Transforming Growth Factor-1 Sensitivity Is Altered in Abl-Myc- and Raf-Myc-Indu - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:59 |只看该作者 |倒序浏览 |打印

作者：John Letterioa, Eva Rudikoffb, Nga Voongc, Steven R. Bauerb作者单位：aCase Western Reserve University, Division of Pediatric Hematology/Oncology, The Ireland Cancer Center, Cleveland, Ohio, USA;bFood and Drug Administration, Center for Biologics Evaluation and Research, Cell and Tissue Therapy Branch, Rockville, Maryland, USA;cNational Institutes of Health, National  # @$ B* \& K: `5 C+ F, P

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      【摘要】
   Understanding the mechanisms leading to transformation of early B-lineage precursors is an important step leading to rational design of new treatments for precursor (pre)-B-cell leukemia. We used normal mouse pre-B cells to determine if and how transforming growth factor (TGF)-&szlig;1 affects these precursors to the B-cell lineage and whether transformed pre-B cells respond to TGF-&szlig;1. We found that normal pre-B cells proliferating in the presence of interleukin (IL)-7 enter cell-cycle arrest after exposure to TGF-&szlig;1. However, clonally related IL-7-independent tumors induced by oncogenes abl myc or raf myc have reduced sensitivity to TGF-&szlig;1. In contrast, tumor cells induced by myc alone remain sensitive to TGF-&szlig;1 growth suppression. These results suggest that lesions in different molecular signaling pathways can lead to loss of TGF-&szlig;1 sensitivity in a single cell type. The approach of using normal pre-B-cell lines and transformation by overexpression of different oncogenes provides a system to compare and contrast molecular pathways that lead to full malignancy. ' T$ v% i8 b7 Z, O8 m
      【关键词】 Transforming growth factor- Pre-B tumors myc abl raf
   INTRODUCTION

Transforming growth factor (TGF)-ß1 is a pleiotropic cytokine that suppresses cell growth and can inhibit or stimulate differentiation. It is a member of a gene superfamily, which includes activins, inhibins, and bone morphogenic proteins . TGF-ß1 is the principal TGF-ß isoform to be expressed in B cells, and it can act in both an autocrine and paracrine fashion to modulate the proliferation, differentiation, and viability of cells in the B lineage. The TGF-ß receptors are expressed throughout B-cell maturation, but the effects of this cytokine are influenced by the state of differentiation, activation, and by other cytokines regulating B-cell activity.

TGF-ß1 affects the B-lineage cells at several stages of development. Disruption of TGF-ß1 gene expression in mice leads to inflammatory responses that include B cell infiltrates in several organs  showed that TGF-ß1 differentially affects different stages of early B-cell development. Progenitor B and early precursor (pre)-B-cell proliferation was inhibited by TGF-ß1, whereas large pre-B cells, although initially inhibited by TGF-ß1, were increased in numbers at days 5 and 7 of culture. These results indicate that early stages of B-cell development are differentially affected by TGF-ß1, depending on their stage of differentiation.1 L5 k$ i! J+ [! Q' L3 P- R: k
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Early B-cell development occurs in the bone marrow and is influenced by cell-cell contact in addition to growth factors such as interleukin (IL)-7 and TGF-ß1. In the current study, we have used a stroma and IL-7-dependent pre-B-cell in vitro culture system to study effects of TGF-ß1 on normal pre-B cells and the loss of TGF-ß1 sensitivity following transformation by different oncogenes. Pre-B cells maintained in these conditions can proliferate indefinitely without differentiation. Both IL-7 and stromal cells are required for pre-B-cell growth. The cells retain the phenotype of normal precursor B cells by a variety of criteria, including cell surface markers and immunoglobulin gene rearrangement status. They can differentiate into antibody secreting cells both in vivo and in vitro, undergo immunoglobulin heavy chain and light chain isotype switching, form germinal centers in vivo, and are not transformed. In vitro differentiation of these cells is triggered either by removal of IL-7 or the stromal cells. Upon IL-7 removal, 80%¨C90% of the cells die by apoptosis, and up to 10%¨C20% of the cells acquire surface immunoglobulin expression .

The process of tumorigenesis is thought to require perturbation of several different oncogenes or tumor suppressors to achieve complete malignant transformation. We previously used the in vitro culture system to show that recombinant retroviruses expressing myc with either abl or raf can rapidly transform pre-B cells . Infected cells lose stromal and IL-7 requirements within 24 hours and are aggressively tumorigenic upon transfer to immunocompetent syngeneic hosts. Both raf-myc and abl-myc retroviruses abrogate the IL-7 requirement of the pre-B cells. However, although the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) 5 pathway is constitutively activated in the abl-myc-transformed pre-B cells, it is not constitutively activated in the raf-myc-transformed cells. These data suggest that multiple pathways can be subverted to lead to tumorigenesis of the same cell type. The question remains whether or not there are other similarities or differences between the subverted growth control pathways that led to tumorigenesis in the myc-abl or myc-raf-transformed pre-B cells.
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Several recent studies of B-lineage tumors point to loss of TGF-ß sensitivity as a common mechanism in B-lineage transformation. Mature B-cell transformation is commonly associated with loss of receptor expression or a loss of responsiveness to the suppressive effects of TGF-ß on growth or viability. However, the mechanisms responsible for loss of TGF-ß signaling can differ and are not well defined. In mouse plasmacytomas, overexpression of intracellular TGF-ß causes intracellular receptor saturation and prevents formation of a signaling receptor on the cell surface, thus rendering the plasmacytoma cells insensitive to endogenous or exogenous TGF-ß . Finally, TGF-ß sensitivity can be abrogated by changes in downstream signal transduction pathways.

In contrast to TGF-ß effects in mature B-lineage tumors, relatively little is known about TGF-ß sensitivity in precursor B-cell tumors. A report that human pre-B-cell leukemia remains susceptible to TGF-ß-induced apoptosis suggests that loss of sensitivity may not be a common pre-B transformation event . In the current study, we investigate the response of normal mouse pre-B cells and their virally transformed counterparts to treatment with TGF-ß and examine the interaction of TGF-ß with IL-7 signal transduction and myc oncogene-mediated transformation.% a  o( a( q, \0 d4 R

MATERIALS AND METHODS; u% ?4 |! Y  o8 s9 R* r0 b3 K

Cell Lines8 s: _. @: Q; r3 K7 v! N
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D-1-3 and B-3/4-3 are normal pre-B-cell lines maintained on irradiated stroma in the presence of IL-7 as described  was used as a positive control in Smad2 Western blots.5 e* G; I2 v. o2 o( L
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Receptor Expression and Function Analysis
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Detection of TGF-ß receptors by affinity labeling with 125I-TGF-ß1 was done as described . Surface expression of TGF-ßRII and IL-7R was performed by flow cytometry. Cell suspensions were stained with monoclonal rat anti-mouse TGF-ßRII (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), washed, and then stained with secondary fluorescein isothiocyanate-labeled mouse anti-rat  light chain (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com/) or were stained with anti-CD127-biotin (anti-mouse IL-7R; BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) followed by streptavidin-phycoerythrin (PE) (BD Pharmingen). Specificity controls included secondary reagents alone, PE-labeled isotype controls (BD Pharmingen), and spleen cells (not shown). Cells were analyzed using a FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and analyzed using FlowJo software (Tree Star, Ashland, OR, http://www.treestar.com/).
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TGF-ß signaling was analyzed by Western blot using mouse monoclonal anti-Smad2 or rabbit anti-phospho-Smad2 and chemiluminescent detection on autoradiography film using Western blot detection systems according to the manufacturer's directions (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com).
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Cell Proliferation Assays( N+ B- i% R6 v4 D4 y

thymidine uptake was determined. Each condition was tested in triplicate wells. Each experiment was repeated a minimum of three times.

Cell-Cycle Analysis

Treated cells were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS), and then fixed in 70% ethanol overnight at ¨C20¡ãC. Cells were pelleted, resuspended in PBS with 0.1% Triton X-100, 0.1 mM EDTA, 50 µg/ml each of RNase A and RNase T, and 50 µg/ml propidium iodide. After 1-hour incubation at room temperature, cells were analyzed on a FACSCalibur (BD Biosciences, San Diego, http://www.bdbiosciences.com).& W8 P. O/ L5 w6 z- c, l% S

RESULTS
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TGF-ß and IL-7 Receptor Expression. k0 C$ k) k6 T+ v2 @

To investigate the potential effects of TGF-ß on in vitro-transformed pre-B-cell lines, we first determined whether they expressed receptors for TGF-ß. Affinity labeling with 125I-TGF-ß1 revealed that normal pre-B-cell lines as well as their transformed progeny express both the type I and type II TGF-ß receptors at the cell surface (Fig. 1A). Flow cytometry also demonstrated that the cell lines used in this study express roughly equivalent levels of TGF-ß RII on the cell surface (Fig. 1B). This result suggested that we could use the in vitro system to compare and contrast effects of TGF-ß on both normal and transformed pre-B-cell lines.

Figure 1. TGF-ß1 and IL-7 receptors are expressed on normal and transformed mouse pre-B cells. (A): Cell lysates from normal pre-B lines (B-1-14, B-3/4-3, D-1-2, and D-1-3), abl-myc (AM) and raf-myc (J3) virus-transformed pre-B lines (both transformed from D-1-3), and P388 (positive control for TGF-ß1 expression) were incubated with 125I-radiolabeled TGF-ß1 in the presence or absence of unlabeled competitor TGF-ß1, then cross-linked and immunoprecipitated with antibody specific for TGF-ßRII and run in SDS-polyacrylamide gel electrophoresis. The gel was dried and exposed to autoradiographic film. The leftmost lane contained labeled standards (MW) with the sizes in kDa shown to the left. The two TGF-ß1 receptor bands, TGF-ßRI and TGF-ßRII, are identified to the right of the panel. (B): Flow cytometry was performed on normal and transformed pre-B cell lines to examine surface expression of TGF-ßRII and IL-7 R on the indicated cell lines, as described in Materials and Methods. Histograms show the level of surface antigen expression for each marker as indicated at the bottom of panel B. Abbreviations: AM, abl-myc; EMM, enhancer µ myc; IL-7, interleukin 7; J3, raf-myc; MW, molecular weight; TGF-ß1, transforming growth factor-ß1.

In addition, we showed that the IL-7 receptor is expressed on each of these cell lines at roughly equivalent levels (Fig. 1B). We previously showed that the normal pre-B-cell lines D-1-3 and B-3/4-3 expressed the IL-7 receptor and that the IL-7 JAK-STAT pathway was stimulated by IL-7 in both abl-myc- and raf-myc-transformed counterparts D-1-3 AM, D-1-3 J3, B-3/4-3 AM, and B-3/4-3 J3 . The EMM lines and tumor were IL-7-dependent, demonstrating intact IL-7 signaling in these lines (see below).6 E+ ]; A6 c9 [) d

TGF-ß and IL-7 Effects on Normal Pre-B Cells5 f3 C9 H$ v# L' x9 e  w) G5 k) O
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Although published literature showed that mouse B-cell differentiation was inhibited by TGF-ß ., P/ ?+ D3 Y; B/ k, ~* P
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Given that normal pre-B-cell lines D-1-3 and B-3/4-3 expressed TGF-ß and IL-7 receptors, we exposed cells to a variety of TGF-ß concentrations in medium with varying IL-7 concentrations. It was not clear how these growth factors would interact in our in vitro system. As seen in Figure 2, these factors have antagonistic but interactive effects on proliferation of in vitro pre-B cells. Dependence on IL-7 concentration is clearly seen when TGF-ß1 is absent. Proliferation is robust and maximal above 50 U/ml IL-7 in the absence of TGF-ß1. At lower concentrations of IL-7, proliferation is concentration-dependent, whereas no proliferation is detected in the absence of IL-7. TGF-ß1 clearly inhibits IL-7-induced pre-B-cell proliferation. At the lowest TGF-ß1 concentration tested, 0.07 ng/ml, there was substantial inhibition of proliferation in IL-7 concentrations at or above 25 U/ml. Between 0.13 and 0.25 ng/ml TGF-ß1, more than 50% reduction in proliferation was observed at all IL-7 concentrations tested. At 0.5, 1, 2, and 4 ng/ml TGF-ß1 the inhibition was profound and almost no proliferation was observed, even at the highest IL-7 concentration of 100 U/ml.2 v" ?2 e; X1 m. g/ p
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Although the presence of the TGF-ß receptors was shown in cross-linking experiments and by flow cytometry (Fig. 1), we also examined the TGF-ß signal transduction pathway and showed that Smad2 was expressed by normal pre-B lines D-1-3 and B-3/4-3 as well as their transformed counterparts (Fig. 2B). In all these lines, exposure to TGF-ß1 resulted in signaling, indicated by phosphorylation of Smad2. These results demonstrate that the IL-7 and TGF-ß signal transduction pathways have opposite effects on pre-B cell proliferation but that these growth factors can moderate the effects of each other in normal pre-B cells cultured in the presence of stromal cells.

Figure 2. TGF-ß1 and IL-7 are antagonists for mouse pre-B-cell proliferation. (A): Normal pre-B-cell lines D-1-3 and B-3/4-3 were added to wells of a 96-well plate containing confluent irradiated (10 Gy) S10 stroma in the presence of various concentrations of IL-7 or TGF-ß1 alone, both, or neither. Each line represents uptake of thymidine (y-axis) after culture for 72 hours at a given concentration of IL-7 (U/ml, legend at the right) plotted against various TGF-ß concentrations (ng/ml, x-axis). Error bars represent SEM, triplicate samples. (B): Cell lysates from normal D-1-3 and B-3/4-3 and their transformed counterparts (AM indicates abl-myc virus, J3 indicates raf-myc virus) were analyzed by Western blot for total SMAD2 and phosphorylated Smad2 with ( ) or without (¨C) treatment with TGF-ß1, as indicated below the panel. Abbreviations: AM, abl-myc; CPM, counts per minute; IL-7, interleukin 7; J3, raf-myc; RIE, rat intestinal epithelium cell line; TGF-ß1, transforming growth factor-ß1.
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Previous reports suggested that TGF-ß could inhibit growth through at least two mechanisms, induction of apoptosis . To determine whether cell-cycle arrest was induced by TGF-ß1, we performed cell-cycle analysis by flow cytometry on propidium iodide-labeled, fixed cells in the presence of IL-7 alone or in IL-7 with TGF-ß. Both normal pre-B cell lines behaved similarly (Fig. 3A). In IL-7 alone, the majority of pre-B cells were in either G0-G1 or S phases. In contrast, when TGF-ß1 was added, the number of cells in G1 almost doubled, and the number of cells in S phase was reduced almost 50% (Fig. 3B). This result shows that TGF-ß1 can inhibit pre-B-cell proliferation by inducing G1 cell-cycle arrest.

Figure 3. TGF-ß1 induces G1 arrest in normal pre-B cells. (A): Pre-B- cell lines B-3/4-3 and D-1-3 were incubated in the presence of 25 U/ml IL-7 and 0.25 ng/ml TGF-ß for 96 hours, then fixed with ethanol overnight at 4¡ãC. Propidium iodide was added and the cells were analyzed for DNA content by flow cytometry on a Becton Dickinson FACSCAN. Data analysis was done using ModFit software (Becton, Dickinson and Company) for determination of 1x, 2x, and intermediate DNA peaks corresponding to G0-G1, G2-M, and S phase, respectively. The histogram plots cell number (y-axis) against fluorescence intensity (x-axis). The G0-G1 peaks are the higher peaks on the histogram, the S-phase cells are identified with diagonal hatches, and the G2-M cells are identified by the rightmost peak. Black triangles below the x-axis show the mean fluorescence of respective peaks. (B): The percentage of cells in each cell cycle phase for each treatment group is shown in the table below the histogram. Abbreviations: IL-7, interleukin 7; TGF-ß1, transforming growth factor-ß1.- ]7 Y1 ~  R4 @
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TGF-ß Effects on Transformed Pre-B Cells

After demonstrating that both normal and abl-myc- or raf-myc-transformed pre-B cells express the TGF-ß receptors and that normal pre-B-cell proliferation is inhibited by TGF-ß, we performed experiments to determine whether or not transformed pre-B-cell lines retained sensitivity to TGF-ß (Fig. 4).

Figure 4. Abl-myc and raf-myc-mediated transformation reduces TGF-ß1 sensitivity. Normal pre-B-cell lines D-1-3, B-3/4-3 and their transformed counterparts (AM indicates abl-myc-transformed, J3 indicates raf-myc-transformed) were added to wells of a 96-well plate in the presence (100 U/ml) or absence of IL-7 in concentrations of TGF-ß ranging from 4 to 0 ng/ml (x-axis) for 72 hours. Each line represents uptake of thymidine (y-axis) in the presence or absence of IL-7 plotted against various TGF-ß concentrations, as shown in the legend at the right. Four percent indicates medium with 4% fetal calf serum but no IL-7. Abbreviations: AM, abl-myc; CPM, counts per minute; IL-7, interleukin 7; J3, raf-myc; TGF-ß1, transforming growth factor-ß1.  s  p9 I: g" G8 C) W! i' T
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In contrast to the substantial growth inhibition induced by TGF-ß on normal pre-B cells, there was substantially less growth inhibition of pre-B cells that were transformed by the combination of abl-myc oncogenes. For B-3/4-3 AM, there was no more than 20% difference in proliferation in the presence or absence of IL-7 at all TGF-ß1 concentrations tested. For D-1-3 AM, there was no more than 50% difference in proliferation in the presence or absence of IL-7 at all TGF-ß1 concentrations tested (Fig. 4).& b5 Y+ k# F! I7 D9 S; o' \

Interestingly, the effects of transformation by raf-myc were different, depending on the cell line. Although D-1-3 J3 proliferates much more in the presence of IL-7 than without, proliferation was inhibited no more than 50% in the presence of TGF-ß1 at all concentrations tested. In fact, in the absence of IL-7, D-1-3 proliferation was almost the same in the absence or in the presence of 4 ng/ml TGF-ß1. In contrast, proliferation of raf-myc transformed B-3/4-3 was inhibited more than 60% even at the lowest TGF-ß1 concentration (0.07 ng/ml) when IL-7 was absent. However, in the presence of 100 U/ml IL-7, B-3/4-3 was virtually unaffected by TGF-ß1 at all concentrations tested. In all experiments, the control normal cell lines were inhibited almost completely at TGF-ß1 concentrations at or above 0.5 ng/ml, as shown previously.* X' u" j! R. s% _
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These results suggest that transformation has affected the antagonistic interaction between TGF-ß- and IL-7-induced signals we observed in normal pre-B cells (Fig. 2). Even at the highest level of TGF-ß, 4 ng/ml, which virtually extinguished normal pre-B cell proliferation, the abl-myc-transformed pre-B- cell lines were minimally affected. For D-1-3 J3 and B-3/4-3 J3, the inhibition by TGF-ß1 was not great in the presence of IL-7. For D-1-3, inhibition by TGF-ß1 in the absence of IL-7 was small, whereas B-3/4-3 remained sensitive to the growth inhibitory effect of TGF-ß1 in the absence, but not the presence, of IL-7.
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The decrease in TGF-ß1-mediated growth inhibition despite the presence of the receptor and evidence of Smad2-mediated signaling suggests that transformation has abrogated responsiveness to further downstream signals from the TGF-ß receptor complex. Both retroviruses used to transform the pre-B lines contained the myc oncogene but differed in expression of raf (J3 virus ), it is possible that myc overexpression alone could be responsible for decreased TGF-ß sensitivity.
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To test this hypothesis, we isolated a pre-B tumor cell line from a myc transgenic mouse strain. Eµ-myc transgenic mice  overexpress the myc oncogene under the control of the immunoglobulin heavy chain intronic enhancer and inevitably develop B-cell lymphomas, primarily from the pre-B-cell stage. We also established a normal pre-B and preneoplastic pre-B line from Eµ-myc transgenic mice as described in Materials and Methods. The EMM lines were established in media with 100 U/ml IL-7. We verified the pre-B phenotype by FACS analysis (not shown), and tested the cell lines for sensitivity to TGF-ß and IL-7. All three EMM lines, including the normal, preneoplastic, and myc-transformed tumor lines, required IL-7 for proliferation, but proliferation was measurably inhibited by TGF-ß1 (Fig. 5). These lines expressed Smad2 and TGF-ß-induced Smad2 phosphorylation (not shown). Interestingly, the preneoplastic line EMM 2, which derived from a Eµ-myc transgenic animal, retained some proliferative capacity even at the highest level of TGF-ß1 tested, 4 ng/ml. This result shows that myc-induced tumorigenesis does not necessarily render a pre-B- cell tumor insensitive to TGF-ß.
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Figure 5. myc overexpression does not abrogate TGF-ß1 sensitivity. Normal (EMM 4), preneoplastic (EMM 2), or tumor-derived (EMM tumor) cell lines were added to wells of a 96-well plate in the presence (100 U/ml) or absence of IL-7 in concentrations of TGF-ß ranging from 4 to 0 ng/ml (x-axis) for 72 hours. Each line represents uptake of thymidine (y-axis) in the presence or absence of IL-7 plotted against various TGF-ß concentrations, as shown in the legend at the right. Four percent indicates medium with 4% fetal calf serum but no IL-7. Abbreviations: CPM, counts per minute; EMM, enhancer µ myc; IL-7, interleukin 7; TGF-ß1, transforming growth factor-ß1.
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DISCUSSION

Previous data showed that TGF-ß effects early stages of normal early B-cell development . In agreement with the studies on polyclonal early B-lineage cells, our results show that normal early pre-B cells express both TGF-ß receptor chains and that TGF-ß1 can suppress IL-7-induced proliferation of normal pre-B cells through induction of growth arrest at the G1 phase of the cell cycle.& w' n; W6 \5 E( n
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We also used in vitro transformed counterparts of these same pre-B-cell lines to study the effects of TGF-ß on malignant counterparts of normal pre-B cells. Although the transformed progeny of these pre-B cells also express the TGF-ß receptors at the cell surface, they are refractory to the inhibitory effects of TGF-ß on cell proliferation. Our data suggest the presence of crosstalk between the IL-7 and TGF-ß signal transduction pathways and that these cytokines provide antagonistic signals. Our data show that transformation by abl-myc or raf-myc oncogene pairs render pre-B cells less sensitive to TGF-ß, despite our observation that TGF-ß induces Smad2 phosphorylation after exposure. Although we demonstrate the ability of abl-myc and raf-myc transformation to unlink the antagonistic interaction between TGF-ß and IL-7-induced signals we observed in normal pre-B cells, our observations indicate that at least part of the TGF-ß signal transduction pathway is intact in these cells and suggests the mechanisms of abrogation are further downstream.7 l3 f( G9 t0 ~
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Previous work showed that TGF-ß treatment of mature B-lineage tumors led to myc downregulation and subsequent apoptosis . Ectopic myc overexpression could reverse this effect, suggesting that overexpression of myc in a variety of B-lineage tumors could supply a mechanism for escape of TGF-ß-mediated inhibition and apoptosis. However, the fact that a pre-B-cell tumor from a myc transgenic mouse retained sensitivity to TGF-ß inhibition of IL-7-dependent proliferation indicates that myc overexpression alone is not sufficient to render a pre-B cell tumor insensitive to TGF-ß, raising the possibility that raf or abl expression alone could lead to TGF-ß insensitivity.7 t( O9 G+ ?1 z% n0 P+ q% b
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Our ability to establish or transform pre-B cells in culture with different oncogenes either singly or in combination could provide a methodical approach to dissection of the molecular pathways of TGF-ß and IL-7 signal transduction and how these pathways are abrogated during transformation. We have previously shown that both abl-myc and raf-myc viruses abrogate the in vitro requirements of pre-B cells for both stromal cells and IL-7 . Thus we have shown that these two viruses rapidly transform pre-B cells in vitro and that both IL-7 and TGF-ß signaling pathways have been affected. Although the mechanism by which stromal dependence has been alleviated has not been determined, this approach could also be used to determine how various oncogenes can lead to release from cell-cell interactions in bone marrow that may normally contribute to B-cell homeostasis.

Summary7 ?3 O' D4 X  w" h9 j) u% H
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We have shown that TGF-ß1 can suppress IL-7-induced proliferation of normal pre-B cells through induction of growth arrest at the G1 phase of the cell cycle. This TGF-ß1 sensitivity is substantially reduced following in vitro transformation by dual oncogene retroviruses expressing either abl and myc or raf and myc. In contrast, pre-B tumors transformed by myc alone remain sensitive to TGF-ß1. These results show that either abl or raf can lead to loss of TGF-ß1 sensitivity in the presence of dysregulated myc, whereas myc alone does not lead to loss of TGF-ß1 sensitivity. The results suggest there is crosstalk between the IL-7 and TGF-ß signal transduction pathways and that there are at least two ways to abrogate both pathways in transformation of precursor B cells.- N1 F/ {# S2 t; j% l" `

DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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This work was supported by Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research and National Cancer Institute intramural funding. We thank Bharat Joshi for critical reading of the manuscript. This work does not represent an official position of the FDA.7 Z6 W; b5 d; V3 w
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