Bcr: a negative regulator of the Bcr-Abl oncoprotein

Abstract

Chronic myelogenous leukemia is typically characterized by the presence of the Philadelphia chromosome (Ph) in which 5′ portions of the BCR gene are fused to a large portion of the ABL gene. Our studies and those of others indicate that Bcr sequences within the Bcr-Abl oncoprotein are critically involved in activating the Abl tyrosine kinase and actively participate in the oncogenic response, which is generated by the Bcr-Abl oncoprotein. We investigated the role of the Bcr protein in the oncogenic effects of Bcr-Abl. Reduction of the level of the Bcr protein by incubating cells with a 3′ BCR anti-sense oligodeoxynucleotide increased the growth rate and survival of hematopoietic cell lines expressing Bcr-Abl. Also, enforced expression of Bcr in Bcr-Abl cell lines strongly reduced transformation efficiency. Induction of Bcr expression drastically reduced the phosphotyrosine content of Bcr-Abl in Rat-1 fibroblasts transformed by P185 BCR-ABL and in hematopoietic cells expressing P210 Bcr-Abl within days following induction of Bcr. Rat-1/P185 cells maintained for three weeks after Bcr induction had dramatically reduced amounts of phosphotyrosine proteins compared to cells in which Bcr expression was repressed by the addition of Tet. In contrast Bcr expression did not decrease the phosphotyrosine content of either v-Src or activated Neu tyrosine kinase. Importantly, the phosphotyrosine content of total P160 BCR (induced plus endogenous) was strongly reduced by inducing expression of Bcr, indicating that the induced Bcr protein was not a target of the tyrosine kinase activity of Bcr-Abl but instead functioned as an inhibitor of Bcr-Abl. These results show that the Bcr protein can function as a negative regulator of Bcr-Abl, but that the inhibitory effects of Bcr are dependent on achieving an elevated level of Bcr expression relative to Bcr-Abl.

Introduction

The BCR gene is the fusion partner in more than 90% of patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). Bcr sequences within Bcr-Abl are critically involved in the oncogenic events that are induced by the Bcr-Abl oncoprotein. In this regard Bcr sequences activate the tyrosine kinase function of Bcr-Abl (Muller et al., 1991; McWhirter and Wang, 1991), and Bcr tyrosine 177 when phosphorylated binds Grb2, an activator of the Ras pathway (Pendergast et al., 1993; Puil et al., 1994). Recently, we have demonstrated that tyrosine residues 328 and 360 within the first exon of the Bcr protein are critically involved in regulating Bcr's serine/threonine kinase activity (Liu et al., 1996b; Wu et al., 1998). Moreover, tyrosine phosphorylation of Bcr first exon sequences inhibits Bcr's serine/threonine protein kinase activity, probably as a direct result of the phosphorylation of these two tyrosine residues (Liu et al., 1996b; Wu et al., 1998). Given the inhibitory effects of Bcr-Abl on the kinase activity of Bcr, we searched for effects of the Bcr protein on the tyrosine kinase activity of Bcr-Abl and its resultant oncogenic activity. In this regard a deletion mutant of Bcr, lacking the oligomerization domain of Bcr (McWhirter et al., 1993) and comprising the bulk of the remaining Bcr first exon sequences [Bcr(64 – 413)], inhibits Abl and Bcr-Abl tyrosine kinases and blocks Bcr-Abl's growth promoting effects (Liu et al., 1996a). Results obtained with a 17 amino acid Bcr peptide (S17K, residues 350 – 366) in the phosphoserine form indicate that these first exon Bcr sequences are able to inhibit the Abl kinase in the phosphoserine form. These inhibitory effects of Bcr first exon sequences raise the possibility that the full-length Bcr protein functions as a conditional negative regulator of the Abl kinase. In this report we show that reduction of Bcr protein concentration in cells by anti-sense oligodeoxynucleotide treatment enhanced the growth rate and survival of Bcr-Abl expressing leukemia cell lines. Enforced expression of Bcr in cells transformed by Bcr-Abl reduced the phosphotyrosine content of the oncoprotein and the other target proteins, and strongly inhibited its oncogenic activity. In contrast the phosphotyrosine content of v-Src and activated Neu were not decreased by induction of Bcr expression.

Results

Reduction of Bcr concentration in hematopoietic cells expressing Bcr-Abl

To investigate the effects of Bcr on Bcr-Abl, we used anti-sense oligodeoxynucleotides directed against 3′ BCR sequences to reduce the level of the Bcr protein without affecting the level of the Bcr-Abl oncoprotein. We selected an oligo down stream of the Bcr-Abl junctions that would not anneal with sequences of genes homologous to Bcr. Treatment of P185 BCR-ABL expressing SUP B15 cells, derived from a patient with Ph-positive acute lymphocytic leukemia (ALL) (Naumovski et al., 1988), with the anti-sense oligo for seven days significantly reduced the level of Bcr protein about 10-fold (Figure 1a) without affecting the concentration of P185 BCR-ABL (Figure 1b). The bands of Bcr were quantitated by use of the PhosphoImager and normalized for the amount of P145 ABL in the extracts as shown in Figure 1B. The level of the Bcr protein was measured by the immune complex kinase assay (Liu et al., 1993) using antibody to the C-terminus of Bcr [anti-Bcr(1256 – 1271)]. This assay is more sensitive and specific than Western blotting with available Bcr antibodies particularly for cells having a low abundance of Bcr like SUP B15 cells (for example K56Z cells, Figure 5a, lane 1). In addition, the Bcr kinase assay estimates levels of functional Bcr, and use of the C-terminal Bcr antibody allows detection of complexes of Bcr associated with Bcr-Abl (Liu et al., 1993). Figure 1a shows that Bcr complexes containing P185 BCR-ABL were greatly reduced in cells treated with anti-sense oligos compared to sense treated cells. Untreated cells gave similar levels of complexes containing Bcr and Bcr-Abl as sense treated cultures (not shown).

Figure 1
figure1

Anti-sense Bcr oligonucleotides decrease the concentration of the Bcr protein. (a) P185 BCR-ABL expressing SUP B15 cells were treated with either 10 μM anti-sense (B15/AS) or 10 μM sense (B15/S) 3′ BCR oligos for 7 days. 6–106 cells were collected and analysed by kinase assays using anti-Bcr(1256 – 1271). [B15/S] denotes SUP B15 cells treated with the sense oligo; [B15/AS] represents SUP B15 cells treated with the anti-sense oligo. IP: BCR 1256-1271 denotes immunoprecipitation with this C-terminal Bcr anti-peptide antibody. Bands were detected and quantitated by PhosphoImager screening. (b) About 1–106 cells shown in a were analysed by Western blotting with anti-Abl 8E9

Figure 5
figure2

Reduction of P210 BCR-ABL tyrosine phosphorylation by induction of Bcr expression in K562 cells. K562 cells were electroporated with 30 μg of EC1214A Bcr. Forty-eight hours later, cells were seeded in soft agar with 400 μg/ml G418 and 1 μg/ml Tet. Several clones were selected after 3 weeks and expanded in media containing G418 and Tet. This figure shows one of the clones (KB-1) expressing Bcr after Tet release. Cells were collected after growing in media with Tet (lane 1), and in the absence of Tet for 1 day (lane 2), 2 days, (lane 3), and 3 days (lane 4). The same amount of cell lysates was loaded in each lane, and blotted with different antibodies as shown in a (anti-Bcr), b (anti-pTyr), and c (anti-Abl 8E9). a and b were run on separate gels; c was a reprobe of a. d, kinetics of inhibition of P210 Bcr-Abl tyrosine phosphorylation by Bcr. Each band in a, b, and c was quantitated by the Personal Densitometer (Molecular Dynamics, Sunnyvale, CA, USA). The specific activity of P210 tyrosine phosphorylation was calculated from densitometer values as follows: PY20 (P210)/8E9 (P210). (e) Kinetics of inhibition of P160 BCR tyrosine phosphorylation by high level expression of exogenous Bcr. The specific activity of P160 BCR tyrosine phosphorylation was calculated from densitometer values as follows: PY20 (P160)/anti-Bcr (P160)

To assess the effects of reduction of Bcr, we treated SUP B15 cells with sense and anti-sense oligos and measured the effects on growth rate for a period of 9 days (Figure 2a). The growth rate was unaffected for the first 5 days; however, growth was stimulated about twofold at day 7 by anti-sense compared to sense. Similar results were obtained with another Bcr-Abl expressing cell line [M3.16 cells (Sirard et al., 1994) that express P210 BCR-ABL] (results not shown). Survival of SUP B15 cells was also enhanced by anti-sense oligos treatment of cells maintained in limiting serum compared to sense-treated cells (Figure 2b). This cell line requires 20% fetal bovine serum for optimal growth. To retard growth we used 5% serum in these cell survival experiments shown in Figure 2b. The enhancement of survival varied from about 2 – 4-fold from day 4 through day 7.

Figure 2
figure3

Reduction of BCR concentration increases growth rate and survival of Bcr-Abl expressing hematopoietic cells. (a) SUP B15 cells were cultured in 20% fetal calf serum RPMI medium with either 3′ BCR anti-sense (10 μM added at day 1) (□, open squares) or sense oligos (10 μM at day 1) (▪, closed squares). The data are the mean of triplicates. (b) SUP B15 cells were maintained in 5% fetal calf serum RPMI and treated with either 3′ BCR anti-sense oligos (□, open squares) at 10 μM or 3′ BCR sense oligos (10 μM) (▪, closed squares). The data are the mean of three replicates

Enforced expression of Bcr reduces the oncogenic activity of Bcr-Abl

Next we questioned whether enforced expression of Bcr in cells transformed by P185 BCR-ABL would interfere with Bcr-Abl oncogenic effects. Rat-1 cells have been used by the Witte group as a means to assess Bcr-Abl's oncogenic effects (Pendergast et al., 1993). Simultaneous transfection of Rat-1 cells with both P185 BCR-ABL DNA and P160 BCR DNA did not yield foci compared to transfection with P185 DNA alone (not shown). To monitor the effects of Bcr on Bcr-Abl transformed cells, Rat-1 cells were transfected with a retrovirus vector (pLXSN) encoding P185 BCR-ABL under conditions for positive selection with G418, yielding transformed foci (Figure 3b). Transformed foci were selected and recloned two more times by limiting dilution. One of these clones (clone #3) expressed high levels of P185 as measured by Western blotting with anti-Abl 8E9 (not shown). Transfection of Bcr DNA into such cells was not informative because the efficiency of transfection without selection was of the order of 1 in 1000 cells (0.1%). Efforts to increase transfection efficiency by several methods failed to improve transfection efficiency above 1%. Therefore, we were forced to use positive selection to monitor the effects of Bcr on Bcr-Abl transformation. For these types of experiments, Rat-1/Bcr-Abl cells were transfected with a second retrovirus vector (pLXSH) encoding BCR (P160) and selected with hygromycin (Figure 3c). Foci formation was strongly inhibited in Bcr expressing Rat-1/Bcr-Abl cells (Figure 3c) compared to vector only transfected cells (compare Figure 3d). Close inspection of the plates revealed that Bcr expression caused cell death and morphological changes in Rat-1/Bcr-Abl cells (Figure 3c). Foci were counted in plates shown in Figure 3c (7 foci) and d (128 foci), indicating more than a 90% reduction in foci as a result of Bcr expression. There was a similar level of inhibition of foci formation (about 85%) in stably transfected cells in which Bcr was expressed from a tetracycline (Tet) repression vector (no Tet added). The foci number was strongly reduced compared to cells in which Bcr expression was repressed by incubation in 0.1 μg/ml of Tet (results not shown).

Figure 3
figure4

The effect of Bcr expression on foci formation induced by Bcr-Abl. Foci were observed in Rat-1 cells transfected with BCR-ABL (15 – 20 foci/μg of DNA) (b), but not vector only (a); stably transfected cells expressing cells BCR-ABL (P185) (clone #3 using G418 selection) were transfected with BCR using hygromycin selection (c); or vector only (d). Foci were photographed at 3 weeks

In other studies, transfection of a hematopoietic cell line expressing Bcr-Abl (KBM-7) with a vector using a LTR promoter (pSRαMSV tkneo) to constitutively express Bcr resulted in non-growing cells after 4 weeks in culture (not shown). KBM-7 is a CML patient cell line that expresses P210 BCR-ABL but lacks Bcr expression (Liu et al., 1993). To better assess the negative effects of Bcr expression on Bcr-Abl, we explored inducible promoters to drive the expression of Bcr. Establishing such a system would allow probing the negative effects of Bcr at early times after induction in a systematic way instead of studying the end stage effects of Bcr. We began these studies with a dexamethesone inducible vector system containing a Neo selection marker (pMEP4, InVitrogen, Carlsbad, CA, USA). We inserted the full length BCR gene into this vector and stably transfected KBM-7 cells in the presence of G418. Unfortunately, these cells expressed Bcr in the absence of the inducer. Nevertheless, low expression of Bcr expression in KBM-7 cells slowed the growth rate as measured by the increase in doubling time from 27 to 35 h compared to vector only transfected cells selected in a similar fashion.

Induction of Bcr in Bcr-Abl transformed Rat-1 cells reduces the phosphotyrosine content of Bcr-Abl

Our colleague Hong-Ji Xu has developed a very efficient system for inducible gene expression using a tetracycline (Tet) responsible system (Hu et al., 1997). However, the method requires selecting clones that have low copy number to prevent the leaky expression observed within the total population of uncloned transfectants, which is due at least in part to high copy number of the gene construct in most of the transfected cells. To evaluate the effects of full length Bcr expression on Bcr-Abl tyrosine phosphorylation in cells, we transfected clone #3 Rat-1/Bcr-Abl cells using hygromycin selection with the Tet repressible promoter construct containing a BCR insert (Hu et al., 1997) in the presence of 0.1 μg/ml of Tet. Uncloned stable transfectants surviving the hygromycin selection showed expression of Bcr in the presence and absence of Tet at early times after transfection. Therefore to enhance the frequency of isolating inducible cell clones, we selected Bcr-Abl transformed foci cell clones that formed in the presence of the 0.1 μg/ml of Tet repressor and hygromycin. Because of the growth inhibitory effects of Bcr on Bcr-Abl cells, only silent or low expression Bcr expressing cells would allow transformed foci to form. Several such foci were obtained that expressed P185 BCR-ABL and no detectable Bcr in the presence of 0.1 μg/ml of Tet but expressed P160 BCR upon release from Tet repression. One such clone (Rat B-1) was selected for further study. To test the effects of Bcr expression on Bcr-Abl tyrosine phosphorylation within cells, we monitored Bcr-Abl tyrosine phosphorylation and Bcr expression following release from Tet repression (Figure 4). Bcr expression was greatly increased within 4 – 6 days (Figure 4a, lanes 2 and 3) after removal of Tet as judged by Western blotting experiments with a Bcr antibody (ant-Bcr 181 – 194) that detects both Bcr-Abl and Bcr proteins. Bcr-Abl tyrosine phosphorylation as measured by anti-phosphotyrosine Western blotting was dramatically reduced at 4 – 6 days (Figure 4b, lanes 1 – 3) after release from Tet repression. Moreover reduction of total phosphotyrosine labeled proteins was also observed, which was greatest at 6 days after induction when the Bcr level reached its highest level (Figure 4b, lane 3), indicating that Bcr expression was responsible for the blockage of tyrosine phosphorylation. Quantitative measurements by densitometry (Table 1) indicated that Bcr-Abl phosphotyrosine content was reduced about 80% at day 6 where Bcr expression was highest. This value was calculated by dividing the intensity of the anti-pTyr Bcr-Abl band by the intensity of the Bcr-Abl band detected by anti-Abl (8E9) (not shown). Interestingly, the specific phosphotyrosine content of total Bcr was reduced by more than 85% (Table 1). This was determined by comparing the mobility of the Bcr band in the anti-Bcr blot in panel A and the 160 kDa phosphotyrosine band in panel B, and by IP/Western studies. The Bcr specific phosphotyrosine value was calculated by dividing the anti-pTyr intensity by the anti-Bcr band intensity of P160 BCR. As controls, vector only cells or cells transfected with the BCR vector but maintained in 0.1 μg/ml of Tet (not shown) had little effect on Bcr-Abl tyrosine phosphorylation. Western blotting with anti-Abl showed little change in the level of Bcr-Abl during this 4 – 6 day time period (not shown).

Figure 4
figure5

The Induction of Bcr expression inhibits Bcr-Abl tyrosine phosphorylation. Clone #3 Rat-1/Bcr-Abl cells were transfected with the BCR vector under control of the Tet repressible promoter (pEC12 14A) using 0.1 ratio of pLXSH. Cell cultures were selected in the presence of hygromycin and 0.1 μg/ml of Tet and foci were isolated. One such clone (Rat B-1) was incubated in the absence of Tet for various times to induce BCR (P160) expression. (a) Western blotting with anti-Bcr(181-194). Lane 1, Rat B-1 P185/TetBCR cells were released from the Tet block for 2 days; lane 2, released for 4 days; lane 3, released for 6 days. The B-1 clone showed no detectable Bcr protein at zero time under these exposure conditions (not shown). (b) Western blotting with anti phosphotyrosine antibody (anti-pY20, Transduction Labs, Lexington, KY, USA). Lane 1, Rat B-1 P185/Tet BCR cells were released for 2 days; lane 2, released for 4 days; lane 3, released for 6 days. (c) Anti-Bcr Western blotting of stably transfected cells. Clone 3 stably transformed Rat-1/Bcr-Abl cells were transfected with the Tet vector encoding Bcr and 1/10th ratio of pLSXH (Hyg) then maintained in hygromycin for three weeks. Lane 1, Rat-1 cells transfected with vector only; lane 2, Rat-1 P185 (B-1) cells stably transfected with the Tet-BCR vector were maintained for three weeks in the absence of Tet; lane 3, Rat-1 P185 (B-1) Tet BCR cells maintained in the presence of Tet for three weeks (0.04 μg/ml); blots were probed with anti-Bcr (181-194). (d) Anti-phosphotyrosine Western blotting. Lanes 1 – 3, same as c. Lane 1, Rat-1 cells transfected with vector; lane 2, Rat-1 P185 Tet BCR stable transfectants maintained for three weeks in the absence of Tet; lane 3, the Rat-1 P185 cells Tet BCR maintained in the presence of Tet (0.04 μg/ml)

Table 1 Reduction of specific phosphotyrosine activity of Bcr-Abl by induction of high levels of Bcr in Rat-1 cells transformed by P185 BCR-ABL

To characterize the long-term effects of Bcr expression on Bcr-Abl induced phosphotyrosine pattern of proteins, we performed a Tet release experiment over a 3-week period (Figure 4c and d). Rat-1/Bcr-Abl clone #3 cells were transfected with Tet vector containing the BCR gene using hygromycin selection. Cells were incubated for three weeks either in the presence or absence of Tet. In the presence of Tet (0.04 μg/ml) in which Bcr expression was reduced 2 – 3-fold (Figure 4c, compare lanes 2 and 3), there was a strong pattern of phosphotyrosine proteins (Figure 4d, lane 3). In the absence of Tet, the intensity of the phosphotyrosine pattern of proteins was greatly reduced (Figure 4d, lane 2) compared to incubation in the presence of Tet (0.04 μg/ml) (lane 3). Similarly, higher concentrations of Tet (0.1 μg/ml) also showed high levels of phosphotyrosine containing proteins (not shown). The phosphotyrosine pattern of cells incubated in absence of Tet (lane 2) (conditions permissive for Bcr expression) was similar in band intensities to Rat-1 cells lacking Bcr-Abl (lane 1). Thus, we conclude that under steady state conditions of elevated Bcr expression, the phosphotyrosine pattern of cellular proteins in Rat-1/Bcr-Abl cells was significantly reduced as a consequence of elevated Bcr expression. These results support our findings shown in Figure 3 in which Bcr expression reduced the foci inducing activity of P185 BCR-ABL. Moreover, the increased expression of Bcr in Bcr-Abl positive Rat-1 cells appeared not to cause any generalized toxic effects during this 3 week period.

Induction of Bcr expression in K562 cells reduces Bcr-Abl tyrosine phosphorylation

Our previous studies indicated that Bcr in the form of a Bcr(64 – 413) inhibited growth of K562 cells (a CML patient cell line expressing P210 BCR-ABL) and inhibited the kinase function of Bcr-Abl (Liu et al., 1996a). To determine the effects of forced expression of P160 BCR on the phosphotyrosine content of P210 Bcr-Abl in these hematopoietic cells, we transfected K562 cells with the BCR EC1214A Tet repressible vector (Hu et al., 1997) and selected K562 cells that retained their ability to grow in soft agar in the presence of 1.0 μg/ml of Tet. Several cell clones were found to express high levels of P160 BCR upon release from the Tet block.

Figure 5 shows the results of induction of Bcr expression in K562 cells with one of these clones (KB-1). In this particular clone Bcr expression was detectable after one day of cell growth in the absence of Tet (Figure 5a, lane 2) and expression peaked at day 3 (lane 3). The same extracts were also Western blotted with either anti-phosphotyrosine or anti-Abl (8E9) (Figure 5b and c). The level of phosphotyrosine containing Bcr-Abl was reduced 50% by induction of Bcr expression (Figure 5b, compare lanes 1 – 4) whereas the level of Bcr-Abl protein did not change significantly (Figure 5c). To quantitate these changes, the specific phosphotyrosine content of the Bcr-Abl oncoprotein was measured (Figure 5d). This value was calculated by dividing the intensity of the anti-pTyr Bcr-Abl band by the intensity of the anti-Abl (8E9) Bcr-Abl band. The results indicate that the phosphotyrosine content of P210 BCR-ABL was reduced about 50% by Bcr expression. Also, the results indicate that there is a threshold of Bcr concentration necessary for inhibition, as highest levels of Bcr (lane 3) were no more inhibitory than lower levels seen in lanes 2 and 4. The high basal level of Bcr-Abl tyrosine phosphorylation in K562 cells overexpressing Bcr (Figure 5) compared to the 80% reduction in the Rat-1 system (Figure 4) is likely due to multiple transforming defects acquired by K562 cells (e.g. lack of p53) besides the Bcr-Abl fusion.

Our previous studies have shown that Bcr is a target for Bcr-Abl (Lu et al., 1993), and several sites of tyrosine phosphorylation have been identified (Puil et al., 1994; Liu et al., 1996b; Wu et al., 1998). Therefore, it was of interest to determine the effects of increased Bcr expression on the level of Bcr tyrosine phosphorylation. Comparison of anti-Bcr blots with anti-phosphotyrosine blots in Figure 5b allowed identification of not only tyrosine phosphorylated P210 BCR-ABL but also tyrosine phosphorylated P160 BCR. The position of the Bcr protein was also confirmed by IP/Western blots (not shown). Of interest, the level of tyrosine phosphorylated Bcr was inhibited by induction of high levels of Bcr (compare the intensities of the P160 BCR band in Figure 5a and b). Because of the drastic increase in Bcr expression by release of the Tet block and the knowledge that Bcr is one of the targets of Bcr-Abl, we determined the change in the specific activity of tyrosine phosphorylated Bcr after release from the Tet block (Figure 5e). Just as with Bcr-Abl, the specific activity of tyrosine phosphorylated Bcr was decreased but more so than that of Bcr-Abl (compare Figure 5d and e). This result argues that induction of high levels of P160 BCR prevented the newly expressed Bcr from being tyrosine phosphorylated by Bcr-Abl. Thus, the majority of Bcr being produced in K562 cells at days 1 – 3 after release from the Tet block is resistant to tyrosine phosphorylation by Bcr-Abl. It is likely that this excess non-tyrosine phosphorylated Bcr would be active as a serine/threonine kinase and based on our results (Liu et al., 1996b; Wu et al., 1998) would therefore function as a Bcr-Abl tyrosine kinase inhibitor (Liu et al., 1996a). Note, that the level of Bcr expression peaked at day 2 (lane 3) after release from the Tet block, yet the level of Bcr-Abl inhibition was maintained at later times (e.g. day 3), suggesting that Bcr had achieved a sufficient level to maintain Bcr-Abl kinase inhibition.

Forced expression of Bcr in Src transformed cells does not inhibit Src tyrosine phosphorylation as measured by anti-phosphotyrosine immunoblotting

We examined the specificity of Bcr's inhibitory effects on a closely related tyrosine kinase, Src. Src has been shown to induce tyrosine phosphorylation of Bcr (Maru et al., 1995). Moreover, studies by Muller et al. (1992) have established that the SH2 domain of v-Src strongly binds to Bcr, consistent with Src directly phosphorylating Bcr. Maru et al. (1995) found that Fps and Fes transformed cells had higher levels of tyrosine phosphorylated Bcr than Scr transformed cells. Of interest, co-expression of Bcr and Fes in human cells stimulated the autophosphorylation of Fes on tyrosine residues (Li and Smithgall, 1996). To determine what effect Bcr expression has on Src tyrosine content, we selected clones of v-Src transformed rat-1 cells transfected with the Tet BCR vector in the presence of G418. One out of six clones had inducible levels of Bcr (Src B-1). Removal of Tet induced significant levels of Bcr expression within 2 days (Figure 6a, lane 3), with highest levels detected at days 3 and 4 after release from the Tet block (Figure 6a, lanes 4 and 5). The concentration of v-Src was measured by anti-Src monoclonal antibody and the results showed that equal numbers of cells had similar levels of pp60 SRC in the cultures following Bcr induction (Figure 6c). The pattern of phosphotyrosine proteins was not significantly affected by Bcr induction nor was the intensity of the pp60 SRC phosphotyrosine band reduced (Figure 6b) as judged by densitometry assays (not shown). Similar experiments were performed on B104-1-1 cells transformed by the activated Neu tyrosine kinase. Two of six selected clones had inducible Bcr expression. Clone Neu B-1 showed induction beginning at day 4 after release from the Tet block. Like the findings with the Src tyrosine kinase, the phosphotyrosine content of the activated Neu tyrosine kinase was not decreased by Bcr induction (not shown). We conclude from these studies and those of Smithgall and colleagues (Li and Smithgall, 1996) that Bcr down regulates the Bcr-Abl tyrosine kinase (and likely the activated c-Abl tyrosine kinase) but activates the Fes tyrosine kinase. In contrast to these effects on Abl and Fes tyrosine kinases, the Src and Neu tyrosine kinases were not significantly affected by Bcr.

Figure 6
figure6

Bcr forced expression in Src transformed Rat cells does not reduce the phosphocontent of p60 SRC. B31 cells transformed with v-Src were transfected with pEC1214A BCR construct and grown in medium containing 400 μg/ml of G418 and 1 μg/ml of Tet for 2 weeks. Six G418-resistant clones were expanded, and Tet release was performed for up to 6 days. Bcr gene expression was monitored by Western blotting with anti-Bcr (181 – 194). One of the six clones (Src B-1) had Bcr inducible expression (a). The same lysates were blotted with anti-Src (C) and with anti-Ptyr (PY20) (b). The phosphotyrosine Src is the minor band migrating just above the 55 kDa marker and beneath the very intense band. Lane 1, no release; lane 2, release from Tet block for 1 day; lane 3, release for 2 days; lane 4, release for 3 days; lane 5, release for 4 days. IP/Western experiments established the identity of the band marked Src

Discussion

In the studies presented here, lowering the level of Bcr enhanced the growth stimulatory and survival effects induced by Bcr-Abl (Figure 2). In contrast, enhancing the level of expression of Bcr blocked the tyrosine phosphorylation of Bcr-Abl and other proteins in fibroblast transformed cells (Figure 4a and b) and in hematopoietic cells (Figure 5). In longer-term studies, sustained Bcr expression in Bcr-Abl transformed fibroblasts dramatically reduced the pattern of phosphotyrosine-containing proteins (Figure 4c and d). Consistent with these long term effects of increased Bcr expression, selection of cells expressing both Bcr and Bcr-Abl reduced the number of transformed foci, demonstrating that Bcr can inhibit Bcr-Abl's oncogenic activity (Figure 3). In contrast to the effects of Bcr on Bcr-Abl, Bcr did not inhibit the tyrosine phosphorylation of Src (Figure 6) and Neu (not shown). The lack of effects of Bcr expression on Src-induced tyrosine phosphorylation is supported by our earlier published study. These studies showed that a phosphoserine Bcr peptide inhibited Bcr-Abl and Abl tyrosine kinases in vitro but did not inhibit the c-Src kinase (Liu et al., 1996a) isolated from a human colon cancer cell line (HT29) (Garcia et al., 1991). Importantly, the Fes tyrosine kinase is activated by co-expression of Bcr in human cells (Li and Smithgall, 1996). These findings taken together indicate that Bcr is a specific negative regulator of Bcr-Abl (and cytoplasmic Abl), although additional studies with other kinases are needed to establish this point.

The experiments with Bcr-Abl transduced cells containing a silent form of Bcr showed quite clearly that increased expression of Bcr by removal of the repressor (Tet) blocked the tyrosine phosphorylation of Bcr-Abl inside cells (Figures 4 and 5). Our studies with Bcr(64 – 413) suggest that the primary reason for the block in tyrosine phosphorylation (Liu et al., 1996a) is binding of phosphoserine containing Bcr to the SH2 domain of Bcr-Abl (Pendergast et al., 1991), resulting in down regulating the activated Abl tyrosine kinase of the oncoprotein (Liu et al., 1996a). However, it is likely that the inhibitory effects of full length Bcr on the oncogenic activity of Bcr-Abl (foci formation) are more complex than just Abl kinase inhibition. In this regard, Bcr possesses other functional properties that may enhance the inhibitory effects of full length Bcr on Bcr-Abl (i.e. Rac Gap of the C-terminal domain of Bcr (Diekmann et al., 1991) and Dbl domains of the central region of Bcr (Ron et al., 1991; Chuang et al., 1995).

Based on our previous studies (Puil et al., 1994; Lu et al., 1993; Ma et al., 1997) we had expected that Bcr would supplement the oncogenic effects of Bcr-Abl. Surprisingly, we found that a deletion mutant of Bcr lacking the oligo domain and comprising the bulk of the first exon sequences [Bcr(64 – 413)] was inhibitory to Abl and Bcr-Abl in vitro (Liu et al., 1996a). A phosphoserine peptide derived from this Bcr domain was also inhibitory to Abl and Bcr-Abl kinases. Moreover, Bcr(64 – 413) was resistant to tyrosine phosphorylation by activated Abl and Bcr-Abl kinases (Liu et al., 1996a) whereas Bcr(1 – 413) was readily tyrosine phosphorylated (Liu et al., 1996a; Lu et al., 1993; Ma et al., 1997). Of interest, Bcr(64 – 413) reduced the level of tyrosine phosphorylation of activated Abl and Bcr-Abl in vivo but Bcr(1 – 413) did not (Liu et al., 1996a). Thus, our findings suggest a hypothesis in which tyrosine phosphorylation of Bcr serves as a molecular switch to turn off the inhibitory effects of Bcr on the Abl tyrosine kinase.

These findings provide evidence for a model for chronic phase CML. Chronic phase CML patients have a relatively benign malignancy resulting in the accumulation of high numbers of myeloid cells in the blood. We propose that the remaining normal Bcr allele expressed in these patients, in the serine phosphorylated form, antagonizes the oncogenic effects of Bcr-Abl by direct inhibition of its tyrosine kinase activity (Liu et al., 1996a) and possibly by other inhibitory functions of Bcr. Furthermore, tyrosine phosphorylation of a portion of the Bcr molecules would neutralize these inhibitory properties of Bcr, possibly by blocking its serine kinase activity (Liu et al., 1996b; Wu et al., 1998); and possibly other activities of Bcr. In chronic phase CML patients, dominant clones of leukemic cells would be those that either express low amounts of Bcr relative to Bcr-Abl or those that express relatively high levels of Bcr-Abl relative to normal levels of Bcr. We have begun to explore the possibility that a form of Bcr that is resistant to tyrosine phosphorylation (Liu et al., 1996a) might have therapeutic potential in CML patients.

Our model would appear to conflict with the recent data published by the Groffen group (Voncken et al., 1995, 1998). The most recent findings (Voncken et al., 1998) indicate that Bcr has no significant role in leukemias induced in BCR-ABL transgenic mice. These experiments were performed by mating BCR knockout mice with BCR-ABL transgenic mice. Two interpretations of these findings are possible that may explain the apparent discrepancy. First, a backup gene or genes may be able to replace the lost function of the intact BCR gene, a finding that is quite common in many gene knockout mice (e.g. SRC knockout mice). Secondly, since the BCR gene knockout was accomplished by inserting foreign sequences into the second exon of Bcr, it raises the possibility that first exon-encoded fragments of Bcr are produced in the knockout mice. Further studies on such mice will shed crucial information on the role of Bcr in CML.

Materials and methods

Cell culture, antibodies, oligos, and vectors

Rat-1 cells were supplied by Mikhail L Gishizky (Sugen, Inc. Redwood, CA, USA). SUP B15 cells were derived from a Ph-positive ALL patient expressing P185 BCR-ABL (Naumovski et al., 1988). The KBM-7 and K562 cell lines were isolated from CML blast crisis patients; KBM-7 cells express P210 BCR-ABL but unlike K562 cells lack a functional Bcr protein (Liu et al., 1993). M3.16 were derived from Mo7e cells after transfection with a vector encoding P210 BCR-ABL (Sirard et al., 1994). B31 cells are a Rat-1 cell line transformed by v-Src (Varmus et al., 1981). B104-1-1 cells are NIH3T3 cells transformed by activated Neu (Hung et al., 1989). Anti-Abl 8E9, anti-phosphotyrosine (pY20, Transduction Labs, Lexington, KY, USA), anti-Bcr(181 – 194) and anti-Bcr(1256 – 1271) were used as described (Liu et al., 1993, 1996a). Src was immunoprecipitated and detected in immunoblotting experiments with monoclonal antibody 327 (Mab 327, Oncogene Science, Uniondale, NY, USA). Neu was detected with anti-Neu (Ab3, Oncogene Science, Uniondale, NY, USA). BCR anti-sense (BCR3351-3368) oligodeoxynucleotides: 5′-ATCATCACCGACACATCC-3′ and BCR sense: 5′-GGATGTGTCGGTGATGAT-3′ were synthesized; two phosphothioesters linkages were placed at each end of the oligo (GenoSys Biotech., Inc. Houston, TX, USA). Retrovirus vectors include: pLXSN (provided by Dr A Dusty Miller, Fred Hutchinson Cancer Research Center, Seattle, WA, USA) containing BCR-ABL (P185); pLXSH (provided by Dr A Dusty Miller, Fred Hutchinson Cancer Research Center, Seattle, WA, USA) containing BCR (P160); pJJ801 encoding Moloney murine leukemia virus; pLNL SLX CMV (supplied by Dr Jean Wang, University of CA at San Diego) encoding BCR (P160); pMEP4 (Invitrogen, Carlsbad, CA, USA) encoding BCR (P160) controlled by a dexamethasone inducible promoter; pEC1214A encoding BCR (P160) controlled by a Tetracycline (Tet) repressible promoter (Hu et al., 1997).

Foci formation

Rat-1 cells (70% confluent 3.5 cm dish) were transfected with 1 μg of a retrovirus vector (pLXSN) encoding P185 BCR-ABL (plus 0.2 μg of helper virus vector pJJ801). After 2 days the cells were split into three 6.0 cm dishes and selected with G418 to observe foci formation. After selecting a stably expressing cell clone (three times by limiting dilution, termed clone #3), cells were transfected with vector (pLXSH) or pLXSH BCR (P160 PCR) for growth in hygromycin (for P160 BCR expression). Foci were allowed to form over several weeks.

Release of Tet repression

Rat-1 cells stably expressing P185 BCR-ABL (clone #3) were transfected with pEC1214A (Hu et al., 1997) encoding P160 BCR using 0.1 ratio of pLXSH. Foci of transformed cells were selected in the presence of hygromycin and expanded; one that expressed P185 BCR-ABL and P160 BCR upon release from Tet expression was selected for further studies (termed clone Rat B-1). Cells were maintained in 0.1 μg/ml of Tet. Release experiments were done by incubating cells for 1 – 6 days for short-term experiments or 3 weeks for long-term experiments in the absence of Tet. Extracts were made in SDS sample buffer and Western blotted with pY20, anti-Bcr and anti-Abl 8E9. Similar experiments were performed on soft agar clones of K562 transfected with pEC1214A (Hu et al., 1997) encoding P160 BCR and Src transformed B31 cells and Neu transformed B104 cells. Src and Neu antibodies described above were used to assess expression of these proteins.

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Acknowledgements

RBA holds the Stringer Chair of Cancer Research. We thank Dr A Dusty Miller of the Fred Hutchison Cancer Research Center for the pLXSN for pLXSH retrovirus vectors and Dr Mikhail L Gishizky from Sugen, Inc. (Redwood, CA, USA) for Rat-1 cells. Special thanks to Gary Gallick and Mien-chie Hung's laboratories for supplying Src and Neu transformed cells, respectively, and for supplying antibodies. This work was supported by grants from the NIH (CA65611, CA49639 and CA16672).

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Correspondence to Ralph B Arlinghaus.

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Wu, Y., Ma, G., Lu, D. et al. Bcr: a negative regulator of the Bcr-Abl oncoprotein. Oncogene 18, 4416–4424 (1999). https://doi.org/10.1038/sj.onc.1202828

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Keywords

  • Bcr
  • Bcr-Abl
  • Bcr-Abl inhibitor
  • tyrosine kinase

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