Introduction

Studies of tumors induced by complex retroviruses that lack typical oncogenes have revealed viral genes that mediate novel and important aspects of cellular growth. Unlike simple retroviruses, which act as acute tumor inducers by the expression of viral oncogenic products or by proviral insertional mutagenesis (Brady et al., 1987), human T-cell lymphotropic virus-1 (HTLV-1) and bovine leukemia virus (BLV) induce tumors after long latency (Burny et al., 1994; Franchini, 1995). While it is not fully understood how these viruses engender malignancy, several lines of evidence link the virally encoded transactivating proteins, Tax_HTLV-1 and Tax_BLV, to cellular transformation. These proteins are unique in that they are essential contributors to both the viral replication and the virus-associated oncogenic potential. A leading hypothesis is that transformation mediated by Tax proteins is related to their activity as transcriptional activators, leading to the disruption of lymphoid cell growth control and resulting in the accumulation of genetic defects, which culminates in overt leukemia.

BLV causes B-cell proliferative disorders in cattle (Burny et al., 1994; Kettmann et al., 1994). Infection results in a chronic disease with B-cell tumors emerging after long latency in less than 5% of infected animals. Experimental transmission to sheep leads to the development of B-cell leukemia in all the infected animals and after shorter latency (Mammerickx et al., 1988; Van den Broeke et al., 1989; Willems et al., 1993). In this respect, the BLV-associated ovine leukemia provides a unique model for studying leukemogenic processes associated with human complex retrovirus-induced diseases. BLV is structurally and functionally related to HTLV-1, which accounts for a significant incidence of T-cell leukemia in endemic areas (Sagata et al., 1985; Watanabe, 1997). Both viruses encode regulatory proteins involved in the infectious potential and the regulation of viral expression (Derse, 1987; Felber et al., 1989; Willems et al., 1994; Franchini, 1995; Kerkhofs et al., 1998). Among these proteins, both Tax_HTLV-1 and Tax_BLV were originally identified as transcriptional transactivators of viral expression through the indirect binding to cis-acting Tax-responsive elements located in the 5' U3 region of the viral long-terminal repeat (Brady et al., 1987; Derse, 1987; Willems et al., 1987). Tax proteins furthermore mediate transformation of rat embryonic fibroblasts in cooperation with Ha-ras, and the injection of these cells induces tumors in nude mice (Pozzatti et al., 1990; Willems et al., 1990). In addition, Tax_HTLV-1 immortalizes human primary T lymphocytes (Grassmann et al., 1992). Altogether, these data suggest a potential role for Tax proteins in leukemogenic processes leading to either T-cell leukemia (HTLV-1) or B-cell proliferative disorders (BLV). A long-lasting controversy in both the HTLV-1 and the BLV pathogenesis, however, is the paucity of viral expression (Haas et al., 1992; Richardson et al., 1997; Rovnak and Casey, 1999). The lack of BLV gene expression including tax is a consistent finding in fully transformed ovine B-cells, but Tax-defective proviruses are not infectious (Van den Broeke et al., 1989, 1999). This has made it difficult to identify discrete mechanisms by which BLV and Tax mediate leukemogenesis. Since Tax expression is not required to maintain the transformed phenotype, it is believed to act at early stages in the multistep process leading to full malignancy.

Tax_HTLV-1 has been extensively studied. Tax_HTLV-1 regulates the expression of several cellular genes, including genes encoding cytokines (interleukin-2 (IL-2)), cytokine receptors ( chain of IL-2 receptor), and proto-oncogenes (c-fos, c-jun, fra-1, c-myc) (Cross et al., 1987; Ballard et al., 1988; Fujii et al., 1988, 1991; Tsuchiya et al., 1993; Semmes et al., 1996). Tax_HTLV-1 targets regulators of cell cycle progression and apoptosis such as cyclin D2, cyclin-dependent kinase (cdk) 4, cdk6, the INK4 family of cdk inhibitors, Bcl-x_L, Bax, DNA polymerase , p53, caspase-8, and caspase-3 (Jeang et al., 1990; Suzuki et al., 1996; Brauweiler et al., 1997; Neuveut et al., 1998; Schmitt et al., 1998; Kawakami et al., 1999; Santiago et al., 1999; Pise-Masison et al., 2000; Mori et al., 2001). Mechanistically, Tax_HTLV-1 was found to act essentially through CREB/ATF and NF-B signaling pathways (Sun and Ballard, 1999). So far, there is no experimental evidence for Tax_BLV-associated cellular mechanisms in B-cell deregulation and transformation. The only data describing an impact on cell growth resulted from studies in nonlymphoid rat cells (Willems et al., 1990). Moreover, a recent study emphasized the discrepancies between this in vitro rat model and in vivo observations in sheep (Twizere et al., 2000). Although the reasons for these conflicting results are unknown, it is likely that B-cell-specific factors are required for the full appreciation of Tax-associated cellular transformation. Our main objective was thus to evaluate the oncogenic potential of the viral Tax transactivator in a relevant cell type. We previously developed primary ovine B-cell cultures and showed that B-cells isolated from a variety of sheep lymphoid tissues including ileal and jejunal Peyer's patches (PP) were permissive to BLV infection (Griebel et al., 1999; Van den Broeke et al., 2001). This culture system, dependent on costimulation with CD154 and chain (_c)-common cytokines, provides an excellent in vitro model for investigating Tax_BLV-associated leukemogenic processes. Here we used a retroviral vector-mediated gene transfer strategy for delivery of Tax_BLV in a pure ovine B-cell population. We provide experimental evidence for the oncogenic potential of Tax_BLV in ovine B-cells, the targets of BLV in experimentally infected sheep. We show that Tax_BLV alters the growth requirements of ovine B-cells, resulting in cytokine-independent B-cell growth. Tax_BLV furthermore enhances B-cell proliferation, alters the cell cycle distribution and mediates protection from apoptotic cell death. Mechanistically, we demonstrate that Tax_BLV expression is accompanied by the upregulation of Bcl-2 and increased nuclear NF-B activity. Our data provide the first evidence to support the oncogenic potential of Tax_BLV in B-cells and suggest an important role for NF-B-dependent cellular pathways in Tax- and BLV-associated B-cell malignancy.

Results

Tax confers cytokine-independent growth to ovine B-cells

The only direct evidence regarding the role of Tax in BLV-associated B-cell leukemia was provided by immortalization and transformation assays in rat embryonic fibroblasts (Willems et al., 1990), but so far there is no similar data available in lymphoid cells. Our main objective was thus to evaluate the oncogenic potential of the transcriptional transactivator in ovine B-cells, a relevant cell type for studying leukemogenic processes. We have previously shown that B-cells isolated from a variety of lymphoid tissues from sheep including ileal and jejunal PP were permissive to BLV infection (Van den Broeke et al., 2001). To examine the impact of Tax in a pure ovine B-cell population, we used ovine B-cell clones derived from the jejunal PP of a lamb (Griebel et al., 2000). While a limited number of BLV-associated bovine tumors are composed of sIgG⁺ B-cells (Vernau et al., 1997), we and others found that leukemic B-cells isolated from sheep were consistently sIgM⁺ (Murakami et al., 1994; Van den Broeke et al., 1997). Therefore, it was deemed most appropriate to first select an sIgM⁺ B-cell clone, referred to as Clone 2 hereafter. The continuous growth of B-cells is dependent on costimulation with CD154 and _c-common cytokines, which protect B-cells from death and support their proliferation (Griebel et al., 1999). The BLV tax cDNA was delivered into Clone-2 B-cells using a Gibbon Ape Leukemia-pseudotyped retroviral vector-mediated gene transfer strategy. Clone-2 cells were transduced with either pLTaxSN that carries the tax cDNA under the control of the MoMuLV LTR promoter (Van den Broeke et al., 1999) or the control vector pNUNL that expresses beta galactosidase (Bagnis et al., 1997), and further cultured in the presence of CD154, IL-2, IL-4, IL-7, IL-15, supplemented with G418 for selection of transduced cells. G418-resistant B-cells were then examined for Tax expression by immunoprecipitation. We found that Tax was present in pLTaxSN-transduced Clone-2 cells (Clone-2-LTaxSN) but at a lower level compared to a BLV-infected B-cell line that constitutively expresses viral proteins (YR2-LTaxSN), while the control B-cell cultures YR2 and Clone-2-NUNL were negative (Figure 1a). The vector was stably integrated as demonstrated by Southern blot (data not shown). To evaluate whether Tax might alter the growth requirements of B-cells, we cultured Clone-2-LTaxSN and control Clone-2 cells in the absence of cytokines, and examined B-cell growth over a 100-day culture period. The Clone-2 and Clone-2-NUNL viable B-cell numbers increased transiently, then declined, and finally all B-cells died within 45 days after cytokine removal. In contrast, the Clone-2-LTaxSN cells displayed a continuous proliferation after cytokine withdrawal (Figure 1b). These cells could be indefinitely maintained in the absence of cytokines but their culture still required CD154 stimulation. Thus, Tax is capable of modulating the growth properties of Clone-2 B-cells from cytokine dependence to cytokine independence, but CD154 stimulation remains an essential factor for continuous growth.

Figure 1.

Tax expression mediates cytokine-independent B-cell growth and enhances viable B-cell numbers in CD154+IL-4 costimulated cultures. (a) CD154+cytokine costimulated B-cell cultures (20 10⁶ cells) were metabolically labeled with ³⁵S-methionine and cysteine for 4 h. Total cell lysates were immunoprecipitated with anti-Tax polyclonal antibodies and subjected to SDS–PAGE. Clone-2-LTaxSN: pLTaxSN-transduced B-cell clone, Clone-2-NUNL: control vector-transduced B-cell clone, YR2-LTaxSN: BLV-expressing B-cell line, YR2: silent control B-cell line. (b) Clone-2 and Clone-2-LTaxSN B-cell cultures were transferred and stimulated with freshly irradiated mCD154 L cells every 4 days, at a ratio of 2 : 5 (L cells : B-cells) in the absence of cytokines. Viable cell numbers were determined by trypan blue exclusion at each passage throughout a 100-day culture period. Data from one experiment representative of three independent assays are shown. (c) Clone-2_L+4 and Clone-2-LTaxSN_L+4 cells were transferred every 3–4 days to fresh medium and costimulated with CD154 and IL-4. Viable cell numbers were determined by trypan blue exclusion throughout a 100-day culture period. Data are shown for one out of three independent experiments

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Tax enhances the viable B-cell numbers, increases proliferation, and alters the cell cycle phase distribution of B-cells

BLV infection alters the B-cell homeostasis with both an increase in B-cell cycling and prolonged survival (Schwartz-Cornil et al., 1997; Stone et al., 2000), but it is not known whether this deregulation is associated with Tax. To further compare the B-cell growth in the presence and the absence of Tax, it was necessary to utilize both CD154 and _c-common cytokines to support the growth of the parental Clone-2 B-cells. We found that CD154 and IL-4 were sufficient to sustain the long-term growth of Clone-2 B-cells, and we further used these conditions to derive Tax-negative and Tax-positive B-cell cultures referred to as Clone-2_L+4 and Clone-2-LTaxSN_L+4, respectively. The Tax protein level in Clone-2-LTaxSN_L+4 cells was similar to that observed in the presence of all the four _c-common cytokines (data not shown). We then determined the viable cell numbers for Clone-2_L+4 and Clone-2-LTaxSN_L+4 at each passage throughout a 90-day culture period. Although CD154 and IL-4 were clearly sufficient to support the continuous growth of Clone-2 cells, in addition, there were significantly higher viable B-cell numbers in Clone-2-LTaxSN_L+4 cultures, as indicated by the 10⁵-fold increase 90 days after culture initiation (Figure 1c). We reproducibly observed this growth advantage in three independent transduction experiments conducted with Clone-2 B-cells, as well as in LTaxSN-transduced sIgG⁺ Clone-1 and Clone-7 B-cell cultures (Griebel et al., 2000) (data not shown). Furthermore, transfer of the pNUNL vector did not result in any significant change, suggesting that Tax and not the vector itself was responsible for the impact on B-cell growth. We consistently found similar differences in all examined B-cell clones, suggesting that any of them may serve as a relevant B-cell model for investigating the oncogenic potential of Tax. All experiments described hereafter were conducted with Clone-2 B-cells.

To examine whether the Tax-associated growth advantage resulted from enhanced B-cell proliferation, we simultaneously examined the viable cell numbers, the proliferative responses, and the cell cycle phase distribution of Clone-2_L+4 and Clone-2-LTaxSN_L+4 cultures in the same experiment. B-cells were collected from 72 h costimulated cultures, reseeded in fresh medium with CD154+IL-4, and analysed after 24, 48, and 72 h. The Clone-2-LTaxSN_L+4 viable cell number reached a fivefold increase after 72 h as compared to a modest twofold expansion for Clone-2_L+4 (Figure 2a). This enhanced viability was accompanied by a higher proliferative response as measured by BrdU incorporation (Figure 2b). The difference in proliferative responses was already visible at 24 h and reached a fivefold induction 72 h after stimulation. We then assessed the cell cycle phase distribution by flow cytometry. The fraction of B-cells in S+G2/M was significantly higher in Clone-2-LTaxSN_L+4 as compared to control cells, with a proportion of 52 and 54% of Clone-2-LTaxSN_L+4 cells and only 36 and 42% of Clone-2_L+4 cells at 24 and 48 h, respectively (Figure 2c). In the 72 h cultures, however, the distribution of Tax-expressing cells was not significantly different from control cells. The fraction of S+G2/M cells in Clone-2-LTaxSN_L+4 declined (41%), while it continued to increase in Clone-2_L+4 (47%). We then repeated the CD154+IL-4 costimulation of equal B-cell numbers collected from these 72 h cultures and repeatedly found an increase in cycling cells at 24 and 48 h, indicating that in the presence of Tax, B-cells have reached saturation after 72 h, while the control cells were still exponentially growing. In addition, we also found that the increase in viable B-cell numbers in Clone-2-LTaxSN_L+4 cultures was significantly reduced 96 h poststimulation, confirming this observation (data not shown). These results indicated that Tax significantly altered the cell cycle distribution of B-cells. Altogether, our observations demonstrate that Tax affects the viable B-cell number, increases DNA synthesis, and acts through mechanisms involved in the control of B-cell cycling.

Figure 2.

Tax supports B-cell growth through increased proliferation and altered cell cycle phase distribution. (a) Cultures were initiated with 1.2 10⁶ B-cells/well and costimulated with CD154 and IL-4. Viable cell numbers were determined by trypan blue exclusion after 24, 48, and 72 h. Data presented are the means.d. of values of three independent cultures. (b) Cultures were initiated with 10⁴ cells/well and carried out for 24, 48, and 72 h prior to pulsing with BrDU as described in Materials and Methods. Incorporation is given as the mean o.d.s.d. of four cultures. (c) The DNA content of Clone-2_L+4 and Clone-2-LTaxSN_L+4 cells was analysed 24, 48, and 72 h after CD154+IL-4 costimulation by propidium iodide staining followed by flow cytometry analysis. Apoptotic and dead cells were excluded from the analysis. The numbers indicate the relative calculated proportions of cells in S+G2/M phases of the cell cycle with relative errors of 1–2% for each. The x-axis indicates fluorescence representing the DNA content. Arrows indicate positions of cells with 2 and 4 N DNA content. Data are shown for one representative experiment from three independent assays

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Tax mediates prolonged survival and rescues B-cells from apoptotic cell death induced by growth factor withdrawal, physical insult, and NF-B inhibition

To further investigate the biologic consequences of Tax expression in ovine B-cells, we examined whether increased viable B-cell numbers were associated with prolonged survival. We first analysed the percentage of apoptotic cells in CD154+IL-4 costimulated cultures by both propidium iodide staining and TUNEL assay. Tax rescued B-cells from spontaneous apoptosis throughout the examined culture period of 72 h, with levels of apoptotic cell death of only 4, 2, and 9% for Clone-2-LTaxSN_L+4 versus 17, 21, and 25% for Clone-2_L+4 after 24, 48, and 72 h, respectively (Figure 3a). These observations suggested that in addition to the impact on B-cell proliferation, Tax might also act through a significant reduction of the level of B-cell death. However, since CD154 and IL-4 signaling have a clear impact on B-cell survival pathways (Wagner et al., 1998; Warren et al., 1999; Van Kooten and Banchereau, 2000; Wick and Berton, 2000), it was necessary to eliminate this interaction in order to appreciate fully the effects of Tax on B-cell death. We thus evaluated the susceptibility to growth factor withdrawal-mediated apoptosis and examined both the viable cell numbers and the levels of apoptotic cell death in the absence of CD154 (Figure 3b). Since parental Clone-2_L+4 B-cells require CD154 stimulation for long-term survival, these experiments were feasible only in short-term cultures. We found that Clone-2-LTaxSN_L+4 viable cell numbers increased throughout the 72-h culture period following CD154 withdrawal, whereas Clone-2_L+4 cell numbers were stationary for 48 h and then significantly decreased (Figure 3b, framed box). The enhanced viable B-cell numbers were correlated with significantly reduced percentages of apoptotic cells with only 5, 7, and 20% for Clone-2-LTaxSN_L+4 versus 28, 45, and 51% for Clone-2_L+4, 24, 48, and 72 h following CD154 withdrawal, respectively (Figure 3b). Despite the profound inhibition of cell proliferation, CD154-deprived Clone-2-LTaxSN_L+4 cells remained largely viable after 1 week in culture. Furthermore, similar experiments conducted after withdrawal of either IL-4 alone or both CD154 and IL-4 confirmed the impact of Tax on B-cell survival (data not shown).

Figure 3.

Tax mediates protection from apoptotic B-cell death induced by growth factor withdrawal, physical insult, and NF-B inhibition. (a) Cultures were initiated with 1.2 10⁶ B-cells/well and costimulated with CD154 and IL-4. Apoptotic cell death in B-cell cultures was examined after 24, 48, and 72 h by TUNEL assay. (b) Cells were cultured in the absence of CD154 and apoptotic cell death was determined as in panel a. The viable B-cell numbers are represented in the framed box. (c) B-cell cultures were -irradiated at doses of 3.4 and 6.8 Gy and then costimulated for 24 h with CD154 and IL-4. Apoptotic b-cell death was determined by cellular DNA profiles and analysed by flow cytometry. (d) B-cells were treated for 6 h with concentrations of 10 or 30 M of Bay-11 and apoptotic cell death was determined by TUNEL assay. Data presented are the means.d. of values of three independent cultures

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We then examined whether Tax might promote resistance to apoptosis induced by physical insult. Gamma irradiation is a potent inducer of apoptotic cell death, caused by DNA damage (Clark et al., 2000; Porter et al., 2000). Clone-2_L+4 and Clone-2-LTaxSN_L+4 were exposed to increasing doses of irradiation and then transferred to fresh medium supplemented with CD154 and IL-4 for 24 h. The percentage of cells undergoing apoptosis was measured by cellular DNA profiles (Figure 3c). Clone-2-LTaxSN_L+4 showed a modest degree of apoptosis with only 3 and 6% of apoptotic cells upon exposure to doses of 3.4 and 6.8 Gy, respectively. In contrast, irradiation caused a significantly higher loss in viability in Clone-2_L+4 with percentages of 15 and 21%, respectively. We found similar ratios when TUNEL was used (data not shown).

Lastly, we investigated whether Tax was capable of reducing B-cell death mediated by Bay-11-7082 (Bay-11), a potent inducer of B-cell apoptosis that acts through the specific inhibition of NF-B (Keller et al., 2000). We treated Clone-2_L+4 and Clone-2-LTaxSN_L+4 with increasing doses of Bay-11 and evaluated the percentages of apoptotic cells by TUNEL assay (Figure 3d). While NF-B inhibition in Clone-2_L+4 induced apoptotic B-cell death in 23 and 60% of B-cells at 10 and 30 M, respectively, we found that Clone-2-LTaxSN_L+4 displayed significantly reduced levels of 4 and 38%, respectively. These observations suggest that Tax is capable of restoring NF-B-driven transcriptional activity in B-cells, thereby leading to increased viability. Altogether, these results clearly demonstrate that Tax mediates prolonged survival of B-cells exposed to apoptotic stimuli such as cytokine withdrawal, irradiation, and NF-B inhibition.

The impact of Tax on B-cell growth is not mediated by secreted factors or mechanisms that involve cell contact

We reasoned that the Tax-associated growth advantage might be mediated by secreted factors released from Clone-2-LTaxSN. To investigate whether there were such factors responsible for altered B-cell growth, we supplemented the culture supernatant of Clone-2_L+4 with 3-day conditioned medium from either Clone-2-LTaxSN_L+4 or Clone-2_L+4. We examined the viable cell numbers at 24, 48, and 72 h in both CD154+IL-4 costimulated and IL-4 deprived cultures and found no significant differences (Table 1). To further address whether cell contact was implicated, we cocultivated Clone-2_L+4 cells with either -irradiated Clone-2-LTaxSN_L+4 cells or Clone-2_L+4 cells, in a medium supplemented with CD154+IL-4 or CD154 alone. Again, we did not observe significant differences in viable B-cell numbers. Finally, the supply of cytokine-independent Clone-2-LTaxSN culture supernatant or coculture with these cytokine-independent B-cells did not support the growth of parental Clone-2 cells in the absence of IL-4 (data not shown). Thus, enhanced B-cell growth and cytokine-independent proliferation were not dependent on secreted growth factors that might have been induced by Tax. Furthermore, our data did not provide any evidence that mechanisms involving cell contact may be implicated in the process.

Table 1 - Enhanced B-cell growth associated with Tax expression is not mediated by secreted factors or mechanisms that involve cell contact.

Full table

Tax expression is accompanied by a higher Bcl-2 protein level

We have demonstrated that Tax expression supported B-cell proliferation and promoted prolonged survival following growth factor withdrawal, physical insult, and NF-B inhibition. The deregulation of pro- and antiapoptotic genes of the Bcl-2 family has been frequently correlated with prolonged lymphocyte survival in vitro and in vivo (Liu et al., 1991; Haury et al., 1993; Motyka and Reynolds, 1995). Furthermore, increased ratios of bcl-2/bax expression were shown to be associated with BLV-induced leukemogenesis in cattle (Reyes and Cockerell, 1998). To assess whether proteins of the Bcl-2 family might be involved in the Tax-mediated rescue signals in ovine B-cells, we examined the expression of proteins that promote (Bax) or protect (Bcl-2, Bcl-x_L) from apoptotic cell death and estimated their relative levels by Western blot. Bax and Bcl-x_L levels did not markedly change upon Tax expression in either CD154+IL-4 costimulated or CD154-deprived cells, but there was significantly more antiapoptotic Bcl-2 protein in Clone-2-LTaxSN_L+4 compared to Clone-2_L+4, with a 3.5-fold increase in costimulated conditions (Figure 4). CD154 withdrawal in itself did not markedly affect the Bax and Bcl-x_L levels, while it provoked a 2.2-fold decrease in the Bcl-2 protein level in Clone-2_L+4. In these conditions, however, Tax expression not only restored but also upregulated the Bcl-2 protein level, as demonstrated by the 6.3-fold increase in Clone-2-LTaxSN_L+4 compared to Clone-2_L+4 (from 0.44 to 2.8). These observations strongly suggest that the upregulation of Bcl-2 in ovine B-cells may mediate rescue signals associated with Tax.

Figure 4.

Tax expression is accompanied by a higher Bcl-2 protein level. Cell lysates were prepared from Clone-2_L+4 and Clone-2-LTaxSN_L+4 cultures 24 h after either CD154+IL-4 costimulation (+/+) or CD154 withdrawal (-/+), and analysed by Western blot ECL immunodetection with antibodies to Bcl-2, Bax, Bcl-x_L, and -tubulin. Equivalents of 2 10⁶ cells were loaded. Densitometric scan values of Bcl-2 bands are indicated as relative levels of ECL immunodetected protein in comparison with the level in costimulated Clone-2_L+4 cells. The blots and densitometric values are shown for one representative experiment from four independent assays. The mean densitometric value of the Bcl-2 protein level in stimulated conditions was 3.2 with a standard deviation of 0.4

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Tax mediates enhanced nuclear NF-kB activity

The NF-B family of transcription factors is involved in the regulation of activation, proliferation, differentiation, and death of numerous cell types including B-cells (Kistler et al., 1998; Horwitz et al., 1999; Keller et al., 2000; Weih et al., 2001). The finding that Tax protects ovine B-cells from apoptotic cell death induced by Bay-11, an NF-B-specific inhibitor suggested that members of the NF-B family may be involved in Tax-associated B-cell survival. To investigate this hypothesis, we examined whether Tax was acting on the nuclear DNA binding activity of NF-B proteins in electrophoretic mobility shift assays (EMSA). Nuclear extracts from CD154 and IL-4 costimulated Clone-2_L+4 cultures displayed significant binding to an NF-B consensus DNA motif, indicating that NF-B was activated in control B-cells and consisted of at least two predominant complexes (Figure 5a). Interestingly, there was a marked increase in nuclear NF-B activity in Clone-2-LTaxSN_L+4 extracts, whereas binding to the Oct-1 consensus sequence occurred at similar levels. Protein/DNA binding was specific for NF-B, as excess unlabeled NF-B oligonucleotide could effectively compete and abrogate binding, whereas mutant oligonucleotide demonstrated no effect. Labeled mutant NF-B DNA motif did not result in complex formation (data not shown). It is well documented that CD154-mediated CD40 triggering in B-cells activates NF-B transcription factors (Kistler et al., 1998). As a consequence, NF-B factors are constitutively present in the nucleus of cultured B-cells. In an effort to exclude the effect of costimulation, we next compared NF-B binding in CD154/IL-4-deprived cultures. As expected, we found that growth factor withdrawal was associated with reduced complex formation in both Clone-2_L+4 and Clone-2-LTaxSN_L+4, but there was clearly higher binding and significant residual NF-B activity in Clone-2-LTaxSN_L+4 (Figure 5a). These findings demonstrate that Tax is capable of partially restoring nuclear NF-B activity in CD154/IL-4-deprived B-cells, suggesting a function in the nuclear translocation of NF-B transcription factors. Finally, we examined the impact of the NF-B-specific inhibitor Bay-11. Although treatment with Bay-11 reduced the nuclear NF-B activity in both Clone-2_L+4 and Clone-2-LTaxSN_L+4, there was clearly less complex inhibition in Clone-2-LTaxSN_L+4, even after a 3-h treatment, whereas binding to the Oct-1 consensus was not modified (Figure 5b). These observations indicate a potential correlation between the higher nuclear NF-B activity observed in B-cells that express Tax and the rescue from Bay-11-mediated B-cell death. In conclusion, our data strongly suggest that Tax-associated protection from apoptotic B-cell death is governed through its capacity to interfere with NF-B-associated signaling pathways.

Figure 5.

Tax mediates increased nuclear NF-B activity in B-cells. (a) Nuclear proteins were extracted from Clone-2_L+4 and Clone-2-LTaxSN_L+4 cells either 48 h after CD154+IL-4 costimulation (+/+) or 48 h after CD154 and IL-4 withdrawal (-/-), and were examined by EMSA for DNA binding using a radiolabeled NF-B consensus oligonucleotide probe. The same extracts were assayed for Oct-1 binding. (b) Clone-2_L+4 and Clone-2-LTaxSN_L+4 cells were either treated with Bay-11 (5 M) for 1 h (+) or cultured without treatment (-). Nuclear extracts were processed as described for panel a

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To determine what members of the NF-B family make up the DNA–protein complexes observed in Clone-2_L+4 and Clone-2-LTaxSN_L+4, EMSA supershift experiments were performed in which antibodies to known NF-B family members were added to the DNA-binding reaction (Figure 6). The only antibody that efficiently shifted all DNA-binding complexes in both Clone-2_L+4 and Clone-2-LTaxSN_L+4 was anti-p50. Furthermore, RelB-specific antibody was clearly reactive as demonstrated by the significant reduction of the second predominant NF-B complex, especially in the presence of Tax, while the other complexes remained unchanged. In contrast, we did not observe any change in complex migration with antibodies to c-Rel and p65, other NF-B members that were shown to be involved in B-cell function. Although these data indicate that p50/p50 homodimers and RelB/p50 heterodimers constitute the major binding complexes in ovine B-cell clones, they do not exclude the possibility of other minor forms of complexes. Taken together, our observations indicate that Tax has an impact on cellular signaling through NF-B with unique consequences on B-cell homeostasis.

Figure 6.

The nuclear NF-B binding complexes are primarily comprised of RelB/p50 heterodimers and p50/p50 homodimers. Nuclear proteins were extracted from Clone-2_L+4 and Clone-2-LTaxSN_L+4 cells 48 h after CD154+IL-4 costimulation and incubated with antibodies to p50, p65, c-Rel, and RelB before the addition of NF-B-specific radiolabeled oligonucleotide. The positions of p50/p50 and RelB/p50 dimers are indicated

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Discussion

In this study, we examined the impact of Tax expression on ovine B-cells, the targets of BLV in experimentally infected sheep, in an attempt to address the role that Tax has in B-cell tumor onset and progression. The most striking observation was the capability of Tax to mediate progression of B-cells from cytokine dependence to cytokine independence. Furthermore, Tax had a clear supportive role in B-cell growth with an impact on both B-cell cycling and survival. Tax was also capable of rescuing B-cells from apoptotic cell death induced by growth factor withdrawal, physical insult, and NF-B inhibition. We provided correlative evidence that higher Bcl-2 protein levels were associated with Tax expression in B-cells. Lastly, we demonstrated that, mechanistically, Tax exerted its effects through the increase of nuclear NF-B activity.

Our results show that in the presence of CD154, Tax expression leads to cytokine-independent growth of ovine B-cell clones. The retroviral-mediated transfer of Tax into B-cells can thus rescue proliferation in the complete absence of signals from the _c-common cytokines. This provides the first experimental evidence that Tax can over-ride the signaling pathways typically controlled by cytokine receptor activation in B-cells. However, CD154 withdrawal did not result in continuous B-cell growth and cells showed signs of arrest, indicating that they were not fully transformed. Thus, Tax is capable of modulating the growth properties of B-cell clones from cytokine dependence to cytokine independence, but CD154 stimulation remains an essential factor for continuous growth. It was previously shown that Tax_HTLV-1 expression in peripheral blood mononuclear cells is sufficient to induce immortalization of T cells, which, however, remain IL-2-dependent, even after extended culture (Grassmann et al., 1992; Akagi et al., 1997). This and our data suggest the involvement of additional factors in ligand-independent transformation for both viruses.

It is clear that normal B-cell cycle control is deregulated during BLV leukemogenesis and our data provide evidence that Tax might be the main protein involved in this process. In our B-cell culture system, Tax had a clear supportive role in B-cell growth. Increased viable B-cell numbers were accompanied by enhanced proliferation, most likely indicating a potential role in DNA synthesis, although so far we cannot rule out a potential impact on DNA repair. While investigating B-cell proliferation, the DNA content of the different B-cell cultures has also been addressed. In the presence of CD154 and IL-4, the effect of Tax expression in B-cell clones was twofold: the cell cycle distribution changed to include a significantly higher proportion of cells in S+G2/M phase, and B-cells furthermore displayed an extended lifespan, as indicated by the significantly reduced levels of apoptotic cell death. A final proof for the role of Tax as a survival factor was provided by experiments conducted with B-cells that were exposed to apoptotic stimuli. Tax rescued B-cells from apoptotic cell death induced by a variety of stimuli, including CD154 withdrawal, physical insult such as irradiation, and treatment with NF-B inhibitors. Our findings suggest that Tax may interfere with the suicide programs of B-cells to extend the lifespan of BLV-infected cells. Prolonged survival may then contribute to both viral persistence and expansion, thereby enhancing the cell susceptibility to additional abnormalities and progression to full malignancy.

We have been unable to demonstrate the implication of potential secreted factors, neither did we identify a mechanism that requires cellular contact. However, we consistently found significantly higher levels of Bcl-2 in the presence of Tax, in both costimulated and deprived conditions. These data suggest that Tax might at least partially exert its effects through the activation of the bcl-2 gene, a proto-oncogene that is frequently expressed in human cancer and leads to prolonged survival of lymphocytes that ordinarily respond to apoptotic signals. Overall, our results clearly indicate that Tax is capable of interacting with cellular pathways that govern the regulation of apoptotic cell death. In this respect, it is interesting to note that Tax was capable of restoring the viability of cells exposed to the NF-B inhibitor Bay-11-7082. Moreover, Tax had a similar impact on the survival of B-cells treated with CAPE and TPCK, which also provoke cell death through impaired NF-B activity (Natarajan et al., 1996; Wu et al., 1996) (data not shown). Altogether, our data provided clear evidence that NF-B activity is necessary for the survival of ovine B-cell clones, and suggest that the impact exerted by Tax on B-cell control might well result from its interaction with NF-B. A critical reason for investigating the Tax-NF-B interplay was thus to understand the contribution of this pathway to B-cell transformation. Members of the NF-B family of transcription factors function pleiotropically in diverse aspects of B-cell growth, differentiation, and death (Kistler et al., 1998; Horwitz et al., 1999; Weih et al., 2001). NF-B was furthermore shown to be critical for the survival of both KSHV- and EBV-transformed B-cells and had a supportive role in cell growth (Cahir-McFarland et al., 2000; Keller et al., 2000). We observed that NF-B was constitutively activated in costimulated ovine B-cell clones and consisted of two predominant complexes, RelB/p50 heterodimers and p50/p50 homodimers, both complexes being strongly upregulated by Tax. Antibodies specific for human p65 and c-Rel were unable to supershift the NF-B complexes, despite the fact that they efficiently recognized ovine p65 and c-Rel in Western blot (data not shown), suggesting that p65 and c-Rel were absent from the nuclear NF-B complexes found in ovine B-cells. RelB was previously shown to be mainly restricted to lymphoid tissue with a strong and predominant nuclear localization in mature B-cells (Carrasco et al., 1993). Interestingly, when coupled to p50, RelB can trigger potent transcriptional activation (Solan et al., 2002). On the other hand, it is often the case that NF-B activation in transformed cell types involves multiple dimer forms including the p50/p50 homodimer (Feinman et al., 1999; Heissmeyer et al., 1999; Keller et al., 2000; Krappmann et al., 2001). Furthermore, although there have been conflicting reports concerning the role p50/p50 plays in transcription, it was recently demonstrated that nuclear p50/p50 homodimer activity in a murine B-cell lymphoma cell line correlated with Bcl-2 expression, indicating that in B-cells p50 homodimers may be capable of transcriptional activation of bcl-2 (Kurland et al., 2001). This and our findings strongly suggest that the impact of Tax on NF-B signaling through the increase of both p50/ p50 and RelB/p50 nuclear activity may be a major contributory mechanism in the activation of cellular genes involved in B-cell homeostatic control. It is well documented that Tax_HTLV-1 increases the nuclear activity of p50/p65, the most frequently observed NF-B form in T cells. Although IB phosphorylation has been defined as a critical regulatory step, there is ongoing debate regarding how Tax_HTLV-1 mediates this effect, with recent evidences linking the transactivator to IB-kinase complex and MAP3Ks (Jeang, 2001). Proof for similar mechanisms in BLV-associated B-cell leukemogenic processes will require further investigation.

In the light of the data presented here and elsewhere, it is tempting to conclude that Tax-associated activation of cellular genes through an NF-B-dependent pathway is essential for tumor progression. On the other hand, several studies support that Tax is not expressed in BLV-associated leukemic cells, suggesting that NF-B activation by Tax is not involved in B-cell malignant transformation. A possible explanation for this discrepancy is that Tax-mediated NF-B activation provides for the initiation of transformation, while it is dispensable to maintain the malignant phenotype. Since in the presence of Tax we saw an increase in nuclear NF-B activity and since we have found upregulation of Bcl-2, it is attractive to speculate on the mechanistic significance of these observations. CD40-mediated survival of human B-cells proceeds through the NF-B-dependent upregulation of Bcl-x_L, an antiapoptotic member of the Bcl-2 family (Lee et al., 1999), and there is extensive data linking NF-B activation to the upregulation of antiapoptotic Bcl-2 family members in different cell types (Kirshenbaum, 2000; Kurland et al., 2001). However, it is not known so far whether the ovine bcl-2 gene is a direct NF-B target gene. The identification of promoter sequences and regulatory B-responsive elements in the ovine bcl-2 counterpart will undoubtedly be the next step in unraveling the mechanisms that link Tax-mediated increase of nuclear NF-B activity and Bcl-2-associated prolonged survival.

Altogether, the data presented here are consistent with a multistep model for malignant B-cell transformation. Although Tax is most likely to be important in the initiation of the process, through its capacity of disrupting normal B-cell homeostatic control, there is compelling evidence that expression of Tax is not sufficient for achieving full malignancy. The subtle changes in B-cell growth, the inability of Tax to transform cells directly, and the need for multiple cooperative changes in growth control mechanisms to induce overt leukemia strongly support this hypothesis. Consistent with these features, BLV causes leukemia after long latency and tumors are clonal. Although Tax is involved in aspects of pathogenesis that are unique to complex retroviruses, the control of Tax expression may have wide-ranging effects that are relevant to other malignancies. This B-cell model therefore provides a unique approach for the investigation of at least two major problems: what is the mechanism by which Tax prolongs the lifespan of B-cells and what additional changes are needed for complete B-cell transformation.

Materials and methods

Cell cultures

Clone-2 (sIgM⁺), Clone-1, and Clone-7 (both sIgG⁺) B-cell clones were established from the jejunal PP of a lamb (Griebel et al., 2000). Cultures were conducted in six-well plates (Falcon), in AIM-V medium (Invitrogen) supplemented with 2% fetal bovine serum (FBS) (Invitrogen), 1 mM sodium pyruvate, 2 mM glutamine, nonessential amino acids, kanamycin (100 g/ml), recombinant human IL-2, -4, -7, and -15 (10 ng/ml) (Peprotech), and -irradiated murine CD154-positive (mCD154) L cells at a 1 : 5 ratio (L cells : B-cells). mCD154 L cells (a gift from Troy Randall) were maintained in OptiMEM medium supplemented with 10% FBS, and -irradiated with 54 Gy (RT-250 therapy generator, Philips, Frankfurt, Germany). B-cell cultures were transferred every 3–4 days to fresh medium, cytokines, and irradiated mCD154 L cells. The ovine leukemia cell lines YR2 and YR2-LTaxSN were maintained in OptiMEM medium supplemented with 10% FBS (Van den Broeke et al., 1999). Retrovirus-producer cell lines PG13LTaxSN and PG13NUNL were used to transduce B-cells by cocultivation as previously described (Van den Broeke et al., 1999). All cultures were incubated at 37°C in 5% CO₂-humidified atmosphere.

Cell viability and proliferation assays

Viable cell numbers were measured directly by trypan blue dye exclusion. Proliferation assays were conducted with 10⁴ cells/well in flat-bottomed 96-well tissue culture plates (Falcon) in a volume of 200 l. At 24, 48, and 72 h after culture initiation, cells were pulsed with BrdU for 4 h and incorporation was quantitated by ELISA (Roche Diagnostics). Apoptotic cell death was assessed by TUNEL assay and DNA profiles. TUNEL was performed according to the manufacturer (Roche Diagnostics) with minor modifications: cells were fixed in 2% paraformaldehyde and stored at -20°C in 70% ethanol before labeling. DNA profiles for both apoptosis and cell cycle analysis were obtained by propidium iodide staining according to the manufacturer (Coulter, Miami, FL, USA). The stained cells were analysed using a FacsCalibur flow cytometer (BD Biosciences). Apoptotic cells were identified as cells with hypodiploid DNA.

Immunoprecipitation and Western blot

B-cells (20 10⁶) were labeled with [³⁵S]methionine (Amersham Pharmacia) for 4 h, lysed in 1 ml of RIPA buffer (50 mM Tris-HCl, 188 mM NaCl, 5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and protease inhibitor cocktail (Roche Diagnostics)) for 30 min at 4°C. Extracts were incubated with a Tax-specific polyclonal antibody (a gift from D Portetelle) for 1 h, followed by incubation with protein A-sepharose CL4B (Amersham Pharmacia Biotech). Immunoprecipitated proteins were analysed on denaturing acrylamide gels followed by autoradiography. For Western blot analysis, 2 10⁶ B-cells were lysed for 30 min at 4°C in 50 l of RIPA buffer, disrupted by passage through a 0.4 mm needle, and centrifuged at 20 000 g for 30 min at 4°C. Supernatants were denatured in SDS–PAGE sample buffer, run on a 15% acrylamide gel, and transferred to Hybond ECL Nitrocellulose (Amersham Pharmacia). Membranes were blocked with 5% blocking agent and probed with antibodies to the following human proteins: Bcl-2 (Dako, Galstrup, Denmark), Bax (Calbiochem, San Diego, CA, USA), Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and -Tubulin (Oncogene, Boston, MS, USA). Detection was performed using HRP-conjugated Ig and chemiluminescence (Amersham Pharmacia).

Electrophoretic mobility shift assays and supershift analyses

Nuclear proteins were isolated and processed according to the procedure described by Badran et al. (2002). Nuclear extracts (10 g) were preincubated for 10 min with 2.5 g poly (dI-dC) (Amersham Pharmacia), then ³²P-labeled oligonucleotide (15 000 c.p.m.) was added for 20 min. NF-B or Oct-1 oligonucleotides (Invitrogen) were end labeled with [-³²P]ATP (>5000 Ci/mmol; Amersham Pharmacia) using T4 polynucleotide kinase (Roche Diagnostics), and purified by gel electrophoresis. For competition, 4 and 20 M excess of unlabeled oligonucleotide was added simultaneously to poly (dI-dC). Samples were separated on 7% acrylamide gels and assessed by autoradiography. For supershift experiments, nuclear proteins were incubated for 1 h at RT with 4 g of human p50 (sc-114), p65 (sc-7151), c-Rel (sc-6955), or RelB (sc-226) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) prior to addition of radiolabeled oligonucleotide.

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Acknowledgements

We thank Troy Randall for the generous gift of mCD154 L cells and Daniel Portetelle for providing the anti-Tax antibodies. This work was supported by funds from the F.N.R.S., the Fonds Medic, the Fondation Bekales, the G.V.P.N., the A.F.M., and the C.I.H.R. MS is supported by an F.R.I.A. F.N.R.S. grant and CVL is Maître de Recherches of the F.N.R.S.