Resistance to TGF-{beta}1 correlates with a reduction of TGF-{beta} type II receptor expression in Burkitt’s lymphoma and Epstein–Barr virus-transformed B lymphoblastoid cell lines

Gareth J. Inman1 and Martin J. Allday1

Section of Virology and Cell Biology and the Ludwig Institute for Cancer Research, Imperial College of Science, Technology and Medicine, St Mary’s Campus, Norfolk Place, London W2 1PG, UK1

Author for correspondence: Martin Allday. Fax +44 20 7724 8586. e-mail m.allday{at}ic.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The pleiotropic cytokine TGF-{beta}1 is a member of a large family of related factors involved in controlling cell proliferation, differentiation and apoptosis. TGF-{beta} ligands interact with a complex of type I and type II transmembrane serine/threonine kinases and they transmit their signals to the nucleus via a family of Smad proteins. A panel of over 20 Burkitt’s lymphoma (BL) cell lines has been compiled including those that are Epstein–Barr virus (EBV) negative, those that carry EBV with a restricted pattern of EBV latent gene expression (group I) and those that express the full range of latent EBV genes (group III), together with selected EBV-transformed lymphoblastoid cell lines (LCLs). Most of the EBV-negative and group I BL cell lines underwent apoptosis or a G1 arrest in response to TGF-{beta}1 treatment. In contrast, group III cell lines and LCLs were completely refractory to these effects of TGF-{beta}1. All of the cell lines expressed the TGF-{beta} pathway Smads and the TGF-{beta} type I receptor. Lack of responsiveness to TGF-{beta}1 appears to correlate with a down-regulation of TGF-{beta} type II receptor expression. Studies of EBV-converted and stably transfected BL cell lines demonstrated that the EBV gene LMP-1 is neither necessary nor sufficient to block the TGF-{beta}1 response.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
TGF-{beta}1 is the prototypical member of a large family of pleiotropic cytokines that exert a wide variety of effects during development and later in adult life, including regulating cell growth, differentiation, apoptosis and matrix organization and biogenesis (Derynck & Feng, 1997 ; Massagué, 1998 ).

Signals from these cytokines are transduced initially by interaction with two different families of cell surface receptors, termed the type I and type II receptors. These receptors are structurally similar, containing small, extracellular cysteine-rich domains and intracellular domains made up mainly of serine/threonine kinase domains. Different TGF-{beta} family ligands interact with distinct combinations of type I and type II receptors to generate signalling specificity (Derynck & Feng, 1997 ; Massagué, 1998 ).

Activation of these receptors has been best characterized in the TGF-{beta} system (Derynck & Feng, 1997 ; Massagué, 1998 ). TGF-{beta}1 binds first to the high-affinity TGF-{beta} type II receptor (TGF-{beta} RII), which is present in the cell membrane as a kinase active homodimer. The type I TGF-{beta} receptor (TGF-{beta} RI), previously present as an inactive homodimer (Gilboa et al., 1998 ), is then recruited by TGF-{beta} RII to form a heterotetrameric complex and also binds TGF-{beta}1. The complex is stabilized further by interaction of the cytoplasmic domains of the TGF-{beta} RII and TGF-{beta} RI (Feng & Derynck, 1996 ). TGF-{beta} RII then phosphorylates and activates the TGF-{beta} RI (Gilboa et al., 1998 ). A third kind of TGF-{beta} receptor, TGF-{beta} RIII, has also been described and it is believed to be involved in ligand presentation to the other two receptors (Derynck & Feng, 1997 ; Massagué, 1998 ).

TGF-{beta} signalling from the cell membrane to the nucleus is mediated by the Smad family of proteins. These can be divided into three different classes: the pathway-restricted Smads, the common-mediator Smads and the inhibitory Smads. Smads 2 and 3 transduce TGF-{beta} and activin signals and are direct substrates for the type I receptor kinases (Heldin et al., 1997 ; Massagué, 1998 ). Following phosphorylation by the type I receptor kinase domains, the pathway-restricted Smads associate to form heterooligomers with each other, homooligomers or heterooligomers with the only common-mediator Smad identified so far, Smad4 (Heldin et al., 1997 ; Massagué, 1998 ). These complexes are probably trimeric in nature and translocate to the nucleus to activate their target genes (Kawabata et al., 1998 ).

Infection of primary B lymphocytes in vitro by Epstein–Barr virus (EBV) readily results in the outgrowth of immortalized lymphoblastoid cell lines (LCLs). EBV in these cells expresses a defined pattern of latent genes, which include the EBV nuclear antigens, EBNA-1, EBNA-2, EBNA-LP, EBNA-3A, EBNA-3B and EBNA-3C, the latent membrane proteins LMP-1, LMP-2A and LMP-2B, two small non-polyadenylated RNAs, EBER1 and EBER2, and the BamHI-A rightward transcripts (BARTs). Genetic analysis has revealed that EBNAs -1, -2, -3A, -3C and -LP and LMP-1 are essential for the efficient immortalization of primary B cells in vitro (Rickinson & Kieff, 1996 ). EBV has also been implicated in the development of human neoplasms, which include nasopharyngeal carcinoma, Hodgkin’s disease, immunoblastic lymphomas in the immunocompromised host and Burkitt’s lymphoma (BL) (Rickinson & Kieff, 1996 ). Freshly isolated BL cell lines and BL biopsies exhibit a so-called group I phenotype (Gregory et al., 1990 ) and are either EBV negative or have a restricted EBV gene expression profile limited to EBNA-1, the EBERs (Rowe et al., 1987 ), LMP-2A and the BARTs (Tao et al., 1998 ). During cultivation in vitro, group I EBV-positive BL cell lines sometimes ‘drift’ to the group III phenotype, expressing the full complement of EBV latent genes expressed in LCLs (Gregory et al., 1990 ; Rowe et al., 1987 ).

Several other groups have previously investigated the effect of TGF-{beta}1 on BL cell lines and LCLs (Bauer et al., 1982 ; Blomhoff et al., 1987 ; Kehrl et al., 1989 ; Kumar et al., 1991 ; Schuster et al., 1991 ; Gauchat et al., 1992 ; Altiok et al., 1993 ; di Renzo et al., 1994 ; Arvanitakis et al., 1995 ; Chaouchi et al., 1995 ; Beckwith et al., 1995 ; MacDonald et al., 1996 ; Saltzman et al., 1998 ; Schrantz et al., 1999 ). These studies have revealed that TGF-{beta} negatively regulates the growth of most of the B cell lines studied and that these effects are not apparent in group III BLs and LCLs. TGF-{beta}1 has also been shown to induce apoptosis in the BL41, Ramos and L3055 BL cell lines (Chaouchi et al., 1995 ; MacDonald et al., 1996 ; Saltzman et al., 1998 ; Schrantz et al., 1999 ). The mechanisms for these effects and their elimination by EBV has remained controversial, however, and in some cases the results are even contradictory (Kumar et al., 1991 ; Arvanitakis et al., 1995 ). In order to clarify some of the issues raised in these studies, we assembled a panel of BL cell lines and LCLs and investigated the biological effects of TGF-{beta}1. We demonstrate that most group I and EBV-negative BL cell lines respond to TGF-{beta} signalling by undergoing apoptosis or growth arrest in the G1 phase of the cell cycle. We show that all cell lines that express the full complement of EBV latent genes are resistant not only to the anti-proliferative effects of TGF-{beta}1, as found previously, but also to the apoptotic effects of this cytokine. We also found that four group I BL cell lines are also resistant to these effects of TGF-{beta}1. Our results indicate that lack of responsiveness does not involve loss of Smad gene expression, but correlates with a down-regulation of the TGF-{beta} RII, which is not due to mutation in the microsatellite instability mutation hot spot. Furthermore, analysis of EBV-converted and stably transfected BL cell lines demonstrated that expression of LMP-1 is not sufficient or necessary to block the TGF-{beta}1 response.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell culture and TGF-{beta}1 treatment.
The Ramos BL cell line and the EBV-converted lines AW-Ramos and EHRB-Ramos were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated foetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin (all from Gibco-BRL). All other cell lines were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated Serum Supreme (BioWhittaker), 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. All cells were maintained at 37 °C in a 10% CO2 humidified incubator. The C-LCL and K-LCL cell lines were derived by in vitro infection with the B95.8 strain of EBV of human primary B cells isolated from peripheral blood and represent separately isolated pools of infected cells (a kind gift of Lindsay Spender, Ludwig Institute for Cancer Research, St Mary’s Hospital, London). They were used at a passage number of less than 20 in all experiments.

Recombinant human TGF-{beta}1 (R&D Systems) was rehydrated in a 4 mM HCl, 1 mg/ml BSA solution at a concentration of 2 µg/ml and used at a final concentration of 5 ng/ml in all experiments except the titration analysis. Control cultures were treated with the appropriate equivalent volume of the TGF-{beta}1 rehydration buffer. For experimental analysis, cells were diluted to a concentration of 3x105 cells/ml 24 h prior to manipulation.

{blacksquare} Proliferation assays.
Aliquots (200 µl) of appropriately treated cells were seeded into 96 well plates and incubated at 37 °C for the period of time indicated. Cells were then pulsed for 2 h with 1 µCi [3H]thymidine (Amersham) and harvested onto glass fibre filters (Camo) by using a Skatron cell harvester. Filters were air-dried and radioactivity was quantified by scintillation counting.

{blacksquare} Cell cycle analysis.
Cell cycle analysis was performed by flow cytometry. Cells were harvested by centrifugation, washed in ice-cold PBS and fixed in 80% ethanol that had been pre-chilled to -20 °C. Fixed cells were stored at 4 °C for up to 1 week. Cells were then repelleted and resuspended at a concentration of approximately 1x106 cells/ml in PBS containing 18 µg/ml propidium iodide and 8 µg/ml RNase A (both from Sigma). After incubation in the dark for at least 1 h, cell cycle profile analysis was performed on 10000–20000 cells on a FACSort flow cytometer by using the Cellquest analysis program (Becton Dickinson).

{blacksquare} Protein content estimation and Western blotting.
Cells were lysed in RIPA lysis buffer (50 mM Tris–HCl, pH 8·0, 150 mM NaCl, 1% NP-40, 0·5% sodium deoxycholate, 0·1% SDS) supplemented with 1 mM PMSF (Sigma) and Complete protease inhibitor cocktail (Boehringer Mannheim). Protein concentration was estimated spectrophotometrically at 750 nm in a Lambda Bio UV/Vis spectrometer (Perkin Elmer) by using the Bio-Rad detergent-compatible assay, exactly as described by the manufacturer. Protein was diluted to a concentration of 2 mg/ml and diluted further in an equal volume of 2x SDS protein sample buffer [60 mM Tris–HCl, pH 6·8, 2% (w/v) SDS, 20% (v/v) glycerol, 2% (v/v) {beta}-mercaptoethanol, bromophenol blue] and loaded onto 7·5 or 10% SDS–PAGE gels. The Western blotting process was carried out as described previously (Allday & Farrell, 1994 ) and proteins were visualized by ECL chemiluminescence (Amersham) as described by the manufacturer. Autoradiograms were then scanned and processed by using a UMAX PowerLook III scanner and Adobe Photoshop software.

{blacksquare} Antibodies.
Sheep anti-mouse Ig conjugated to horseradish peroxidase (HRP) (Amersham), goat anti-rabbit Ig–HRP (Dako), anti-Smad2 MAb (Transduction Laboratories), anti-LMP-1 MAb S12 (Mann et al., 1985 ), anti-cyclin D2 MAb (G132-43, Pharmingen), anti-poly(ADP-ribose) polymerase (PARP) polyclonal antibody (Boehringer Mannheim), anti-TGF-{beta} RI polyclonal antibody (V-22, Santa Cruz Biotechnology) and anti-TGF-{beta} RI polyclonal antibody VPN (Franzen et al., 1993 ) were all used as recommended by the suppliers.

{blacksquare} RT–PCR.
Total RNA was prepared by using RNAzol B (Biogenesis) according to the manufacturer’s instructions. First-strand cDNA was prepared from 1 µg total RNA by using the AMV reverse transcriptase system (Promega) exactly as described by the manufacturer. Ten per cent of this cDNA was used in the PCR assays. Primer sequences and PCR conditions for Smads 2, 3 and 4 and the control ribosomal protein 36B4 (Rich & Steitz, 1987 ) can be obtained from the authors.

{blacksquare} Northern blotting.
Twenty µg of each total RNA sample was separated on 1% agarose gels and transferred to nitrocellulose. The filters were hybridized under standard conditions (Sambrook et al., 1989 ). Gel-purified probes were labelled with 32P by random priming of full-length cDNA fragments of TGF-{beta} RI (Franzen et al., 1993 ) and TGF-{beta} RII (Lin et al., 1992 ) by using the Rediprime II system (Amersham) exactly as described by the manufacturer. Probes were then purified on NICK columns (Pharmacia Biotech) before use.

{blacksquare} 125I-TGF-{beta}1 chemical cross-linking.
125I-TGF-{beta}1 (Amersham) chemical cross-linking was performed essentially as described previously (Franzen et al., 1993 ). Cells (1x107) were washed twice in ice-cold PBS-B (PBS containing 0·9 mM CaCl2, 0·49 mM MgCl2 and 1 mg/ml BSA) and resuspended at a concentration of 1x106 cells/ml in PBS-B containing 2·3 µCi 125I-TGF-{beta}1. Cells were then incubated on ice for 3 h with shaking and then washed twice in PBS-B and once in PBS-B without BSA. Next, cells were resuspended at 2x106 cells/ml in PBS-B without BSA supplemented with 0·28 mM disuccinimidyl suberate cross-linking reagent (DSS, Pierce) and incubated on ice with shaking for 30 min. Cross-linking was stopped by washing in detachment buffer (10 mM Tris–HCl, pH 7·4, 1 mM EDTA, 10% glycerol, 0·3 mM PMSF). Cell pellets were then lysed in 500 µl lysis buffer (125 mM NaCl, 10 mM Tris–HCl, pH 7·4, 1 mM EDTA, 1% Triton X-100, 0·3 mM PMSF, 1% Trasylol) for 40 min on ice. Lysates were sonicated for 5 min at 4 °C and centrifuged at 13000 g for 15 min at 4 °C. Ten µl of VPN antiserum was then added to the supernatants, which were incubated overnight at 4 °C with rotation. Forty µl protein A–Sepharose lysis buffer slurry was added and lysates were incubated for 30 min at 4 °C with rotation. The protein A–Sepharose beads were washed three times in lysis buffer and then resuspended in 40 µl 2x SDS protein sample buffer. Samples were then analysed by 10% SDS–PAGE. Gels were dried onto Whatman 3MM paper and analysed by phosphorimaging by using the Storm 850 phosphorimager and Imagequant software (Molecular Dynamics).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
TGF-{beta}1 inhibits DNA synthesis in most EBV-negative and group I BL cell lines, but not in LCLs or group III BL cell lines
In order to investigate the biological effects of TGF-{beta}1 on human B cell lines, we assembled a panel of LCL and BL cell lines (see Table 1).


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Table 1. Effects of TGF-{beta}1 on cell lines used in this study

 
Titration analysis employing the TGF-{beta}1-sensitive cell line L3055 (MacDonald et al., 1996 ) indicated that concentrations of TGF-{beta}1 of 0·1 ng/ml or higher resulted in a marked decrease in [3H]thymidine incorporation after 12 h treatment (data not shown). TGF-{beta}1 was used at 5 ng/ml throughout the remainder of the study on the panel of BL cell lines. Six of seven EBV-negative BL cell lines exhibited a marked decrease in DNA synthesis compared with control cultures, which continued to proliferate (Table 1; examples shown in Fig. 1a). Similarly, seven of nine group I BL cell lines were responsive to TGF-{beta}1 treatment in this assay (Table 1; examples shown in Fig. 1a). Treatment of the EBV-negative BL DG75, the high-grade lymphoma BJAB and the group I Mak-I and Akata cell lines had no effect on DNA synthesis. TGF-{beta}1-treated and control cultures of these cell lines continued to proliferate at the same rate (Table 1; examples shown in Fig. 1b). In contrast to these findings, we found that all LCLs, EBV-converted and group III BL cell lines were resistant to TGF-{beta}1-mediated inhibition of DNA synthesis (Table 1; examples shown in Fig. 1b).



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Fig. 1. DNA synthesis responses to TGF-{beta}1 treatment. DNA synthesis was measured in the presence ({diamondsuit}) or absence ({blacksquare}) of TGF-{beta}1 by pulsing the cells with 1 µCi [3H]thymidine for 2 h at the times indicated, as described in Methods. Assays were performed in triplicate and standard errors are shown. Each assay was performed at least three times and representative examples are shown. (a) Examples of cell lines responsive to TGF-{beta}1. (b) Examples of cell lines that did not respond to TGF-{beta}1 in this assay.

 
TGF-{beta}1 induces apoptosis or G1 arrest in sensitive BL cell lines and both of these effects are blocked by full EBV latent gene expression
TGF-{beta}1 typically causes a G1 arrest in epithelial cells (Polyak, 1996 ) and primary B cells stimulated with mitogens (Smeland et al., 1987 ). In order to monitor the effects of TGF-{beta}1 on the cell cycle in our panel of cell lines, flow cytometric analysis (FACS) was performed. Treatment of the group I Chep-BL cell line with TGF-{beta}1 for 48 h resulted in a pronounced G1 arrest (Fig. 2a). The increase in the number of cells present in the G1 phase was accompanied by a drop in the number of cells present in the S and G2/M phases of the cell cycle (Fig. 2a). This TGF-{beta}1-mediated G1 arrest was also observed in the EBV-negative CA46 cell line (data not shown; see Table 1). Treatment of the EBV-negative BL41 and L3055 and the group I BL MUTU-I cell lines with TGF-{beta}1 for 48 h resulted in a dramatic increase in the number of cells containing a sub-G1 DNA content, accompanied by a corresponding drop in the number of cells present in the G2/M and S phases of the cell cycle (Fig. 2a). These effects were also observed in all the other cell lines that were found to be sensitive to TGF-{beta}1 in the DNA synthesis assay (see Table 1). In contrast to these findings, we observed that all cell lines tested that did not respond in the DNA synthesis assay exhibited no changes in cell cycle profile following TGF-{beta}1 treatment (Table 1 and examples shown in Fig. 2b).



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Fig. 2. Cell cycle analysis. Samples of the cell lines indicated were analysed for DNA content by FACS analysis. The percentages of cells in each phase of the cell cycle are shown. Assays were performed at least twice and representative examples are shown. (a) TGF-{beta}1-sensitive cell lines. (b) TGF-{beta}1-resistant cell lines.

 
The appearance of a sub-diploid distribution in FACS analysis is generally characteristic of cells undergoing apoptosis (Allday et al., 1995 ; Milner et al., 1998 ). Recent work from many labs has demonstrated that the cysteine proteases of the ICE/CED3 family (the caspases) play a critical role in the apoptotic process (Cryns & Yuan, 1998 ). Activation of CPP32 (caspase 3) is increased markedly in many cells undergoing apoptosis and the cleavage of one of its substrates, PARP, has been used as an indicator of its activity (Lazebnik et al., 1994 ). Cell extracts prepared from cultures of the panel of cell lines with and without treatment with TGF-{beta}1 for 48 h were analysed for cleavage of PARP by Western blotting (data summarized in Table 1; examples shown in Fig. 6). We found a perfect correlation between the appearance of the 89 kDa C-terminal PARP fragment and the appearance of a sub-diploid distribution in FACS analysis (Table 1), confirming that TGF-{beta}1 can induce apoptosis in these cell lines. Consistent with the observation that TGF-{beta}1 induced G1 arrest in the CA46 and Chep-BL cell lines, PARP cleavage analysis revealed no evidence of TGF-{beta}1-induced apoptosis (Table 1). Once again, we observed that the BL41-B95.8 cell line and the group III BLs and LCLs were resistant to the effects of TGF-{beta}1, since no PARP cleavage was visible following treatment of these cell lines with this cytokine (Table 1). This demonstrated clearly that full latent EBV gene expression correlates with a blockage in the TGF-{beta}1-mediated apoptotic response.



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Fig. 6. LMP-1 is neither sufficient nor necessary to block the TGF-{beta}1 response. (a)–(c) Samples of the cell lines indicated were washed, fixed and resuspended in a propidium iodide–RNase A–PBS solution after 48 h incubation in the presence (+TGF-{beta}1) or absence (-TGF-{beta}1) of 5 ng/ml TGF-{beta}1. DNA content was measured by FACS analysis. The percentages of the cells in each phase of the cell cycle are shown. Assays were performed at least twice and representative examples are shown. (d) Total cellular RIPA lysates were prepared from the cell lines indicated after incubation for 48 h in the presence (+) or absence (-) of 5 ng/ml TGF-{beta}1. Fifty µg of each lysate was separated by 10% SDS–PAGE and Western blotting analysis was performed for PARP, LMP-1 and cyclin D2 as indicated.

 
LCLs, EBV-negative, group I and group III BL cell lines express Smad2, Smad3 and Smad4
Since some of the cell lines were resistant to TGF-{beta}-mediated signalling, we sought to identify the mechanism(s) of this resistance by studying components of the TGF-{beta} signal transduction pathway. Smad2, Smad3 and Smad4 have been demonstrated to be key intracellular signalling molecules in the TGF-{beta}1 signal transduction pathway (Massagué, 1998 ; Heldin et al., 1997 ). Mutation or deletion of Smad2 and Smad4 [which occurs frequently in the C-terminal Smad homology region 2 (MH2) of these Smads] has been observed in some human tumours and has been demonstrated to block TGF-{beta}1 responses (Heldin et al., 1997 ). In order to determine whether Smads involved in TGF-{beta}1-mediated signalling were expressed in the panel of cell lines, Western blotting analysis with an anti-Smad2 MAb and RT–PCR analysis designed to amplify the MH2 domain of Smad2, Smad3 and Smad4 was performed. All the cell lines tested were found to express these Smads (data summarized in Table 2).


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Table 2. Expression of TGF-{beta} pathway components

 
TGF-{beta}1 sensitivity correlates with TGF-{beta} RII expression levels
TGF-{beta} RI acts immediately upstream of the Smad proteins. Recently, it has been demonstrated that genetic or expression defects in TGF-{beta} RI and TGF-{beta} RII can be responsible for TGF-{beta} resistance (Massagué, 1998 ). Kumar et al. (1991) have reported that LCLs may have reduced levels of TGF-{beta} RI and TGF-{beta} RII expression; however, this is controversial, since functional levels of expression have also been reported (Arvanitakis et al., 1995 ).

Chemical cross-linking analysis with 125I-TGF-{beta}1 was used to determine the cell surface expression levels of the TGF-{beta} receptors. We observed a correlation between overall TGF-{beta} receptor cell surface expression levels and TGF-{beta} sensitivity (Fig. 3; Tables 1 and 2). The TGF-{beta}1-sensitive cell lines CA46, BL41 and MUTU-I showed clearly detectable levels of TGF-{beta} RI, TGF-{beta} RII and TGF-{beta} RIII. The TGF-{beta}-resistant BL cell lines DG75 and MUTU-III and the high-grade lymphoma BJAB exhibited a marked down-regulation of expression of all these receptors (Fig. 3). Strikingly, the Akata cell line had no detectable expression of any of the TGF-{beta} receptors. The LCLs PD-LCL, JM-SAV-LCL and K-LCL also have very low levels of expression of these receptors. The TGF-{beta}1-resistant MUTU-III group III BL cell line was derived from the TGF-{beta}1-sensitive MUTU-I cell line, following a phenotypic drift in culture due to switching on of the full latent EBV gene expression programme (Gregory et al., 1990 ). The cross-linking analysis demonstrated clearly that a down-regulation of TGF-{beta} receptor cell surface expression accompanies this change in EBV gene expression (Fig. 3). Similarly, conversion of the EBV-negative BL cell line BL41 to a group III phenotype by infection with EBV to generate the BL41-B95.8 cell line results in a similar reduction in TGF-{beta} receptor expression (Fig. 3).



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Fig. 3. Cell surface TGF-{beta} receptor expression. TGF-{beta} receptor complexes were analysed by cross-linking 125I-TGF-{beta}1 chemically to the cell surface membranes of the cell lines indicated. The three TGF-{beta} receptor complexes (RI, RII and RIII) are indicated and were visualized by phosphorimaging.

 
In an attempt to delineate the mechanisms responsible for this reduction in cell surface TGF-{beta} receptor expression, Northern and Western blotting analysis were performed. All cell lines were found to express similar amounts of TGF-{beta} RI mRNA, as judged by Northern blotting (Table 2; examples shown in Fig. 4a). Western blotting analysis with a TGF-{beta} RI-specific antiserum (V-22, Santa-Cruz) was consistent with these data, showing that all the cell lines tested expressed TGF-{beta} RI protein (Table 2; examples shown in Fig. 4b). We observed a slight reduction in expression in the X50-7 LCL, PD-LCL, BL72 and BL41-B95.8 cell lines relative to the other cell lines tested (Fig. 4b).



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Fig. 4. TGF-{beta} RI expression. (a) Northern blot analysis of TGF-{beta} RI expression. Twenty µg total RNA prepared from cycling cultures of the cell lines indicated was separated by 1% agarose gel electrophoresis, transferred to nitrocellulose filters and probed with [32P]dCTP-labelled full-length TGF-{beta} RI cDNA. (b) Western blot analysis of TGF-{beta} RI expression. Total cellular RIPA lysates were prepared from the cell lines indicated. Fifty µg of each lysate was separated by 10% SDS–PAGE and subjected to Western blot analysis with a polyclonal anti-TGF-{beta} RI antiserum.

 
It has not been possible to detect TGF-{beta} RII-specific protein expression reliably by Western blot analysis with commercially available antibodies (data not shown). However, Northern blot analysis for TGF-{beta} RII RNA revealed a striking correlation between TGF-{beta}1 sensitivity and levels of this mRNA. All TGF-{beta}1-sensitive cell lines showed readily detectable TGF-{beta} RII transcripts (Fig. 5). The TGF-{beta}-resistant high-grade lymphoma BJAB and BL cell lines DG75, Akata and BL72 showed markedly reduced TGF-{beta} RII expression. Similarly, all LCLs had very low levels of this transcript. Comparisons between BL41 and BL41-B95.8 and MUTU-I and MUTU-III again indicated that a switch to full latent gene expression correlates with down-regulation of TGF-{beta} RII at the RNA level (Fig. 5). These changes were not due to uneven loading, as shown by rRNA in the ethidium bromide-stained gels (Fig. 5). Generally, sensitivity to TGF-{beta}1 appears to correlate well with the expression of the type II receptor (see Tables 1 and 2). The exception is the group I Mak-I cell line, which was insensitive to the effects of TGF-{beta}1 but appeared to express normal levels of TGF-{beta} RI and TGF-{beta} RII at the RNA, protein and cell surface levels (Figs 3–5; Tables 1 and 2). Experiments are in progress to determine the reason for TGF-{beta}1 resistance in this cell line.



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Fig. 5. TGF-{beta} RII expression. Two examples of Northern blot analysis of TGF-{beta} RII expression are shown (a, b). Twenty µg total RNA prepared from cycling cultures of the cell lines indicated was separated by 1% agarose gel electrophoresis, transferred to nitrocellulose filters, probed with [32P]dCTP-labelled full-length TGF-{beta} RII cDNA and exposed to autoradiography. Loading levels of RNA on the Northern blots were determined by staining the agarose gels with ethidium bromide. Photographs of these gels are shown below their corresponding Northern blots.

 
The polyadenine repeat of the TGF-{beta} RII gene is not mutated in TGF-{beta}1-resistant cell lines
The TGF-{beta} RII gene contains a 10 bp polyadenine repeat in the coding region of the extracellular domain at nucleotide 709. This region has been demonstrated to contain one or two base-pair deletions in most RER-positive sporadic and HNPCC colon and gastric tumours (Massagué, 1998 ). Tumours and cell lines containing these mutations have been shown to contain reduced levels of TGF-{beta} RII RNA (Markowitz et al., 1995 ; Jiang et al., 1997 ), which are due to a decrease in RNA stability (Jiang et al., 1997 ). Recently, it was shown that the DG75 cell line exhibits a microsatellite instability phenotype that results in mutation of the pro-apoptotic bax gene (Brimmell et al., 1998 ). Genomic DNA was isolated from the Akata, DG75, BJAB, X50-7, BL30 and L3055 cell lines and exon 3 of TGF-{beta} RII (which contains the polyadenine repeat; Takenoshita et al., 1996 ) was amplified by PCR. Direct DNA sequencing of the PCR products showed that none of these cell lines contained mutations in the polyadenine repeat (data not shown).

LMP-1 is not necessary or sufficient to block the TGF-{beta}1 response
It has been reported previously that LMP-1 can block the TGF-{beta}1-mediated induction of growth arrest in stably transfected BL41 cells (Arvanitakis et al., 1995 ). In order to investigate this further, we tested the effects of TGF-{beta}1 on the same BL41 cell clones (BL41MTLM5 and BL41MTLM11) and on BL41 cells infected with wild-type EBV (BL41-B95.8) or infected with the P3HR1 virus strain (BL41-P3HR1), which lacks expression of EBNA-2 and LMP-1. This study was extended further to include the Ramos cell line and two P3HR1 EBV-converted versions of this cell line, EHRB-Ramos and AW-Ramos. FACS analysis and Western blotting analysis for PARP cleavage demonstrated that the BL41, BL41gpt2 (empty vector control), BL41MTLM5, BL41MTLM11 and Ramos cell lines all exhibited an apoptotic response after 48 h TGF-{beta}1 treatment (Fig. 6ad). As shown previously, the BL41-B95.8 cell line was completely refractory to TGF-{beta}1. Interestingly, the BL41-P3HR1 cell line exhibited a G1 arrest in response to TGF-{beta}1, implying that restricted EBV latent gene expression is sufficient to block the apoptotic response to TGF-{beta}1. TGF-{beta}1 treatment had no effect on the EHRB-Ramos or AW-Ramos cell lines. Western blot analysis showed that apoptosis in response to TGF-{beta}1 was not blocked by LMP-1 expression alone but was blocked in P3HR1-infected cell lines, which do not express LMP-1 (Fig. 6). It has also been reported that LMP-1 expression can induce cyclin D2 in the BL41 cell line and that this is sufficient to alleviate the anti-proliferative effects of TGF-{beta}1 (Arvanitakis et al., 1995 ). Western blotting showed that none of these cell lines expressed cyclin D2 protein (Fig. 6d), as has been demonstrated by others at the mRNA level by RT–PCR (Pokrovskaja et al., 1996 ).


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The pleiotropic cytokine TGF-{beta}1 exerts a diverse range of effects on different cell types, inhibiting proliferation in some and inducing apoptosis in others (Massagué, 1998 ; Heldin et al., 1997 ; Polyak, 1996 ). Perturbation of the TGF-{beta}1 signal transduction pathway has been implicated in the development of many human cancers, notably those of the pancreas and gastrointestinal tract (Massagué, 1998 ). Studies in knockout mice have also indicated that genes involved in TGF-{beta}1 signalling can act as tumour suppressors in this organism (Zhu et al., 1998 ; Takaku et al., 1998 ).

Previous reports have suggested that some BL cell lines may retain responsiveness to TGF-{beta}1 signalling and undergo a reduction in DNA synthesis (Kumar et al., 1991 ; Altiok et al., 1993 ; Arvanitakis et al., 1995 ; Beckwith et al., 1995 ; Smeland et al., 1987 ) and that the BL41 (Chaouchi et al., 1995 ; Schrantz et al., 1999 ), Ramos (Saltzman et al., 1998 ) and L3055 (MacDonald et al., 1996 ) cell lines undergo apoptosis in response to this cytokine. We have found that most BL cell lines that retain the phenotype of tumour biopsies (EBV-negative and group I with respect to EBV gene expression) respond to TGF-{beta}1 signalling by a reduction in DNA synthesis and that the majority of these undergo apoptosis following TGF-{beta} treatment. These tumour cell lines may retain the phenotypic response of some normal B cells, which have been reported to undergo apoptosis in response to TGF-{beta}1 when isolated from peripheral blood (Lomo et al., 1995 ; Douglas et al., 1997 ). CA46 and Chep-BL cell lines undergo a G1 arrest in response to TGF-{beta}1. This G1 arrest has been observed previously in CA46 cells (Beckwith et al., 1995 ). The lack of an apoptotic response in CA46 cells is probably due to a defect in the apoptotic pathway, since CA46 cells have also been observed to be resistant to apoptosis induced by anti-IgM treatment (Kaptein et al., 1996 ) and cisplatin (our unpublished observations). Indeed, it has been shown recently that CA46 cells have a mutated bax gene (Gutierrez et al., 1999 ), which may indicate that bax could be involved in TGF-{beta}1-mediated apoptosis in BL cell lines. Chep-BL cells undergo apoptosis in response to ionizing radiation (Milner et al., 1997 ), which indicates that the induction of the apoptotic pathway by TGF-{beta}1 may involve a separate mechanism. Indeed, it is interesting to note that, in some myeloid leukaemia cell lines, induction of apoptosis by TGF-{beta}1 is p53 independent (Selvakumaran et al., 1994 ). This is also clearly the case in BL cells, as induction of apoptosis by TGF-{beta}1 in these cells does not correlate with p53 status (Farrell et al., 1991 ).

The finding that the majority of BL cell lines undergo apoptosis in response to TGF-{beta} stimulation provides an interesting contrast to murine plasmacytomas induced in susceptible BALB/c mice. All of these tumours exhibit a functional loss of TGF-{beta} receptor expression (Amoroso et al., 1998 ). Such lines are often described as a murine equivalent of BL and, like BL cell lines, are B cells in origin and contain a deregulated c-myc gene. However, clearly unlike BL, their evolution requires inhibition of the TGF-{beta} response. Loss of susceptibility to TGF-{beta} may play some part in the generation of some BL tumours, as we found that the DG75, Akata and Mak-I cell lines are completely refractory to the growth-inhibitory effects of TGF-{beta}1. Resistance of Akata cells to TGF-{beta} has also been reported by others (di Renzo et al., 1994 ).

We observed that all BL cell lines and LCLs that express the full complement of EBV latent genes were not only resistant to TGF-{beta}1-mediated inhibition of DNA synthesis, as noted previously by others (see Introduction), but they were also completely refractory to the TGF-{beta}1-mediated induction of apoptosis. Measurement of TGF-{beta}1 cell surface receptor expression indicated that, in these cells and the DG75, Akata and BJAB cell lines, TGF-{beta}1 resistance could be due to down-regulation of TGF-{beta} RI, TGF-{beta} RII and TGF-{beta} RIII. However, these cells express normal amounts of TGF-{beta} RI RNA and protein, but exhibit a marked decrease in TGF-{beta} RII RNA expression. These data are consistent with binding of 125I-TGF-{beta}1 to TGF-{beta} RII (Wrana et al., 1994 ). If TGF-{beta} RII is down-regulated, TGF-{beta} RI and TGF-{beta} RII will not be detected on the cell surface in 125I-TGF-{beta} cross-linking assays. Similar results were obtained with RER-positive gastrointestinal tumours (Markowitz et al., 1995 ; Jiang et al., 1997 ). In these gastrointestinal tumours, it was shown that the TGF-{beta} RII RNA is destabilized because of small deletions or additions in the microsatellite polyadenine tract in exon 3 of this gene (Jiang et al., 1997 ); however, a similar mechanism is unlikely to be operating in BL cell lines, since no mutations were detected.

The correlation of EBV latent gene expression with TGF-{beta} RII RNA levels suggests that it is likely that the actions of one or more EBV latent proteins may lead to down-regulation of TGF-{beta} RII. Sharma and co-workers have suggested that LMP-1 gene expression is responsible for blocking TGF-{beta} signalling by up-regulating cyclin D2, but that this does not correlate with TGF-{beta} RII expression (Arvanitakis et al., 1995 ). This was in contrast to a previous report (Kumar et al., 1991 ) and the data presented here, which show clearly that down-regulation of TGF-{beta} RII correlates with lack of sensitivity to TGF-{beta}1. We found that LMP-1 could not block TGF-{beta}1 responses or up-regulate cyclin D2 by itself and, indeed, that infection of the TGF-{beta}1-sensitive Ramos cell lines with P3HR1 virus, which lacks LMP-1 expression, was sufficient to block TGF-{beta}-induced responses without inducing cyclin D2. P3HR1 virus infection appears, at least in part, to inhibit responses to TGF-{beta}1. We are currently investigating whether this could be due to the action of one or more of the limited number of latent virus genes expressed by this virus.


   Acknowledgments
 
We would like to thank Gillian Parker for technical assistance. We are very grateful to Chris Gregory, Alan Rickinson and Lindsay Spender for providing us with cell lines. We are also indebted to Serhiy Souchelnytskyi, Peter ten Dijke and Carl-Henrik Heldin for providing us with the VPN antiserum, TGF-{beta} RI and TGF-{beta} RII cDNAs and invaluable advice. This work was supported by Wellcome Trust project grants 047383 and 050096 to M.J.A.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
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Received 5 October 1999; accepted 2 February 2000.