We have analyzed the Fas-mediated death pathway in a panel of 11 Epstein-Barr virus (EBV)-negative and 10 EBV-positive Burkitt’s lymphoma (BL) cell lines. We show that the increased expression of Fas in EBV-positive cell lines is mediated via LMP-1. Four of the 21 BL cell lines are readily responsive to Fas-mediated cell death signals. Of the remaining 17 cell lines, 10 can be sensitized by up-regulating Fas either via exogenous expression of LMP-1 or via treatment with CD40L. These same cell lines can also be sensitized by treatment with cycloheximide (CHX), which, however, does not result in up-regulation of Fas. Neither up-regulation of Fas, nor treatment with CHX, restore Fas sensitivity in seven BL cell lines. Further analyses indicated that 5 of the 7 cell lines (and none of the 14 responsive cell lines) were also compromised in the integrity/expression of the proapoptotic gene Bax. Thus, in most BL cell lines, the Fas pathway seems to be inhibited, although the mechanism of inhibition varies. The correlation between Bax mutation and irreversible (by CD40L or CHX) Fas resistance raises the possibility, for the first time, that Bax may play a critical function in Fas-mediated cell death in BL.

The Fas-FasL3 system plays a central role in the selection of the lymphoid repertoire. The Fas receptor CD95, also known as APO-1, is a Mr, 45,000 cell surface receptor expressed in a broad variety of cell lineages (1). The expression of the FasL, on the other hand, is restricted to activated T cells and cells from certain immunologically privileged anatomical sites (2, 3).

The contribution of the Fas-mediated cell death pathway in controlling peripheral T cell immune responses is well characterized (4). Fas is also involved in the elimination of self-antigen-activated B cells in the GC (5, 6, 7). Germline Fas mutations in both mice and humans result in autoimmune lymphoproliferative disorders (8). An increased frequency of plasmacytomas has also been observed in these mice (9).

The presence of Fas receptors in normal and neoplastic cells from various lineages suggests that many neoplasms are potentially susceptible to apoptosis via engagement of Fas. Conversely, the inhibition of Fas-mediated apoptosis may be an important component of neoplasia.

Triggering of Fas by FasL activates a cascade of downstream molecular events that culminate in apoptosis (4). The characterization of the components of this signal transduction pathway demonstrated that CD95 contains a death domain that orchestrates the assembly of a signaling complex (death-inducing signaling complex), which includes an adapter molecule, Fadd, and the protease Flice. Formation of the death-inducing signaling complex leads to activation of a proapoptotic protease cascade involving enzymes that, like Flice, cleave at aspartate residues (caspases; Refs. 10 and 11).

The proapoptotic protein Bax (12), which is common to several apoptotic pathways, potentiates death in response to multiple stimuli. Bax mutations have been implicated in resistance to apoptosis after withdrawal of growth factors (13), γ-irradiation (14), dexamethasone (15), and chemotherapeutic agents (16). There is no data, however, that implicates a direct role for Bax in Fas-mediated apoptosis. Reports that Bax may also participate in activation of caspases (17) suggest such a role. Bax is specifically expressed in GC cells (7), and the elimination of B cells in the GC is Fas-dependent, suggesting that both Fas and Bax may play a role in B cell apoptosis. Consistent with this, Bax-deficient mice display hyperplasia of both thymocytes and B cells (18), indicating that some aspect of B cell homeostasis is, indeed, regulated by Bax.

It has been proposed that BL develops from pro/pre-B cells that carry a deregulated c-myc juxtaposed to immunoglobulin heavy or light chain sequences (19). Like pre-B cells, BL cells undergo apoptosis after cross-linking of surface immunoglobulin (20). The BL phenotype, however, is similar to that of a GC B cell.

Although previous studies (21) have demonstrated that the presence of EBV up-regulates the expression of CD95 in BL cells, the mechanisms of this up-regulation have not been clarified. In the present study, we show that this induction is dependent on the expression of the EBV latent gene LMP-1. However, regardless of CD95 expression, Falk et al.(21) had also suggested that, unlike EBV immortalized normal B cells, EBV-positive BL, in general, are resistant to Fas-mediated cell death. In contrast, Daniel et al.(22) concluded that although the EBV-negative BL cell lines are resistant to Fas, EBV-positive cell lines are moderately to highly sensitive. Additional studies also suggested that Fas sensitivity in some BL cell lines could be modulated by CHX or by CD40 L (23, 24). We have reassessed the integrity of the Fas death pathway in a larger panel of BL in the presence and absence of CHX or CD40L. We show here that, in general, BL cells possess an intact Fas signal transduction pathway, but are Fas-resistant. This resistance can be reversed either by up-regulation (by CD40L) of Fas or by treatment with CHX. In one-third of BL cell lines, however, Fas resistance remains irreversible. A frequent lesion in these cell lines is compromised Bax function. These observations, coupled with differential expression of Bax in GC cells, raise the possibility that Bax may be necessary for Fas-mediated apoptosis in GC/BL cells.

Cell Lines.

Twenty-one BL cell lines were used: 10 EBV positive (KK124, AS283A, PA682PB, Raji, Akata, PA682PE, Namalwa, SE686, PA682BM, and Daudi) and 11 EBV negative (BL30, BL41, ST486, EW36, Ramos, LW878, JD38, Louckes, JLP119, CA46, and BML895).

Isogenic BL41 cell lines derived by infection with P3HR1 or B95.8EBV strains were also used. LMP-1 stable transfectants of BL41, a gift of Dr. E. Kieff (Harvard Medical School, Boston, MA), were maintained in guanosine triphosphate selection medium (25). A spontaneously immortalized EBV cell line, VDSO, was used as a control. All cell lines were grown at 37°C in RPMI 1640 supplemented with 15% FCS, in an atmosphere containing 5% CO2. With the exception of BML895, all BL cell lines used in this study have been characterized previously (26).

Proliferation Assay.

Cells were seeded at 2 × 105 cells/ml in flat-bottomed 96-well plates with 100 ng/ml CH11 anti-Fas MoAb (Medical and Biological Laboratories Co. Ltd.,, Watertown, MA) or an isotype control IgM MoAb, and 5 μg/ml protein A (Sigma Chemical Co., St. Louis, MO) were added at the time of plating. Plates were incubated for 24 h and then labeled for 16 h with [3H]thymidine (10 μCi/well, 6.7Ci/mmol; 1 Ci = 37 GBq). The concentration of anti-Fas MoAb and the kinetics of cell death were derived from preliminary titration experiments using 5–500 ng/ml CH11 over a period of 12–48 h. Cells were harvested onto glass fiber filter paper, and the incorporated radioactivity was measured by liquid scintillation counting. All samples were measured in sextuplicate in two independent experiments.

Cytotoxicity Assay.

Cells were seeded at 2 × 105 cells/ml in flat-bottomed 96-well plates with 100 ng/ml CH11 anti-Fas MoAb, and 5 μg/ml protein A were added at the time of plating. Plates were incubated for 24 h, harvested, and stained with Hoechst 33342 and Propidium Iodide, as described previously (27). Cells were examined by fluorescence microscopy and viable apoptotic and nonapoptotic cells counted. A minimum of 200 total cells/sample/assay were counted. Samples were measured in duplicate. In addition to the morphological assessment of apoptosis, cytotoxicity assays using trypan blue exclusion were also performed after exposure of the cell lines to anti-Fas MoAb (without protein A) for a period of 14–18 h. There was a very good concordance between these two assays and the proliferation assay. In additional experiments, cells were also preincubated for 48 h with CD40L (Immunex Corp., Seattle, WA) or incubated with 10 μg/ml of CHX for 3 h, washed, and equal aliquots were either incubated with no CH11 or with 100 ng/ml CH11, as described above. The ability of CD40L and CHX to potentiate Fas-induced death was determined by comparing percentage of apoptosis or percentage of inhibition of growth in the presence and absence of CD40L or CHX.

Northern Blots.

RNA was prepared with RNAzol B (Tel-Test Inc., Friendswood, TX). Total RNA (20 μg) was run in a 1.2% agarose gel with formaldehyde, transferred to a nylon membrane, and hybridized to 32P-radiolabeled probes. We used the full-length human cDNA of the Fas and the Bax genes as probes. To assess the amount of RNA loaded for each cell line, the membrane was stripped (0.1% SDS at 90°C) and rehybridized with a glyceraldehyde-3-phosphate dehydrogenase probe.

RT-PCR Analysis.

To differentiate among alternate spliced forms of Fas mRNA (28), and specifically to determine whether any soluble Fas variant (29) is expressed in BL, we carried out RT-PCR analysis. Total RNA (5 μg) was reverse-transcribed using a poly-T primer. The cDNA was then amplified using three primer pairs, as described by Dirks et al.(23). The 340-bp amplification product from exon 9 of Fas cDNA is present in all variant transcripts (28). Amplification of cDNA using a second set of primers yields a 470-bp product from two variant transcripts, which retain exon 4. These two variants can be further distinguished by the amplified product of the third primer pair, which yields a product of 360 bp from the full-length Fas transcript, but yields a smaller transcript of 297 bp if it has spliced out the transmembrane domain.

FACS Analysis.

Mouse MoAb ZB-4 against human CD95 was obtained from MBL. Viable cells were stained indirectly with ZB-4 in 100 μl of PBS containing 1% FCS and 0.1% sodium azide and followed with FITC-conjugated goat Abs to mouse immunoglobulin (Becton Dickinson Immunocytometry Systems, San Diego CA). Irrelevant isotype-matched immunoglobulin was used as a control. Stained cells were analyzed using FACScan flow cytometry equipped with a Cell Quest data analysis program (Becton Dickinson Immunocytometry Systems), as described previously (30). A minimum of 10,000 events for each sample was collected.

Western Blots.

Protein extracts from equal amounts of cells were prepared by standard procedure and electrophoresed in 4–15% acrylamide Tris-HCl minigels (Bio-Rad, Hercules, CA). After electrotransfer to nitrocellulose membranes, the blots were incubated with a polyclonal antibody for Bax (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed with a horseradish peroxidase-coupled secondary antibody using the Enhanced Chemiluminescence detection system (Amersham Corp., Arlington Heights, IL).

SSCP and Sequencing.

The integrity of the Bax gene was analyzed by SSCP and confirmed by sequencing. RT-PCR of two overlapping fragments encompassing the full-length cDNA were amplified in the presence of 32P-dCTP and run in a nondenaturing 6% acrylamide gel with 10% glycerol. Autoradiographs were exposed for a few hours. A 94-bp region of exon 3 containing a poly-G tract was also amplified using DNA from the same 21 cell lines. These PCR products were analyzed for their poly-G content by PAGE. Sequence analyses were performed both directly from PCR-amplified products and from cloned cDNAs in a TA vector (Invitrogen, San Diego, CA) using the Sequenase 2.0 kit (United States Biochemical Corp., Cleveland, OH) in the presence of 35S-dATP.

BL Cell Lines Express Fas Antigen.

To investigate Fas-mediated apoptosis in BL, we first screened the panel of 21 BL cell lines for expression of Fas antigen by FACS analysis (Fig. 1 A). All 11 EBV-negative cell lines expressed moderate to low levels of Fas antigen on the cell surface. In contrast, 7 of the 10 EBV-positive cell lines expressed high levels of Fas.

We performed Northern blot analysis on all of the cell lines except LW878. Band intensities of the Fas transcripts were consistent with the levels of the surface expression of Fas for each of the BL cell lines. A representation of the Fas RNA expression in 16 cell lines is shown in Fig. 1,B. Thus, Fas mRNA expression was lower in EBV-negative cell lines than in most EBV-positive cell lines. As seen in Fig. 1 B, two prominent Fas transcripts, of 2.5 and 1.9 Kb, were detected in all BL cell lines and represent transcripts resulting from the use of both regular and alternate polyadenylation signals at positions 2518–2523 and 1831–1836 in the Fas gene, respectively (1).

Expression of the EBV-encoded LMP-1 Protein Correlates with High-Density Fas Antigen Expression.

The difference in the levels of Fas expression in the EBV-positive and EBV-negative BL cells suggested a possible relationship between the presence of EBV and the expression of Fas in BL cells. To assess this, we used isogenic BL cell lines derived from an EBV-negative BL (BL41) by in vitro infection with either the defective strain of EBV P3HRI or the wild type B95–8 strain. The B95–8 infected derivative expresses the full complement of EBV latent genes, whereas the P3HR1-infected BL41 cell line fails to express EBNA-2 and LMP-1. Both FACS (Fig. 2) and Northern analyses (data not shown) of these EBV-converted lines indicated a moderate increase in Fas expression in BL41-B95.8 cells, but not in BL41-P3HRI cells, suggesting a role for either EBNA-2 or LMP-1 in the up-regulation of Fas.

LMP-1 is expressed in all of the EBV-positive BL cell lines reported here, with the exception of Namalwa, Daudi, and Akata (31). These three cell lines expressed low levels of Fas, suggesting that increased expression of Fas in EBV-positive cell lines may be a function of LMP-1 expression. To assess this, we compared Fas levels in BL41 clones stably transfected with LMP-1 or with vector alone. As seen in Fig. 2, exogenous expression of LMP-1 was sufficient to result in a 20-fold increase in Fas expression in BL41.

BL Cell Lines Are Insensitive to Fas-mediated Apoptosis.

To determine the capacity of Fas to transduce an apoptotic signal in BL cells, we tested the ability of anti-Fas MoAb CH11 to inhibit growth as measured by thymidine incorporation. As shown in Fig. 3,A, only 4 (all EBV positive) of the 21 cell lines (20%) demonstrated an inhibition of growth. Similar data were also obtained when Fas-dependent cell death was assessed by trypan blue dye exclusion. Hoechst and propidium iodide staining of BL cells exposed to anti-Fas MoAb CH11 confirmed that the growth inhibition observed for SE686, KK124, PA682PB, and AS238A reflected apoptotic cell death (Fig. 3 B).

Although the four Fas-sensitive BL cell lines expressed a high level of Fas on the surface, not all of the cell lines that expressed a high density of Fas were sensitive to CH11.

CHX Enhances Fas-mediated Apoptosis in BL Cell Lines that Express Low Levels of Fas.

Resistance to Fas-dependent cell death can be mediated through proteins that block the Fas signaling pathway. This resistance can be reversed by blocking de novo protein synthesis (23). The ability of CHX to abrogate Fas resistance in BL cell lines was demonstrated in 10 of the 17 cell lines resistant to apoptosis induced by anti-Fas MoAb CH11 (Fig. 4). Engagement of the Fas receptor was necessary for the induction of cell death because CHX alone did not cause apoptosis. Treatment with CHX failed to increase Fas expression in BL cell lines (data not shown), suggesting that the low level of CD95 on the surface of resistant cells was sufficient to induce Fas-mediated apoptosis in the absence of proteins that block Fas death signaling. Sensitivity to Fas, in the presence or absence of CHX, allowed us to classify the cell lines into three phenotypes. Fas-sensitive group A included SE686, KK124, AS283A, and PA682PB. Conditionally Fas-sensitive (only in the presence of CHX) group B cell lines included PA682BM, Akata, Namalwa, BL30, BL41, ST486, EW36, Ramos, JD38, and JLP119. Fas-insensitive group C BL cells were represented by PA682PE, Raji, Louckes, LW878, CA46, BML895, and Daudi.

Increased Fas Expression Enhances the Susceptibility of Group B Cells to Fas-mediated Apoptosis, Whereas Group C Cells Remain Resistant.

To assess whether the level of Fas expression is a major determinant of sensitivity to Fas-mediated apoptosis, we analyzed isogenic lines of BL41 (a group B cell line) that were induced to express high levels of Fas either by infection with the B95–8 strain of EBV or by transfection with the LMP-1 expression vector (Fig. 2). In these BL41 cell lines, the up-regulation of Fas expression correlated directly with the transition to Fas sensitivity (Fig. 5). The limited sensitivity to anti-Fas treatment in BL41/B95.8 reflects the moderate level of Fas expression in these cells.

In previous studies, Falk et al.(21), however, concluded that up-regulation of Fas in EBV-negative BL cell lines by conversion to EBV positivity did not restore Fas sensitivity. Because recent data have demonstrated that CD40 ligation induces Fas expression in B lymphocytes and enhances Fas-mediated apoptosis (24), we decided to use CD40 interaction to expand this analysis. Groups B and C BL cell lines were incubated with 2 μg/ml soluble CD40L for 48 h and exposed either to anti-Fas MoAb CH11 or control antibody. As shown in Fig. 6,A, Fas expression was up-regulated in representative LMP-1-negative BL cells after exposure to soluble CD40L. Thymidine incorporation assays revealed that CD40 ligation and treatment with anti-Fas MoAb CH11 inhibited proliferation in group B cell lines (Fig. 6 B). This growth inhibition ranged from 23% inhibition in EW36 to 94% inhibition in ST486, a similar level of resistance to Fas observed for EBV-positive BL cell lines. In contrast, group C BL cell lines remained Fas-resistant despite an increase of up to 40-fold in the relative expression of Fas.

Fas-resistant Cell Lines Exhibit Alterations in Bax.

To understand the molecular basis of the Fas-resistant group C phenotype, we first examined our panel of BL cell lines for mutations in the Fas gene by SSCP analysis. None of the 21 BL cell lines carried mutations in Fas (data not shown). Fas resistance can also result from expression of a soluble isoform of Fas that lacks a transmembrane domain (32). Since this soluble isoform can be easily detected by RT-PCR analysis, we analyzed Fas expression in the 21 cell lines as described in “Materials and Methods.” This analysis failed to show expression of soluble Fas in any of the BL cell lines (data not shown), suggesting that resistance to Fas-mediated apoptosis was not due to perturbations in Fas splicing.

Because Fas-induced cell death strictly depends on the activation of a cascade of caspases (33), Fas resistance could also result from abnormalities of caspases, a precedent for which exists in the low expression of caspase 3 observed in MCF-7 (34). Assessment of caspase levels by Western blot analysis showed that irrespective of their Fas-sensitivity, all cell lines from groups A, B, and C expressed similar amounts of caspases 3 (CPP32) and 8 (Flice).

The inability to detect alterations in characterized components of the Fas death pathway prompted an analysis of other proteins involved in apoptosis. The proapoptotic protein Bax functions in several apoptotic pathways and has also been suggested to potentiate activation of caspases (17). Bax mutations have been recently described in hematopoietic cell lines, including the group C cell line Daudi (35). To determine whether Bax played a role in the Fas resistance encountered in group C cell lines, we assessed the integrity of this gene in the panel of 21 BL cell lines by SSCP analysis and subsequent sequencing. Fig. 7 shows a representative panel from the SSCP analysis of the Bax locus. No Bax alterations were detected in any of the 14 cell lines from groups A and B. However, four of the seven cell lines in group C appeared to carry abnormally migrating Bax bands; two of these are shown in Fig. 7. Sequencing was performed for several of the normally migrating amplimers and of all of the abnormal amplimers. Normally migrating amplimers carried a wild type Bax sequence. Sequencing of abnormally migrating amplimers confirmed that four cell lines carried mutations in Bax. Daudi carried a heterozygous mutation in a conserved residue in the BH1 domain of Bax (G to T at nucleotide 323; Fig. 8). Two additional cell lines carried frameshift mutations resulting from either an addition or subtraction of a G residue in the poly G tract. These mutations were identical to those previously described in colon tumors with a mutator phenotype (36). BML895 had an additional G residue in one allele and lacked a G in the other allele (Fig. 8), whereas LW878 had an extra G in only one allele. The fourth cell line, CA46, carried a hemi/homozygous deletion of 11 bp in the first exon-first intron boundary of the Bax gene, resulting in a spliced version of Bax that is out-of-frame (Fig. 8).

Examination of Bax expression in all 21 cell lines by Northern blot analysis (Fig. 9,A) revealed very low levels of Bax mRNA in Raji, another group C cell line. Western blot analysis (Fig. 9,B) confirmed that BML895 and CA46 did not express any Bax protein, and Raji expressed barely detectable levels, consistent with the Northern analysis (Fig. 9 B). In addition, these analyses also demonstrated that the cell line LW878, which carried a frameshift mutation in only one allele of Bax, expressed Bax at levels even below those detected for Raji. The remaining cell lines displayed similar levels of Bax.

In addition to the low or no expression of Bax in four cell lines, Bax in Daudi is nonfunctional, having been reported to behave as a dominant negative mutation (37). We conclude that five of seven group C cell lines are deficient in Bax. These data, summarized in Table 1, show a statistically significant (Fisher’s exact test, P < 0.001) association between the deficiency in Bax and resistance to Fas-mediated apoptosis, suggesting that Bax may play a role in Fas-mediated cell death.

BL, a high-grade B-cell lymphoma characterized by a translocation involving the c-myc and immunoglobulin genes, has phenotypic features that resemble a GC B cell. Therefore, BL cells express Bcl-6, are positive for CD38 and CD77, carry somatic mutations in the immunoglobulin hypervariable regions, and express low Bcl-2 and moderate to high Bcl-XL(38).4

GC B cells that compete ineffectively for Ab are destined to die by apoptosis, whereas those that synthesize high affinity Ab are rescued by antigen, CD40L, IL4, and possibly other costimulatory molecules (5, 6, 7). The propensity of the GC B cells to undergo apoptosis is also reflected by the pattern of expression of the apoptotic genes in various B cell subsets. Fas and Bax are equally expressed in centrocytes and centroblasts (7). It would thus follow that, in the absence of antigenic rescue, BL cells, like their normal counterparts in the GC, should be susceptible to apoptosis. Indeed, apoptosis is frequently observed in primary BL biopsies (38), and the clinical development of the tumor is probably a result of a shift in the equilibrium between life and death, such that the fraction of cells dying is less than the fraction of cells replicating and surviving. Whether the observed apoptosis in tumors in vivo is Fas-mediated remains unknown. Because the Fas-mediated death pathway plays a functional role in the elimination of B cells in the GC, we focused this study on analyzing the integrity of this pathway in BL. Our results indicate that in the majority of BL cell lines, the Fas-dependent cell death pathway is either down-regulated (group B) or compromised (group C), whereas only a small fraction of BL cell lines are Fas-sensitive (group A).

Fas, which is expressed at high levels in the CD77-positive GC B cells (39), is in general expressed in low, but detectable amounts in EBV-negative BL cells (Fig. 1). It would, thus, seem that down-regulation of Fas is one means whereby BL cells escape Fas-mediated cell death. In EBV-positive BL cell lines, however, we demonstrated that the levels of both Fas transcript and protein are generally higher than in EBV-negative cell lines. This is similar to the observations previously reported by Falk et al.(21). We have further extended these observations by demonstrating that the increase in CD95 in EBV-positive cells is related to the expression of LMP-1 (Fig. 2). It is interesting to note that the expression of LMP-1 in BL cells is severely restricted in vivo. In its restriction of LMP-1 expression, BL is an exception to other EBV-containing tumors (40). It is possible that this restriction of LMP-1 aids the BL cell in escaping Fas-mediated apoptosis; this possibility is supported by our observations that cross-linking of the Fas receptor causes apoptosis only in those BL cells (group A) that express LMP-1 and, hence, high amounts of Fas. Since LMP-1 also up-regulates Bcl-2 (41), our data are consistent with the inability of Bcl-2 to protect Fas-mediated apoptosis in BL cells.

The potential importance of down-regulating Fas expression in BL cells is highlighted by the observation that group B cell lines contain an intact Fas-mediated death pathway, as evidenced by the ability of anti-Fas MoAb to transduce a death signal in the presence of CHX or CD40L (Figs. 4 and 6). The inability of these group B cell lines to transduce a death signal by anti-Fas treatment alone probably reflects active inhibition mediated by antiapoptotic protein(s). CHX may either inhibit the synthesis of these proteins or reverse Fas resistance by influencing posttranslational modifications of proteins. Several candidate inhibitory proteins that could fulfill this function have been described, including Fas-associated phosphatase-1, FAP-1 (42), and Flice inhibitory protein FLIP (43). The observation that up-regulation of Fas expression and treatment with CHX were both effective in converting the same panel of group B cell lines to Fas sensitivity is significant. This finding suggests that the inhibitory mechanism in group B cell lines can be overcome either by increasing (via up-regulating Fas expression) the activation of downstream proteins, or by diminishing (via CHX) the inhibitory influence, whatever the mechanism of the latter may be. In this context, the ability of CD40L to increase Fas expression and sensitize BL to apoptosis, may provide a strategy that can be exploited for therapy.

Seven of 21 lines were resistant to Fas-mediated cell death even after incubation with CHX or treatment with CD40L (group C). Whether these cell lines acquired, secondary to lymphomagenesis, an additional lesion in the development of Fas resistance or whether this lesion was necessary for the clinical development of the lymphoma remains to be answered. It may, however, be revealing that among these seven resistant cell lines two EBV-negative cell lines (Louckes and LW878) are exceptional because they express moderate amounts of Fas, which, at least in ST486 (Fig. 6), was sufficient to induce Fas-mediated cell death. If the amount of CD95 expression on these two cell lines reflects those of the primary tumors, the development of additional blocks to Fas-apoptosis is consistent with our observation that in the face of moderate to high Fas expression, the labile inhibitory proteins that may account for resistance in other BL cells are not enough to abrogate the death pathway.

A common lesion in the group C cell lines was a deficiency in Bax expression, detected in five of seven Fas-resistant cell lines (Fig. 9). This resulted from: (a) frameshift mutations in both alleles (BML 895); (b) a frameshift mutation and loss of the wild type allele (CA46; Fig. 8), both resulting in loss of Bax protein expression; (c) a frameshift mutation in one allele and reduced Bax expression from the other allele (LW878); (d) a dominant negative mutation in the BH1 region of Bax (Daudi); and, finally, (e) unusually low level of Bax protein and mRNA (Raji). In addition to the previously described mutation in Daudi, a frameshift mutation in Bax has also been described recently in another EBV-negative cell line, DG-75 (44). These studies have, however, not correlated the integrity of Bax with Fas-sensitivity in BL. Preliminary data from analysis of primary BL biopsies indicate that loss of Bax expression is not confined to cell lines and also occurs in primary tumors (45).

The hypothesis that Bax plays a role in Fas-mediated cell death is supported by other observations in the literature. Thus, deficiencies in Bax have been associated with Fas resistance in two of four Fas-resistant prostatic carcinoma cell lines (46), and the effectiveness of signaling through the Fas receptor in myeloma cell lines has been shown to directly correlate with the relative expression level of Bax (47). Cerebellar granule cells from Bax-deficient mice are compromised in their ability to activate caspases, suggesting that Bax may be capable of influencing Fas-mediated signals at the level of caspase processing (17). A recent study by Scaffidi et al.(34) suggests the presence of two alternative downstream cascades after Fas stimulation, one that directly activates caspase 8 (group 1) and the other one that proceeds through the mitochondria (group 2). Coincidentally, both the cell lines with group 2 phenotype carry Bax mutations.

To directly test our hypothesis that resistance to Fas-mediated apoptosis is a result of mutations in Bax, we have attempted to obtain stable clones expressing Bax from several of the Bax-deficient BL cell lines. Transfectants with wild type Bax cDNA were easily obtained from Bax-positive BL cells, however, this cDNA caused at least a 10-fold suppression of clonogenicity in Bax-negative cell lines, and none of the resulting clones expressed Bax. No differences in clonogenicity were noted with either the vector control or with a mutant Bax. Several alternative approaches can be used to further define the role of Bax in Fas-mediated apoptosis in BL, including depletion of Bax in Bax-positive cell lines, using, for instance, antisense strategies. We have attempted to transiently restore Bax in Fas-resistant cell lines, and preliminary data (48), indeed, support the hypothesis that Bax is an essential element in Fas-mediated apoptosis in BL.

We are now analyzing events downstream of the stimulation of the Fas receptor to determine where Bax fits into the hierarchy of molecular signals that result in cell death.

Fig. 1.

Expression of Fas in BL cell lines. A, reactivity of anti-Fas MoAb (ZB-4) with BL cell lines and VDSO (cell line used as control), as analyzed by flow cytometry. Bound MoAbs were detected with FITC-labeled goat Abs to mouse immunoglobulin. Results are expressed as the mean fluorescence intensity minus the background (irrelevant Ab control). B, Northern blot analysis of Fas. Top, two transcripts of 2.5 and 1.9 Kb, detected with a full-length cDNA probe. Bottom, the same blot after stripping and rehybridizing with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to compare the amount of RNA loaded in each lane. Lane 1, VDSO; Lane 2, KK124; Lane 3, Raji; Lane 4, CA46; Lane 5, ST486; Lane 6, JD38; Lane 7, BL41; Lane 8, PA682PB; Lane 9, PA682BM; Lane 10, PA682PE; Lane 11, AS283A; Lane 12, BML895; Lane 13, Daudi; Lane 14, EW36; Lane 15, Akata; Lane 16, Louckes.

Fig. 1.

Expression of Fas in BL cell lines. A, reactivity of anti-Fas MoAb (ZB-4) with BL cell lines and VDSO (cell line used as control), as analyzed by flow cytometry. Bound MoAbs were detected with FITC-labeled goat Abs to mouse immunoglobulin. Results are expressed as the mean fluorescence intensity minus the background (irrelevant Ab control). B, Northern blot analysis of Fas. Top, two transcripts of 2.5 and 1.9 Kb, detected with a full-length cDNA probe. Bottom, the same blot after stripping and rehybridizing with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to compare the amount of RNA loaded in each lane. Lane 1, VDSO; Lane 2, KK124; Lane 3, Raji; Lane 4, CA46; Lane 5, ST486; Lane 6, JD38; Lane 7, BL41; Lane 8, PA682PB; Lane 9, PA682BM; Lane 10, PA682PE; Lane 11, AS283A; Lane 12, BML895; Lane 13, Daudi; Lane 14, EW36; Lane 15, Akata; Lane 16, Louckes.

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Fig. 2.

Expression of Fas in EBV-converted BL41 cells and BL41 cells transfected with LMP-1, as analyzed by flow cytometry. The results are plotted on a logarithmic scale. Filled area, Fas expression; unfilled area, irrelevant IgM.

Fig. 2.

Expression of Fas in EBV-converted BL41 cells and BL41 cells transfected with LMP-1, as analyzed by flow cytometry. The results are plotted on a logarithmic scale. Filled area, Fas expression; unfilled area, irrelevant IgM.

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Fig. 3.

Response of BL cell lines to treatment with anti-Fas MoAb. VDSO cell line was used as a Fas-sensitive control. A, anti-Fas-induced inhibition of proliferation. BL cells were grown in the presence of anti-Fas antibody (CH11), and proliferation was measured in a [3H]thymidine assay after 24 h. Data were compared with control samples grown in the presence of control Ab. Results are expressed as the percentage inhibition of [3H]thymidine incorporation in CH11-treated cells compared with cells grown in control Ab. B, anti-Fas-induced apoptosis. BL cells were grown in the presence or absence of anti-Fas antibody (CH11) stained with Hoechst 33342 and PI (Sigma Chemical Co.) and checked microscopically for morphological changes characteristic of apoptosis. The percentage of cells that are undergoing apoptosis out of the total number of viable cells is shown.

Fig. 3.

Response of BL cell lines to treatment with anti-Fas MoAb. VDSO cell line was used as a Fas-sensitive control. A, anti-Fas-induced inhibition of proliferation. BL cells were grown in the presence of anti-Fas antibody (CH11), and proliferation was measured in a [3H]thymidine assay after 24 h. Data were compared with control samples grown in the presence of control Ab. Results are expressed as the percentage inhibition of [3H]thymidine incorporation in CH11-treated cells compared with cells grown in control Ab. B, anti-Fas-induced apoptosis. BL cells were grown in the presence or absence of anti-Fas antibody (CH11) stained with Hoechst 33342 and PI (Sigma Chemical Co.) and checked microscopically for morphological changes characteristic of apoptosis. The percentage of cells that are undergoing apoptosis out of the total number of viable cells is shown.

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Fig. 4.

Response of BL cell lines to treatment with anti-Fas MoAb CH11 in the presence and absence of CHX. Percentage of apoptosis was measured after staining with Hoechst and PI. Cells were grown in the presence and absence of CH11 and after a 3-h pretreatment with 10 μg/ml CHX or no pretreatment. Results are expressed as percentage of apoptosis over no CH11 controls (□) or percentage of increased apoptosis over cumulative apoptosis measured in no CH11 and CHX controls (▪).

Fig. 4.

Response of BL cell lines to treatment with anti-Fas MoAb CH11 in the presence and absence of CHX. Percentage of apoptosis was measured after staining with Hoechst and PI. Cells were grown in the presence and absence of CH11 and after a 3-h pretreatment with 10 μg/ml CHX or no pretreatment. Results are expressed as percentage of apoptosis over no CH11 controls (□) or percentage of increased apoptosis over cumulative apoptosis measured in no CH11 and CHX controls (▪).

Close modal
Fig. 5.

Anti-Fas-induced inhibition of proliferation in isogenic lines of BL41. BL41 cell lines [parental, P3HR-1-converted, B95-8 converted, LMP-1 transfected, or vector alone (GTP) transfected] were grown in the presence of anti-Fas antibody (CH11), and proliferation was measured in a [3H]thymidine assay after 24 h. Data were compared with samples grown in the presence of control Ab. Results are expressed as percentage inhibition of [3H]thymidine incorporation compared with cells grown with control Ab.

Fig. 5.

Anti-Fas-induced inhibition of proliferation in isogenic lines of BL41. BL41 cell lines [parental, P3HR-1-converted, B95-8 converted, LMP-1 transfected, or vector alone (GTP) transfected] were grown in the presence of anti-Fas antibody (CH11), and proliferation was measured in a [3H]thymidine assay after 24 h. Data were compared with samples grown in the presence of control Ab. Results are expressed as percentage inhibition of [3H]thymidine incorporation compared with cells grown with control Ab.

Close modal
Fig. 6.

A, expression of Fas in cells treated with CD40L. BL cells were exposed to soluble CD40L for 2 days and analyzed for Fas expression by flow cytometry. Filled area, Fas expression in the presence of CD40L; unfilled area, Fas expression in the absence of CD40L. B, anti-Fas-induced inhibition of proliferation in groups B and C cells treated with CD40L. BL cells were exposed to soluble CD40L for 2 days before adding anti-Fas MoAb. After 24 h, proliferation was measured in a [3H]thymidine assay.

Fig. 6.

A, expression of Fas in cells treated with CD40L. BL cells were exposed to soluble CD40L for 2 days and analyzed for Fas expression by flow cytometry. Filled area, Fas expression in the presence of CD40L; unfilled area, Fas expression in the absence of CD40L. B, anti-Fas-induced inhibition of proliferation in groups B and C cells treated with CD40L. BL cells were exposed to soluble CD40L for 2 days before adding anti-Fas MoAb. After 24 h, proliferation was measured in a [3H]thymidine assay.

Close modal
Fig. 7.

SSCP analyses of the Bax gene. Top, a 94-bp fragment on exon 3, containing a stretch of 8 G. +, positive control (colon cancer cell line HCT with 7 Gs in one allele); BL, blank (no DNA) control. Note the mutations in Lane 2. Bottom, a 290-bp fragment corresponding to the 3′ region of the Bax open reading frame. The only mutation detected is in Lane 16. ND, nondenatured PCR product; , VDSO (a wild type control); Lane 1, Raji; Lane 2, BML895; Lane 3, EW36; Lane 4, PA682BM; Lane 5, PA682PE; Lane 6, PA682PB; Lane 7, CA46; Lane 8, Namalwa; Lane 9, BL30; Lane 10, SE686; Lane 11, JD38; Lane 12, BL41; Lane 13, ST486; Lane 14, AS283A; Lane 15, Louckes; Lane 16, Daudi; Lane 17, JLP119.

Fig. 7.

SSCP analyses of the Bax gene. Top, a 94-bp fragment on exon 3, containing a stretch of 8 G. +, positive control (colon cancer cell line HCT with 7 Gs in one allele); BL, blank (no DNA) control. Note the mutations in Lane 2. Bottom, a 290-bp fragment corresponding to the 3′ region of the Bax open reading frame. The only mutation detected is in Lane 16. ND, nondenatured PCR product; , VDSO (a wild type control); Lane 1, Raji; Lane 2, BML895; Lane 3, EW36; Lane 4, PA682BM; Lane 5, PA682PE; Lane 6, PA682PB; Lane 7, CA46; Lane 8, Namalwa; Lane 9, BL30; Lane 10, SE686; Lane 11, JD38; Lane 12, BL41; Lane 13, ST486; Lane 14, AS283A; Lane 15, Louckes; Lane 16, Daudi; Lane 17, JLP119.

Close modal
Fig. 8.

Partial sequence analysis of the Bax gene. Left, the exon 1-intron 1 boundary from CA46 and from a wild type (WT) control. The sequence shown can be read in the right, and the bracket indicates the 11 bp deleted in CA46. Middle, the point mutation observed in subcloned cDNA from Daudi RNA; Clone 1, the mutant allele; Clone 2, the wild type allele; *, the T that substitutes a G. Right, two representative clones of BML895 cDNA. Clone 1 contains 7 Gs corresponding to one allele, and Clone 2 contains 9 Gs corresponding to the other allele.

Fig. 8.

Partial sequence analysis of the Bax gene. Left, the exon 1-intron 1 boundary from CA46 and from a wild type (WT) control. The sequence shown can be read in the right, and the bracket indicates the 11 bp deleted in CA46. Middle, the point mutation observed in subcloned cDNA from Daudi RNA; Clone 1, the mutant allele; Clone 2, the wild type allele; *, the T that substitutes a G. Right, two representative clones of BML895 cDNA. Clone 1 contains 7 Gs corresponding to one allele, and Clone 2 contains 9 Gs corresponding to the other allele.

Close modal
Fig. 9.

Bax expression determined by Northern blot (A) and by Western Blot (B). A, two transcripts of 1.5 and 1 Kb are seen in all BL cell lines, although very low signal levels in Lanes 3, 4, and 16. B, the Mr 21,000 Bax protein is detected in most cell lines, except in Lanes 4 and 12; note that Lanes 3 and 17 are very weak. Bax protein in Lane 17 was detected only after a 4-fold overexposure compared with the rest. Lane1, VDSO; Lane 2, KK124; Lane 3, Raji; Lane 4, CA46; Lane 5, SE686; Lane 6, JD38; Lane 7, BL41; Lane 8, PA682PB; Lane 9, PA682BM; Lane 10, PA682PE; Lane 11, AS283A; Lane 12, BML895; Lane 13, Daudi; Lane 14, EW36; Lane 15, Akata; Lane 16, Louckes; Lane 17, LW878.

Fig. 9.

Bax expression determined by Northern blot (A) and by Western Blot (B). A, two transcripts of 1.5 and 1 Kb are seen in all BL cell lines, although very low signal levels in Lanes 3, 4, and 16. B, the Mr 21,000 Bax protein is detected in most cell lines, except in Lanes 4 and 12; note that Lanes 3 and 17 are very weak. Bax protein in Lane 17 was detected only after a 4-fold overexposure compared with the rest. Lane1, VDSO; Lane 2, KK124; Lane 3, Raji; Lane 4, CA46; Lane 5, SE686; Lane 6, JD38; Lane 7, BL41; Lane 8, PA682PB; Lane 9, PA682BM; Lane 10, PA682PE; Lane 11, AS283A; Lane 12, BML895; Lane 13, Daudi; Lane 14, EW36; Lane 15, Akata; Lane 16, Louckes; Lane 17, LW878.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3

The abbreviations used are: FasL, Fas ligand; GC, germinal center; BL, Burkitt’s lymphoma; CHX, cycloheximide; RT-PCR, reverse transcription-PCR; FACS, fluorescence-activated cell sorting; SSCP, single-strand conformational polymorphism; EBV, Epstein-Barr virus; Ab, antibody; MoAb; monoclonal antibody.

4K.

Naresh, H. Venkatesh, S. Kumar, M. Raffeld, K. Bhatia, and I. T. Magrath, unpublished data.

Table 1

EBV genome positivity, Fas expression, Fas sensitivity, and the integrity/expression of Bax in BL

Cell lineEBVFas levelFas responseBax proteinBax gene
SE686 High Wta 
KK124 High Wt 
AS283A High Wt 
PA682PB High Wt 
PA682BM High Conditional Wt 
Akata Low Conditional Wt 
Namalwa Low Conditional Wt 
BL30 − Low Conditional Wt 
BL41 − Low Conditional Wt 
ST486 − Low Conditional Wt 
EW36 − Low Conditional Wt 
Ramos − Mod Conditional Wt 
JD38 − Mod Conditional Wt 
JLP119 − Mod Conditional Wt 
Raji High − Very low Wt 
PA682PE High − Wt 
Daudi Low − Mutant 
LW878 − Mod − − Mutant 
Louckes − Mod − Wt 
CA46 − Low − − Mutant 
BML895 − Low − − Mutant 
Cell lineEBVFas levelFas responseBax proteinBax gene
SE686 High Wta 
KK124 High Wt 
AS283A High Wt 
PA682PB High Wt 
PA682BM High Conditional Wt 
Akata Low Conditional Wt 
Namalwa Low Conditional Wt 
BL30 − Low Conditional Wt 
BL41 − Low Conditional Wt 
ST486 − Low Conditional Wt 
EW36 − Low Conditional Wt 
Ramos − Mod Conditional Wt 
JD38 − Mod Conditional Wt 
JLP119 − Mod Conditional Wt 
Raji High − Very low Wt 
PA682PE High − Wt 
Daudi Low − Mutant 
LW878 − Mod − − Mutant 
Louckes − Mod − Wt 
CA46 − Low − − Mutant 
BML895 − Low − − Mutant 
a

Wt, wild type; Conditional, Fas responsive in the presence of CD40L or CHX; Mod, moderate.

We thankfully acknowledge the contributions made by Dr. C. Zacharchuck, in helpful discussions and in the critical reading of this manuscript, and by Dr. G. Gupta in helping with statistical analysis. We thank Immunex Corp. (Seattle, WA) for providing us with human recombinant CD40L through a material transfer agreement between Immunex Corp. and Dr. G. Tosato.

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