁵ cells were grown in 10 ml of IMDM (GIBCO-BRL) supplemented with 10% heat inactivated fetal bovine serum (FBS) in the presence of media, 10 g/ml AZT, ddC, or d4T, 1000 u/ml IFN- (or 10 ng/ml soluble TRAIL) (provided by Immunex Corporation, Seattle, WA, USA), 10 g/ml AZT (or ddC or d4T) and 1000 u/ml IFN- (or 10 ng/ml soluble TRAIL), for 48 h in 25 cm flasks. 2.0´10⁵ cells were removed from the flask and washed twice with 5 ml of PBS, resuspended in 0.1 ml of binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl₂) and stained with 5 l of annexin V-FITC (PharMingen, San Diego, CA, USA) and 2.5 l of 0.5 mg/ml P.I. for 15 min. 0.4 ml of binding buffer was added to the suspension. Cells were then analysed using a FACScan^TM flow cytometer (Becton Dickinson, San José, CA, USA).

For caspase inhibitor studies, cells (1.0´10⁵ cells/ml, 50 ml) were grown in IMDM (GIBCO-BRL) supplemented with 10% heat inactivated FBS in the presence of media, 10 g/ml AZT, 1000 u/ml IFN-, 10 g/ml AZT and 1000 u/ml IFN-, 10 g/ml AZT and 1000 u/ml IFN- with 10 M of Ac-DEVD-CHO (PharMingen) for 8 h. An additional 10 M of Ac-DEVD-CHO was then added to the Ac-DEVD-CHO treated cells and incubated for an additional 24 h. Cells were then harvested, lysed, and subjected to Western blot analysis in a 12% SDS-PAGE. Caspase 3 and caspase 8 protein bands were detected using mAb (PharMingen). This caspase 8 antibody detects only the uncleaved procaspase form.

Cell viability assays

To determine the effects of antiviral nucleosides and IFN- on the different lines, 0.5´10⁵ cells were grown in 1.0 ml of IMDM supplemented with 10% heat inactivated FBS (in triplicate) in the presence of media, 10 g/ml AZT (3TC or d4T), 1000 u/ml IFN-, 10 g/ml AZT and 1000 u/ml IFN- for 48 h. Control experiments were also performed with etoposide at a concentration of 10 g/ml for 24 h. Cells were harvested and viability was determined by Trypan blue exclusion. To determine the effects of thymidine on AZT and IFN- mediated apoptosis, cells were cultured as described above for 48 h in the presence of 10 or 100 g/ml thymidine and viability determined by Trypan blue exclusion. To study the effect of inhibitory DR-4 and DR-5 fusion proteins on AZT and IFN- mediated apoptosis in PEL, a triplicate set of 2.0´10⁴ BCBL-1 PEL cells were grown in 100 L of 10% FBS IMDM media, in the presence of 5.0 ng/ml of rhTRAIL-R2:Fc (Alexis Corporation, San Diego, CA, USA), 2.0 g/ml of rhTRAIL-R1:Fc (Alexis Corporation) and/or 1.0 g/ml of ZB4, a CD-95 blocker, (Immunotech, Beckman/Coulter Corporation, Brea, CA, USA) for 8 h. Ten g/ml AZT and 1000 u/ml IFN- (or 10 ng/ml soluble TRAIL in place of IFN-) were then added to the media for another 48 h. As a control assay, 100 ng/ml (a cytotoxic dose) of soluble TRAIL (Immunex) was used with and without soluble blockers. Cells were harvested and viability determined by Trypan exclusion.

Western blot analysis

1.0´10⁵ cells/ml were grown in 50 ml IMDM (GIBCO-BRL) supplemented with 10% heat inactivated FBS in the presence of 10 g/ml AZT, 1000 u/ml IFN-, 10 g/ml AZT and 1000 u/ml IFN-, 10 g/ml etoposide or medium for 48 h. Cells were harvested and lysed in lysis buffer (50 mM TRIS-HCl pH 7.5, 0.5% NP-40, 10% glycerol, 250 mM NaCl, and 5 mM EDTA). Forty g of total protein per lane was loaded and subjected to electrophoresis in a 15% acrylamide SDS-PAGE. Proteins were transferred from the acrylamide gel to nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk, then probed with 1 g/ml mAb anti-caspase-8, anti-caspase 3 (PharMingen), or mAb anti-FADD (Transduction Laboratory, Lexington, KY, USA) complexed with anti-mouse-HRP (Amersham Pharmacia Biotech, Uppsala, Sweden) and visualized using ECL stain (Amersham).

Flow cytometry for TRAIL, DR-5 and DcR2

1.0´10⁵ cells were grown in 10 ml of IMDM (GIBCO-BRL) supplemented with 10% FBS in the presence of media or 1000 u/ml IFN-. Cells were harvested after 24 h, washed once with 1.0 ml of PBS containing 0.5% BSA, pelleted at 1000 g, and resuspended in 50 l of PBS. 2.0´10⁵ cells in 20 l volume was used for detection of surface TRAIL protein. Nonspecific binding sites were blocked by the addition of 20 l of human IgG (4.1 mg/ml, Sigma Chemical Co., St. Louis, MO, USA) and incubated on ice for 1 h. Two l of mAb anti-TRAIL (2.0 mg/ml, Immunex M181) or Isotype mouse IgG (2.0 mg/ml, Sigma Chemical Co.) were then added to the suspension and incubated for another hour. After three washes with 1.0 ml of PBS containing 0.5% BSA, cells were resuspended in 50 l of sheep anti-mouse IgG FITC conjugated (1 : 50, Sigma Chemical Co.) and incubated in ice for 30 min. After three washes with 1.0 ml of PBS, cells were analysed using a FACScan^TM flow cytometer (Becton Dickinson).

For DR-5 surface expression, 1.0´10⁶ cells were washed three times in ice cold PBS. Cells were then incubated in 100 g of human IgG (Sigma) in PBS in a total volume of 50 l for 60 min. The cells were washed once with 1.0 ml of 0.1% BSA in PBS and resuspended in 20 l of 4.0 g DR-5 mAb (Immunex, M413) and 0.1% BSA in PBS and incubated on ice for 60 min. After three washes with 0.1% BSA in PBS, 50 l of sheep anti-mouse-FITC labeled (1 : 100 dilution in 0.1% BSA in PBS; Sigma) was added to the cells and incubated for 30 min. Cells were then washed two times with 1.0 ml of PBS, then resuspended in 0.5 ml of binding buffer and analysed using a FACScan^TM flow cytometer (Becton Dickinson).

The expression level of DcR2 on the surface of transfected and wild type PEL cells was evaluated by flow cytometry. Cells were stained with 50 g/ml of goat anti-DcR2 affinity purified polyclonal antibody (R&D), followed by staining with 1 : 200 diluted donkey anti-goat FITC labeled antibody (Jackson Immunoresearch Laboratories). As a control, cells were stained with the second antibody only. To reduce non-specific binding, stainings were done in the excess of human IgG.

RNAse protection assay

1.0´10⁵ cells/ml were grown in 50 ml IMDM (GIBCO-BRL) supplemented with 10% heat inactivated FBS in the presence of 10 g/ml AZT, 1000 u/ml IFN-, 10 g/ml AZT and 1000 u/ml IFN-, 10 g/ml etoposide or medium for 8 h. Cells were harvested and total RNA's extracted using a RNAeasy purification kit (QIAGEN Genomics, Inc., Bothell, WA, USA). Twenty g of RNA's were used per reaction. RPAs were done using RiboQuant RPA (PharMingen) kit. Assays were done according to manufacturer's instructions using customized sets of probes synthesized by PharMingen.

Stable DNA transfections

Cells were transfected with dominant negative FADD (19) in a 35 mm dish using NovaFECTOR cationic lipid (VennNova LLC, Pompano Beach, FL, USA). 1.0´10⁶ of BCBL-1 cells were grown to log phase in IMDM media supplemented with 10% heat inactivated FBS. Cells were then washed twice with 10 ml of serum-free IMDM media and centrifuged at 1000 g prior to the addition of DNA/lipid complex. Four g of pcDNA3 (Invitrogen, Carlsbad, CA, USA) or pcDNA3-FADD DN were complexed with 20 g of lipid for 15 min at room temperature in 1.0 ml of serum-free IMDM media. Cell pellets were then resuspended with the 1.0 ml of DNA/lipid complex and incubated in 5% CO₂ at 37°C for 4 h. One ml of IMDM supplemented with 20% heat inactivated FBS was added to the dish and incubated for 16 h. The full-length TRAIL receptor 4 (decoy receptor 2), DcR2 was amplified by RT-PCR from poly-A of PHA-stimulated peripheral blood lymphocytes and cloned into episomal expression vector pBMG-Neo. The construct was introduced into BCBL cells by electroporation. Cells were then transferred to a 75 cm flask and grown in 50 ml of IMDM supplemented with 10% heat inactivated FBS and 500 g/ml G418 for selection of positive transfectants. Fresh medium was changed every 4 days for a duration of 4 weeks. Single clones were selected by limiting dilutions. Dominant negative FADD transfectants were confirmed by Western blot using mouse mAb anti-FADD (Transduction Laboratories). The expression of DcR2 transfectants was assessed by flow cytometry.

Acknowledgements

The authors wish to thank Drs Parkash Gill (University of Southern California) and Scott Kaufmann (Mayo clinic, Rochester, MN) for their helpful suggestions. This work was supported by grants CA82274 (WJ Harrington Jr and LH Boise), CA77837 (WJ Harrington Jr and LH Boise), CA86431 (GN Barber), CA80228 and CA39201 (ER Podack) from the National Institutes of Health.

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Figure 1 (a) AZT and IFN- synergize to induce apoptosis in HHV-8+ PEL (BC-3, BCBL-1) while EBV+ BL cells (BL-5, BL-7) undergo apoptosis with AZT alone. BC-3 and BCBL-1 (HHV-8+ PEL's) and BL-5 and BL-7 (EBV+ BL's) were treated in medium (Panels A), AZT 10 g/ml (Panels B), IFN- 1000 u/ml (Panels C), or AZT 10 g/ml plus IFN- 1000 u/ml (Panels D) for 48 h. Cells were then analysed for apoptosis by PI annexin staining and FACs analysis. Upper and lower right quadrants are the apoptotic populations. (b) AZT and IFN- are synergistically cytotoxic in HHV-8+ PEL but not EBV- BL. PEL cells BCBL-1 and BC-3, EBV+ BL cells BL-5 and BL-7, and EBV- Ramos cells were cultured for 48 h in medium (control), IFN- 1000 u/ml, AZT 10 g/ml, and IFN- 1000 u/ml plus AZT 10 g/ml. Cell viability was then determined by Trypan blue exclusion. Bars represent per cent viable cells

Figure 2 (a) TRAIL mRNA is induced by IFN- but not by AZT or Etoposide in HHV-8+ PEL. PEL cells (BC-3, Lanes 1-5) and EBV+cells (BL-5, Lanes 6-10) were treated in the following manner for 8 h and examined for TRAIL mRNA expression by ribonuclease protection assay. Lane 1=BC-3 in medium. Lane 2=BC-3 in IFN- 1000 u/ml. Lane 3=BC-3 in AZT 10 g/ml. Lane 4=BC-3 in IFN- 1000 u/ml and AZT 10 g/ml. Lane 5=BC-3 in etoposide 10 g/ml. Lane 6=BL-5 in medium. Lane 7=BL-5 in IFN- 1000 u/ml. Lane 8=BL-5 in AZT 10 g/ml. Lane 9=BL-5 in IFN- 1000 u/ml and AZT 10 g/ml. Lane 10=BL-5 in etoposide 10 g/ml. (b) IFN- induces a high level of expression of TRAIL mRNA in HHV-8+ PEL but not EBV+ BL and EBV- BL. PEL lines and primary tumor isolates (BCBL-1, BC-3, BC-1, BCBL-LN, BC-2, BC-5), EBV+ BL (P3HR-1), EBV+AIDS BL lines (BL-8, SM-1, BL-5, BL-7), and EBV- BL lines (Ramos, BJAB) were treated with media (C) or IFN- 1000 u/ml (I) for 8 h. mRNA was extracted and assayed by ribonuclease protection assay for expression of DR-4, DR-5 and TRAIL. GAPDH expression was used as an internal control. (c) IFN- induces TRAIL surface expression in HHV-8+ PEL but not EBV+ BL. EBV+ BL: BL-5 and BL-7 cells (Panels 1 and 2) and HHV-8+ PEL cells: BCBL-1 and BC-3 (Panels 3 and 4) were treated for 24 h with IFN- 1000 u/ml or media and assayed for TRAIL expression by FACs analysis. Shaded area equals isotype control (mouse IgG1). Dotted line are cells treated with media and solid line are cells treated with IFN-

Figure 3 (a) HHV-8+ PEL cells express TRAIL receptors. PEL cells (BCBL-1) were grown in IMDM media enriched with 10% FCS and surface expression of DR-5 (dotted line) and DR-4 (solid line) was determined by FACs analysis. Shaded area is mouse isotype control (IgG1). (b) Transfection of BCBL-1 cells with FADD-DN. BCBL-1 cells were transfected with FADD-DN by electroporation. Subclones were derived after G418 selection. Subclones (BCBL-2A4, BCBL-3A3, BCBL-3B2, BCBL-3C2, and BCBL-1A2), BCBL-Neo (transfected with neo-resistance gene only), and BCBL-1 parental cells were assayed for expression of FADD and FADD-DN by Western blot. (c) BCBL-1 FADD-DN cells are resistant to AZT and IFN- but sensitive to VP-16. BCBL-1, BCBL-1 Neo transfectants and BCBL-1 FADD-DN clones (BCBL-2A4, BCBL-3C2, BCBL-1A2) were treated for 48 h with medium, AZT 10 g/ml, IFN- 1000 u/ml, AZT 10 g/ml plus IFN- 1000 u/ml, or VP-16 10 g/ml (for 24 h) and cell death was measured by Trypan blue exclusion. The figure is representative of 2 experiments. (d) BCBL-1 FADD-DN cells express TRAIL in response to IFN-. BCBL-1A2 cells were treated with medium (C) or IFN- (I) for 8 h and caspase 8, DR-4 and DR-5 expression levels were detected by RPA

Figure 4 (a) Activation of caspases 3 and 8 by AZT and IFN- in PEL cells is blocked by the caspase inhibitor DEVD and expression of FADD DN. PEL cells; BC-3 (top panel), BCBL-1 (middle panel) and BCBL-1A2 (FADD-DN cells) (lower panel) were cultured for 48 h in media (C), IFN- 1000 u/ml (I), AZT 10 g/ml (A), AZT 10 g/ml plus IFN- 1000 u/ml (A+I), AZT 10 g/ml and IFN- 1000 u/ml plus the caspase inhibitor DEVD 10 M (A+I+D) and assayed by Western blot for caspase 3 and 8. The caspase 8 antibody used only detects the uncleaved pro-caspase form. (b) Soluble TRAIL receptors inhibit AZT and IFN- mediated cytotoxicity in PEL cells. BCBL-1 (HHV-8+ PEL cells) were treated in triplicate with the following conditions: medium (Control); AZT 10 g/ml+IFN- 1000 u/ml; soluble DR-4 2 g/ml+soluble DR-5 5 ng/ml; pre-treatment for 8 h with soluble DR-4 2 g/ml and soluble DR-5 5 ng/ml followed by treatment for 48 h with AZT 10 g/ml+IFN- 1000 u/ml; pre-treatment for 8 h with soluble DR-4 2 g/ml+soluble DR-5 5 ng/ml+ZB4 1 g/ml followed by treatment for 48 h with AZT 10 g/ml and IFN- 1000 u/ml; TRAIL 100 ng/ml; pre-treatment for 8 h with soluble DR-4 2 g/ml+soluble DR-5 5 ng/ml followed by treatment for 48 h with TRAIL 100 ng/ml. Cytotoxicity was measured after 48 h by Trypan blue exclusion. (c) DcR2 expression in BCBL-1 blocks apoptosis induced by AZT and IFN-. Top panel: Flow cytometry profiles of DcR2 expression in transfected BCBL-1 cells (bold line) versus wild type (thin line). Dotted line is isotype control. Bottom panel: Wild type and BCBL-1 cells were treated with IFN- 1000 u/ml, AZT 10 g/ml or IFN- 1000 u/ml plus AZT 10 g/ml and viability was measured by Trypan blue exclusion

Figure 5 (a) TRAIL substitutes for IFN- to potentiate apoptosis in AZT treated PEL cells. BCBL-1 cells were treated under the following conditions for 48 h and apoptosis was measured by Annexin/PI staining. Panel A=BCBL-1 cells in medium. Panel B=BCBL-1 cells in AZT 10 g/ml. Panel C=BCBL-1 cells in AZT 10 g/ml and IFN- 1000 u/ml. Panel D=BCBL-1 cells treated with soluble TRAIL 10 ng/ml. Panel E=BCBL-1 cells treated with AZT 10 g/ml plus soluble TRAIL 10 ng/ml. The per cent of apoptotic cells in each panel is shown in Panel F, (b) AZT potentiates TRAIL mediated cytotoxicity in BCBL-1. BCBL-1 and BCBL-1 FADD-DN (BCBL-1A2) were treated in media, AZT 10 g/ml, AZT 10 g/ml plus IFN- 1000 u/ml, TRAIL 10 ng/ml, or AZT 10 g/ml plus TRAIL 10 ng/ml for 48 h and per cent viability was determined by Trypan blue exclusion. Bars represent per cent viable cells. The data is representative of at least two experiments

Figure 6 (a) Antivirals nucleosides ddC and d4T do not synergize with IFN- to induce apoptosis in PEL. BCBL-1 and BC-3 cells were cultured for 48 h in media (control), IFN- 1000 u/ml, AZT 10 g/ml, AZT 10 g/ml and IFN- 1000 u/ml, ddC 10 g/ml, ddC 10 g/ml and IFN- 1000 u/ml, d4T 10 g/ml, or d4T 10 g/ml and IFN- 1000 u/ml. Cell viability was determined by Trypan blue exclusion. Bars represent per cent viable cells. (b) Inhibition of AZT or AZT+IFN- induced apoptosis by Thymidine in BL-7 (EBV+ BL) and BCBL-1 (HHV-8+ PEL). BL-7 cells (Top panel) were cultured for 48 h in medium (control), AZT 10 g/ml, Thymidine 10 g/ml, AZT 10 g/ml plus Thymidine 10 g/ml, Thymidine 100 g/ml or AZT 10 g/ml plus Thymidine 100 g/ml. BCBL-1 cells (lower panel) were cultured for 48 h in medium (control), AZT 10 g/ml plus IFN- 1000 u/ml, Thymidine 10 g/ml, AZT 10 g/ml plus IFN- 1000 u/ml plus Thymidine 10 g/ml, Thymidine 100 g/ml, AZT 10 g/ml plus IFN- 1000 u/ml plus Thymidine 100 g/ml