Section of Virology and Cell Biology and the Ludwig Institute for Cancer Research, Imperial College of Science, Technology and Medicine, St Marys 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- 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- system (Derynck & Feng, 1997
; Massagué, 1998
). TGF-
1 binds first to the high-affinity TGF-
type II receptor (TGF-
RII), which is present in the cell membrane as a kinase active homodimer. The type I TGF-
receptor (TGF-
RI), previously present as an inactive homodimer (Gilboa et al., 1998
), is then recruited by TGF-
RII to form a heterotetrameric complex and also binds TGF-
1. The complex is stabilized further by interaction of the cytoplasmic domains of the TGF-
RII and TGF-
RI (Feng & Derynck, 1996
). TGF-
RII then phosphorylates and activates the TGF-
RI (Gilboa et al., 1998
). A third kind of TGF-
receptor, TGF-
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- 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-
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 EpsteinBarr 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, Hodgkins disease, immunoblastic lymphomas in the immunocompromised host and Burkitts 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-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-
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-
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-
1. We demonstrate that most group I and EBV-negative BL cell lines respond to TGF-
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-
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-
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-
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-
1 response.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant human TGF-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-
1 rehydration buffer. For experimental analysis, cells were diluted to a concentration of 3x105 cells/ml 24 h prior to manipulation.
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.
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 1000020000 cells on a FACSort flow cytometer by using the Cellquest analysis program (Becton Dickinson).
Protein content estimation and Western blotting.
Cells were lysed in RIPA lysis buffer (50 mM TrisHCl, 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 TrisHCl, pH 6·8, 2% (w/v) SDS, 20% (v/v) glycerol, 2% (v/v) -mercaptoethanol, bromophenol blue] and loaded onto 7·5 or 10% SDSPAGE 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.
Antibodies.
Sheep anti-mouse Ig conjugated to horseradish peroxidase (HRP) (Amersham), goat anti-rabbit IgHRP (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-
RI polyclonal antibody (V-22, Santa Cruz Biotechnology) and anti-TGF-
RI polyclonal antibody VPN (Franzen et al., 1993
) were all used as recommended by the suppliers.
RTPCR.
Total RNA was prepared by using RNAzol B (Biogenesis) according to the manufacturers 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.
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-
RI (Franzen et al., 1993
) and TGF-
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.
125I-TGF-
1 chemical cross-linking.
125I-TGF-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-
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 TrisHCl, 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 TrisHCl, 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 ASepharose lysis buffer slurry was added and lysates were incubated for 30 min at 4 °C with rotation. The protein ASepharose 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% SDSPAGE. Gels were dried onto Whatman 3MM paper and analysed by phosphorimaging by using the Storm 850 phosphorimager and Imagequant software (Molecular Dynamics).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
Chemical cross-linking analysis with 125I-TGF-1 was used to determine the cell surface expression levels of the TGF-
receptors. We observed a correlation between overall TGF-
receptor cell surface expression levels and TGF-
sensitivity (Fig. 3
; Tables 1
and 2
). The TGF-
1-sensitive cell lines CA46, BL41 and MUTU-I showed clearly detectable levels of TGF-
RI, TGF-
RII and TGF-
RIII. The TGF-
-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-
receptors. The LCLs PD-LCL, JM-SAV-LCL and K-LCL also have very low levels of expression of these receptors. The TGF-
1-resistant MUTU-III group III BL cell line was derived from the TGF-
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-
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-
receptor expression (Fig. 3
).
|
|
|
LMP-1 is not necessary or sufficient to block the TGF-1 response
It has been reported previously that LMP-1 can block the TGF-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-
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-
1 treatment (Fig. 6a
d
). As shown previously, the BL41-B95.8 cell line was completely refractory to TGF-
1. Interestingly, the BL41-P3HR1 cell line exhibited a G1 arrest in response to TGF-
1, implying that restricted EBV latent gene expression is sufficient to block the apoptotic response to TGF-
1. TGF-
1 treatment had no effect on the EHRB-Ramos or AW-Ramos cell lines. Western blot analysis showed that apoptosis in response to TGF-
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-
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 RTPCR (Pokrovskaja et al., 1996
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous reports have suggested that some BL cell lines may retain responsiveness to TGF-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-
1 signalling by a reduction in DNA synthesis and that the majority of these undergo apoptosis following TGF-
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-
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-
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-
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-
1 may involve a separate mechanism. Indeed, it is interesting to note that, in some myeloid leukaemia cell lines, induction of apoptosis by TGF-
1 is p53 independent (Selvakumaran et al., 1994
). This is also clearly the case in BL cells, as induction of apoptosis by TGF-
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- stimulation provides an interesting contrast to murine plasmacytomas induced in susceptible BALB/c mice. All of these tumours exhibit a functional loss of TGF-
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-
response. Loss of susceptibility to TGF-
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-
1. Resistance of Akata cells to TGF-
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-1-mediated inhibition of DNA synthesis, as noted previously by others (see Introduction), but they were also completely refractory to the TGF-
1-mediated induction of apoptosis. Measurement of TGF-
1 cell surface receptor expression indicated that, in these cells and the DG75, Akata and BJAB cell lines, TGF-
1 resistance could be due to down-regulation of TGF-
RI, TGF-
RII and TGF-
RIII. However, these cells express normal amounts of TGF-
RI RNA and protein, but exhibit a marked decrease in TGF-
RII RNA expression. These data are consistent with binding of 125I-TGF-
1 to TGF-
RII (Wrana et al., 1994
). If TGF-
RII is down-regulated, TGF-
RI and TGF-
RII will not be detected on the cell surface in 125I-TGF-
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-
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- 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-
RII. Sharma and co-workers have suggested that LMP-1 gene expression is responsible for blocking TGF-
signalling by up-regulating cyclin D2, but that this does not correlate with TGF-
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-
RII correlates with lack of sensitivity to TGF-
1. We found that LMP-1 could not block TGF-
1 responses or up-regulate cyclin D2 by itself and, indeed, that infection of the TGF-
1-sensitive Ramos cell lines with P3HR1 virus, which lacks LMP-1 expression, was sufficient to block TGF-
-induced responses without inducing cyclin D2. P3HR1 virus infection appears, at least in part, to inhibit responses to TGF-
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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allday, M. J., Sinclair, A., Parker, G., Crawford, D. H. & Farrell, P. J. (1995). EpsteinBarr virus efficiently immortalizes human B cells without neutralizing the function of p53.EMBO Journal 14, 1382-1391.[Abstract]
Altiok, A., Ehlin-Henriksson, B. & Klein, E. (1993). Correlation between the growth-inhibitory effect of TGF-beta 1 and phenotypic characteristics in a panel of B-cell lines.International Journal of Cancer 55, 137-140.
Amoroso, S. R., Huang, N., Roberts, A. B., Potter, M. & Letterio, J. J. (1998). Consistent loss of functional transforming growth factor beta receptor expression in murine plasmacytomas.Proceedings of the National Academy of Sciences, USA 95, 189-194.
Arvanitakis, L., Yaseen, N. & Sharma, S. (1995). Latent membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-beta 1-mediated growth inhibition in EBV-positive B cells.Journal of Immunology 155, 1047-1056.[Abstract]
Bauer, G., Hofler, P. & Simon, M. (1982). EpsteinBarr virus induction by a serum factor. Characterization of the purified factor and the mechanism of its activation.Journal of Biological Chemistry 257, 11411-11415.
Beckwith, M., Ruscetti, F. W., Sing, G. K., Urba, W. J. & Longo, D. L. (1995). Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53.Blood 85, 2461-2470.
Blomhoff, H. K., Smeland, E., Mustafa, A. S., Godal, T. & Ohlsson, R. (1987). EpsteinBarr virus mediates a switch in responsiveness to transforming growth factor, type beta, in cells of the B cell lineage.European Journal of Immunology 17, 299-301.[Medline]
Brimmell, M., Mendiola, R., Mangion, J. & Packham, G. (1998). BAX frameshift mutations in cell lines derived from human haemopoietic malignancies are associated with resistance to apoptosis and microsatellite instability.Oncogene 16, 1803-1812.[Medline]
Chaouchi, N., Arvanitakis, L., Auffredou, M. T., Blanchard, D. A., Vazquez, A. & Sharma, S. (1995). Characterization of transforming growth factor-beta 1 induced apoptosis in normal human B cells and lymphoma B cell lines.Oncogene 11, 1615-1622.[Medline]
Cryns, V. & Yuan, J. (1998). Proteases to die for.Genes & Development 12, 1551-1570.
Derynck, R. & Feng, X. H. (1997). TGF-beta receptor signaling.Biochimica et Biophysica Acta 1333, F105-F150.[Medline]
di Renzo, L., Altiok, A., Klein, G. & Klein, E. (1994). Endogenous TGF-beta contributes to the induction of the EBV lytic cycle in two Burkitt lymphoma cell lines.International Journal of Cancer 57, 914-919.
Douglas, R. S., Capocasale, R. J., Lamb, R. J., Nowell, P. C. & Moore, J. S. (1997). Chronic lymphocytic leukemia B cells are resistant to the apoptotic effects of transforming growth factor-beta.Blood 89, 941-947.
Farrell, P. J., Allan, G. J., Shanahan, F., Vousden, K. H. & Crook, T. (1991). p53 is frequently mutated in Burkitts lymphoma cell lines.EMBO Journal 10, 2879-2887.[Abstract]
Feng, X. H. & Derynck, R. (1996). Ligand-independent activation of transforming growth factor (TGF) beta signaling pathways by heteromeric cytoplasmic domains of TGF-beta receptors.Journal of Biological Chemistry 271, 13123-13129.
Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C.-H. & Miyazono, K. (1993). Cloning of a TGF beta type I receptor that forms a heteromeric complex with the TGF beta type II receptor.Cell 75, 681-692.[Medline]
Gauchat, J. F., Gascan, H., de Waal Malefyt, R. & de Vries, J. E. (1992). Regulation of germ-line epsilon transcription and induction of epsilon switching in cloned EBV-transformed and malignant human B cell lines by cytokines and CD4+ T cells.Journal of Immunology 148, 2291-2299.
Gilboa, L., Wells, R. G., Lodish, H. F. & Henis, Y. I. (1998). Oligomeric structure of type I and type II transforming growth factor receptors: homodimers form in the ER and persist at the plasma membrane.Journal of Cell Biology 140, 767-777.
Gregory, C. D., Rowe, M. & Rickinson, A. B. (1990). Different EpsteinBarr virusB cell interactions in phenotypically distinct clones of a Burkitts lymphoma cell line.Journal of General Virology 71, 1481-1495.[Abstract]
Gutierrez, M. I., Cherney, B., Hussain, A., Mostowski, H., Tosato, G., Magrath, I. & Bhatia, K. (1999). Bax is frequently compromised in Burkitts lymphomas with irreversible resistance to Fas-induced apoptosis.Cancer Research 59, 696-703.
Heldin, C.-H., Miyazono, K. & ten Dijke, P. (1997). TGF- signalling from cell membrane to nucleus through SMAD proteins.Nature 390, 465-471.[Medline]
Jiang, W., Tillekeratne, M. P. M., Brattain, M. G. & Banerji, S. S. (1997). Decreased stability of transforming growth factor type II receptor mRNA in RER+ human colon carcinoma cells.Biochemistry 36, 14786-14793.
Kaptein, J. S., Lin, C.-K. E., Wang, C. L., Nguyen, T. T., Kalunta, C. I., Park, E., Chen, F.-S. & Lad, P. M. (1996). Anti-IgM-mediated regulation of c-myc and its possible relationship to apoptosis.Journal of Biological Chemistry 271, 18875-18884.
Kawabata, M., Inoue, H., Hanyu, A., Imamura, T. & Miyazono, K. (1998). Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.EMBO Journal 17, 4056-4065.
Kehrl, J. H., Taylor, A. S., Delsing, G. A., Roberts, A. B., Sporn, M. B. & Fauci, A. S. (1989). Further studies of the role of transforming growth factor-beta in human B cell function.Journal of Immunology 143, 1868-1874.
Kumar, A., Rogers, T., Maizel, A. & Sharma, S. (1991). Loss of transforming growth factor beta 1 receptors and its effects on the growth of EBV-transformed human B cells.Journal of Immunology 147, 998-1006.
Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G. & Earnshaw, W. C. (1994). Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.Nature 371, 346-347.[Medline]
Lin, H. Y., Wang, X. F., Ng-Eaton, E., Weinberg, R. A. & Lodish, H. F. (1992). Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase.Cell 68, 775-785.[Medline]
Lomo, J., Blomhoff, H. K., Beiske, K., Stokke, T. & Smeland, E. B. (1995). TGF-beta 1 and cyclic AMP promote apoptosis in resting human B lymphocytes.Journal of Immunology 154, 1634-1643.
MacDonald, I., Wang, H., Grand, R., Armitage, R. J., Fanslow, W. C., Gregory, C. D. & Gordon, J. (1996). Transforming growth factor-beta 1 cooperates with anti-immunoglobulin for the induction of apoptosis in group I (biopsy-like) Burkitt lymphoma cell lines.Blood 87, 1147-1154.
Mann, K. P., Staunton, D. & Thorley-Lawson, D. A. (1985). EpsteinBarr virus-encoded protein found in plasma membranes of transformed cells.Journal of Virology 55, 710-720.[Medline]
Markowitz, S., Wang, J., Myeroff, L., Parsons, R., Sun, L., Lutterbaugh, J., Fan, R. S., Zborowska, E., Kinzler, K. W., Vogelstein, B. and others (1995). Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 268, 13361338.[Medline]
Massagué, J. (1998). TGF- signal transduction.Annual Review of Biochemistry 67, 753-791.[Medline]
Milner, A. E., Grand, R. J., Vaughan, A. T., Armitage, R. J. & Gregory, C. D. (1997). Differential effects of BCL-2 on survival and proliferation of human B-lymphoma cells following gamma-irradiation.Oncogene 15, 1815-1822.[Medline]
Milner, A. E., Levens, J. M. & Gregory, C. D. (1998). Flow cytometric methods of analyzing apoptotic cells.Methods in Molecular Biology 80, 347-354.[Medline]
Pokrovskaja, K., Ehlin-Henriksson, B., Bartkova, J., Bartek, J., Scuderi, R., Szekely, L., Wiman, K. G. & Klein, G. (1996). Phenotype-related differences in the expression of D-type cyclins in human B cell-derived lines.Cell Growth and Differentiation 7, 1723-1732.[Abstract]
Polyak, K. (1996). Negative regulation of cell growth by TGF beta.Biochimica et Biophysica Acta 1242, 185-199.[Medline]
Rich, B. E. & Steitz, J. A. (1987). Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.Molecular and Cellular Biology 7, 4065-4074.[Medline]
Rickinson, A. B. & Kieff, E. (1996). EpsteinBarr virus. In Fields Virology, pp. 2397-2446. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Rowe, M., Rowe, D. T., Gregory, C. D., Young, L. S., Farrell, P. J., Rupani, H. & Rickinson, A. B. (1987). Differences in B cell growth phenotype reflect novel patterns of EpsteinBarr virus latent gene expression in Burkitts lymphoma cells.EMBO Journal 6, 2743-2751.[Abstract]
Saltzman, A., Munro, R., Searfoss, G., Franks, C., Jaye, M. & Ivashchenko, Y. (1998). Transforming growth factor--mediated apoptosis in Ramos B-lymphoma cell line is accompanied by caspase activation and BclX1 downregulation.Experimental Cell Research 242, 244-254.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schrantz, N., Blanchard, D. A., Auffredou, M. T., Sharma, S., Leca, G. & Vazquez, A. (1999). Role of caspases and possible involvement of retinoblastoma protein during TGF-mediated apoptosis of human B lymphoblasts.Oncogene 18, 3511-3519.[Medline]
Schuster, C., Chasserot-Golaz, S. & Beck, G. (1991). Activation of EpsteinBarr virus promoters by a growth-factor and a glucocorticoid.FEBS Letters 284, 82-86.[Medline]
Selvakumaran, M., Lin, H. K., Miyashita, T., Wang, H. G., Krajewski, S., Reed, J. C., Hoffman, B. & Liebermann, D. (1994). Immediate early up-regulation of bax expression by p53 but not TGF beta 1: a paradigm for distinct apoptotic pathways.Oncogene 9, 1791-1798.[Medline]
Smeland, E. B., Blomhoff, H. K., Holte, H., Ruud, E., Beiske, K., Funderud, S., Godal, T. & Ohlsson, R. (1987). Transforming growth factor type beta (TGF beta) inhibits G1 to S transition, but not activation of human B lymphocytes.Experimental Cell Research 171, 213-222.[Medline]
Takaku, K., Oshima, M., Miyoshi, H., Matsui, M., Seldin, M. F. & Taketo, M. M. (1998). Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes.Cell 92, 645-656.[Medline]
Takenoshita, S., Hagiwara, K., Nagashima, M., Gemma, A., Bennett, W. P. & Harris, C. C. (1996). The genomic structure of the gene encoding the human transforming growth factor type II receptor (TGF-
RII).Genomics 36, 341-344.[Medline]
Tao, Q., Robertson, K. D., Manns, A., Hildesheim, A. & Ambinder, R. F. (1998). EpsteinBarr virus (EBV) in endemic Burkitts lymphoma: molecular analysis of primary tumor tissue.Blood 91, 1373-1381.
Wrana, J. L., Attisano, L., Wieser, R., Ventura, F. & Massagué, J. (1994). Mechanism of activation of the TGF- receptor.Nature 370, 341-347.[Medline]
Zhu, Y., Richardson, J. A., Parada, L. F. & Graff, J. M. (1998). Smad3 mutant mice develop metastatic colorectal cancer.Cell 94, 703-714.[Medline]
Received 5 October 1999;
accepted 2 February 2000.