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in ALL, the fusion protein is 185 kDa and contains the first 426 aa of Bcr. These Bcr ... is present in the Gag–v-Abl fusion protein of A-MuLV, while. Bcr-Abl lacks such a site. ...... and S. Oroszlan. 1984. Myristylation of gag-onc fusion proteins in.
MOLECULAR AND CELLULAR BIOLOGY, Mar. 1995, p. 1286–1293 0270-7306/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 15, No. 3

The Human Leukemia Oncogene bcr-abl Abrogates the Anchorage Requirement but Not the Growth Factor Requirement for Proliferation MARK W. RENSHAW, JOHN R. MCWHIRTER,

AND

JEAN Y. J. WANG*

Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0347 Received 20 September 1994/Returned for modification 7 November 1994/Accepted 28 November 1994

Proliferation of normal cells in a multicellular organism requires not only growth factors but also the proper attachment to the extracellular matrix. A hallmark of neoplastic transformation is the loss of anchorage dependence which usually accompanies the loss of growth factor requirement. The Bcr-Abl tyrosine kinase of human leukemias is shown here to abrogate only the anchorage, not the growth factor, requirement. Bcr-Abltransformed cells grow in soft agar but do not proliferate in serum-free media. Bcr-Abl does not activate the mitogenic pathway, as indicated by its inability to induce enhancers such as the serum response element or the tetradecanoyl phorbol acetate response element (TRE). However, Bcr-Abl can alleviate the anchorage requirement for the induction of the TRE enhancer; i.e., it allows serum to activate the TRE in detached cells. This activity is dependent on the association of an active Bcr-Abl tyrosine kinase with the actin filaments. Despite its association with the adapter protein Grb2, Bcr-Abl’s effect on the TRE enhancer is not blocked by dominant negative Ras or Raf. The finding that Bcr-Abl tyrosine kinase abrogates only anchorage dependence may have important implications on the pathogenesis of chronic myelogenous leukemia. mechanism by which bcr-abl induces the semitransformed phenotype of the chronic phase is not understood. The Bcr-Abl protein contains many functional domains, several of which are required for transformation. Two regions in Bcr are essential. The first 63 aa of Bcr contain a coiled-coil tetramerization domain which activates the tyrosine kinase as well as the F-actin binding function of Abl (37, 38). The second Bcr region, between aa 176 and 242, contains a tyrosine residue at position 177 which becomes phosphorylated and binds to the SH2 domain of the adapter protein Grb2 (45, 46). Mutation of tyrosine 177 to phenylalanine abolishes Grb2 binding and reduces the ability to transform Rat-1 fibroblasts. Three regions in Abl, the SH2 domain, the tyrosine kinase domain, and the F-actin binding domain, are required for transformation of Rat-1/myc cells (39). The abrogation of interleukin-3 dependence in BAF3 cells, however, requires only the tyrosine kinase and F-actin binding functions, not the SH2 domain, of Abl (39). Another oncogenic derivative of Abl is the Gag–v-Abl fusion protein of the Abelson murine leukemia virus (A-MuLV). The Bcr-Abl and v-Abl proteins differ in two ways. The SH3 domain of Abl is intact in Bcr-Abl but deleted in v-Abl. An N-terminal myristoylation site from the retroviral Gag protein is present in the Gag–v-Abl fusion protein of A-MuLV, while Bcr-Abl lacks such a site. In transformed Rat-1 cells, Bcr-Abl is colocalized exclusively with actin filaments, whereas Gag–vAbl was diffused throughout the cytoplasm (39). The differences between Bcr-Abl and Gag-v-Abl should affect the transforming activities; however, this has not been observed with the previously described transformation assays for Bcr-Abl. While studying the transforming function of Gag–v-Abl, we found that this oncogenic tyrosine kinase transforms only cells with a permissive context (47). We have isolated two stable subclones of NIH 3T3 cells, the P- and the N-3T3 clones, on the basis of their responses to the v-Abl tyrosine kinase. AMuLV induces serum- and anchorage-independent growth in the P-3T3 cells but causes G1 arrest in the N-3T3 cells (47). It

Human chronic myelogenous leukemia (CML) and a subset of acute lymphocytic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph1), a translocation in which the breakpoint cluster region (bcr) gene on chromosome 22 becomes fused to the c-abl proto-oncogene on chromosome 9 (6, 12, 30). In CML, a 210-kDa Bcr-Abl protein is produced containing either the first 927 or 902 amino acids (aa) of Bcr, while in ALL, the fusion protein is 185 kDa and contains the first 426 aa of Bcr. These Bcr amino acids are fused with 1,097 aa of c-Abl, resulting in a chimeric protein with deregulated tyrosine kinase activity (8). The oncogenic potential of Bcr-Abl can be demonstrated in several different assays. Expression of Bcr-Abl in Rat-1 fibroblasts, which also overproduce the c-Myc protein, leads to growth in soft agar (32, 33). Bcr-Abl can also abrogate the interleukin-3 requirement of murine BAF3 cells (7). Infection of primary bone marrow cells with p210bcr-abl retrovirus leads to the clonal outgrowth of pre-B-lymphoid cells that express high levels of the fusion protein (20, 35, 63). However, these p210bcr-abl-expressing clones showed a wide variation in tumorigenicity, suggesting that Bcr-Abl alone is not sufficient to induce oncogenic transformation (20, 35, 36). Animal models for CML have been developed through the reconstitution of lethally irradiated mice with bone marrow cells infected with Bcr-Abl-bearing retrovirus (60). The reconstituted mice developed several different types of hematological malignancies, the most frequent of which was a myeloproliferative CML-like syndrome (10, 28). Massive splenomegaly characteristic of CML, as well as a predominance of mature functional granulocytes in peripheral blood, was observed. Thus, bcr-abl could stimulate the expansion of the myeloid compartment but did not stop the myeloid differentiation. This observation is consistent with the chronic phase of the human CML (5, 8). The * Corresponding author. Mailing address: Department of Biology and Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0347. Phone: (619) 5346253. Fax: (619) 534-0555. Electronic mail address: [email protected]. 1286

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TABLE 1. Cell context-dependent transforming capabilities of v-abl and bcr-abl Plasmid

pSLX pSLX-v-abl pSLX-bcr-abl pSLX pSLX-v-abl pSLX-bcr-abl a b c

Cell line

N-3T3 N-3T3 N-3T3 P-3T3 P-3T3 P-3T3

No. of foci (avg 6 SE) inb:

Transfection efficiencya (avg 6 SE)

10% CS

0.5% CS

Growth in soft agarc (avg 6 SE)

1,498 6 801 1,140 6 622 860 6 454 1,210 6 145 1,006 6 156 1,375 6 248

0 585 6 32 0 0 2,252 6 698 683 6 162

0 0 0 0 2,103 6 642 38 6 28

0 0 0 0 2,270 6 988 755 6 254

Measured as the number of neomycin-resistant colonies selected in medium containing G418 (1 mg/ml) per microgram of plasmid DNA. Number of foci formed per microgram of plasmid DNA in medium containing 10 or 0.5% calf serum (CS). Number of colonies per microgram of plasmid DNA able to grow in soft agar.

was previously reported that bcr-abl did not transform NIH 3T3 cells (9). Since Gag–v-Abl transforms only the P-3T3 cells, we examined if bcr-abl can transform this subclone of NIH 3T3 cells. We show here that Bcr-Abl could indeed transform the P-3T3 cells. Unlike v-abl, bcr-abl can abrogate only the anchorage requirement for growth. The fact that bcr-abl can only induce anchorage independence may account for the semitransformed phenotype in the chronic phase of CML. MATERIALS AND METHODS Cell culture. NIH 3T3 (Lewis clone 7) and COS cells were cultured in Dulbecco’s modified Eagle’s medium with 10% defined-supplemented bovine calf serum (HyClone), 200 U of penicillin per ml, and 200 mg of streptomycin per ml. Neomycin-resistant colonies were selected in 1 mg of G418 per ml. Growth factor-independent clones were selected in 0.5% serum. Soft agar colonies were selected in medium containing 0.3% Bacto agar (Difco). Growth factor-independent foci were selected in Dulbecco’s modified Eagle’s medium with 0.5% calf serum. Cell proliferation was measured by the incorporation of [3H]thymidine after a 1-h pulse-labeling with 1 mCi/ml as previously described (40). The P-3T3 and N-3T3 clones were isolated and characterized as previously described (47). Plasmid constructs. Expression plasmids used in this study were previously described (37–39). The p210 coding fragment was cleaved from pSP65 Bcr-Abl p210 (19) and cloned into the retroviral vector pSLX CMV (51). pCH110 was purchased from Pharmacia. Plasmid p2XTRE contains four AP-1 sites in front of the luciferase gene of p19 LUC (58). pJUN contains positions 21700 to 1740 of the c-jun promoter in p19 LUC. pFL3 contains 2730 to 11 of the c-fos promoter (13) in p19 LUC. Plasmid KSRSPA expresses a dominant negative c-Raf protein with a mutation in the ATP binding site at Lys-301. Plasmid pZIPNeo SVRas N17 expresses a dominant negative c-Ras protein, S17N. Cell transfection. NIH 3T3 cells were transfected by the calcium phosphate precipitation method previously described (23). COS cells were transiently transfected by the DEAE-dextran method as previously described (38). In transformation assays, cells were transfected with 1 mg of the bcr-abl or v-abl construct and 10 mg of pZAP, the replication-competent Moloney viral DNA (21, 53). Transfection efficiency was determined by G418 resistance. In transient transfection assays, duplicate sets of plates were cotransfected with 1 mg of pCH110, 1 mg of the luciferase reporter plasmid, 5 mg of the pSLX CMV construct, and 5 mg of pZAP or the dominant negative Ras or Raf plasmid, pZIPNeo SVRas N17 or pRSPA cRaf 301. The cells were made quiescent 12 h posttransfection, stimulated 24 h later with 10% calf serum for 4 h, and then harvested for the luciferase assay. Detached cells were prepared by trypsinization prior to stimulation with serum. Briefly, the monolayer was rinsed with phosphate-buffered saline and incubated with ATV trypsin (Irvine Scientific) at 378C for 2 min. Trypsinized cells were resuspended in 3 ml of medium containing 0.1% serum and 0.2% bovine serum albumin (fraction 5) and placed in a Falcon 2059 tube. Calf serum (0.3 ml) was added to the tube, which was then attached to a platform shaker at a 458 angle and incubated for 4 h at 378C. To perform the luciferase assay, cells were lysed by sonication in 100 ml of 100 mM KH2PO4–1 mM dithiothreitol. Insoluble material was removed by spinning in a microcentrifuge for 10 min. Luciferase and b-galactosidase activities were measured from equal amounts of protein as previously described (14, 27). The ratio of the two activities was then expressed as fold activation over that of the unstimulated attached cells. Virus. bcr-abl or v-abl virus was harvested from transformed P-3T3 cells generated as described above. The titer was determined as the number of focusforming units per milliliter for A-MuLV and soft agar colony-forming units per milliliter for bcr-abl virus. For infection, cells were incubated for 2 h with a 1:1 mixture of viral stock and fresh medium containing hexadimethrine bromide (Sigma) at 8 mg/ml.

Immunoprecipitations and immunoblotting. Cells were lysed at approximately 3 3 106 cells per ml in immunoprecipitation lysis buffer (50 mM Tris [pH 8.0], 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], 0.004% NaN3, 10 mM sodium PPi [pH 7.0], 50 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 13 protease inhibitor cocktail [10 mM benzamidine-HCl, 10 mg each of phenanthroline, aprotinin, leupeptin, and pepstatin per ml]). Lysates were precleared by ultracentrifugation at 100,000 3 g for 30 min at 48C and incubated for 3 h with either monoclonal anti-Abl 8E9 or polyclonal anti-Gag or anti-Bcr antiserum (37, 48). Immune complexes were precipitated for 1 h with formaldehyde-fixed Staphylococcus aureus beads as previously described (59). Beads were washed four times in immunoprecipitation lysis buffer and boiled in SDS-polyacrylamide gel electrophoresis sample buffer (67 mM Tris-HCl [pH 6.8], 10 mM EDTA, 2% SDS, 10% glycerol, 0.3% 2-mercaptoethanol, 0.03% bromophenol blue) by boiling for 15 min. Proteins were separated on SDS-polyacrylamide gels, and immunoblotting with anti-Grb2 (Transduction Laboratories), antiphosphotyrosine, anti-Abl, or anti-Bcr antibodies was performed as previously described (38, 48, 59).

RESULTS Transformation by bcr-abl requires a permissive cell context. Despite a previous report that Bcr-Abl did not transform NIH 3T3 cells (9), we found that this activated tyrosine kinase could induce foci in the P-3T3 cells (Table 1). The P-3T3 subclone comprised only about 5% of the total NIH 3T3 population (47). With the N-3T3 clone, which represents the majority of NIH 3T3 cells, expression of bcr-abl did not lead to focus formation (Table 1). The inability of bcr-abl to transform 3T3 cells in earlier experiments performed by Daley et al. (9) could be due to the use of N-3T3 type cells. In the focusforming assay, differences between v-abl and bcr-abl could be detected (Table 1). The v-abl oncogene generated foci in both cell types, with a higher focus-forming efficiency in the P-3T3 cells (Table 1). The bcr-abl gene, on the other hand, generated foci only in the P-3T3 cells and at threefold-lower efficiency than v-abl (Table 1). The bcr-abl-induced foci were smaller and did not show as dramatic a shape change as the v-abl-transformed cells. This could be due to a lower level of expression of Bcr-Abl than v-Abl in these 3T3 cells (Fig. 1A) and is consistent with previous reports that bcr-abl is a weaker transforming agent than v-abl (9, 11, 35). Bcr-Abl abrogates the anchorage but not the serum requirement for growth. To determine if the bcr-abl-transformed cells acquired anchorage independence, we tested their ability to grow in soft agar. The bcr-abl-transformed P-3T3 cells formed soft agar colonies at the same frequency as they formed foci in 10% serum (Table 1). This result showed that every morphologically transformed P-3T3 cell was capable of anchorageindependent growth. In contrast, neither bcr-abl or v-abl induces soft agar colonies in the N-3T3 cells (Table 1). To determine if the transformed cells showed a reduced requirement for growth factors, they were also tested for focus formation in 0.5% serum. Interestingly, while v-abl-transformed foci grew in 0.5% serum at the same frequency as in 10%

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FIG. 1. Expression of Bcr-Abl and Gag–v-Abl in transfected P-3T3 cells. (A) Immunoblots of lysates from pooled P-3T3 cells transfected with the pSLX CMV vector (lane 1), pSLX–v-abl (lane 2), or pSLX-bcr-abl (lane 3) or from COS cells transiently transfected with pSVL-bcr-abl. Stably transfected P-3T3 cells were selected as G418-resistant colonies; about 100 such colonies were pooled, and their lysates were analyzed. The samples were probed with antibodies for Abl, phosphotyrosine (Ptyr), or Bcr as indicated. Note that the Bcr-Abl in the stable clones of P-3T3 cells has the same size as the control generated upon transient transfection. (B) Immunoblots of lysates of individual P-3T3 clones isolated after transfection with the indicated plasmids. The vector (lane 1)- or the bcr-abl (lane 3)-transfected clone was isolated as a G418-resistant colony. The v-abl-transfected clone (lane 2) was isolated as a fully transformed colony. The gag-bcr-ablexpressing clone (lane 4) was selected as a focus in 0.5% serum. The control bcr-abl (lane 5) was as described in for panel A. Total lysates were probed with anti-Abl, anti-Ptyr, or anti-Bcr antibodies. To determine if the protein contains the retroviral Gag sequences, lysates were first reacted with anti-Gag and the immunoprecipitated proteins were then probed with anti-Abl. Note that the Gag–v-Abl and the Gag-Bcr-Abl proteins but not c-Abl or Bcr-Abl protein were immunoprecipitated with the anti-Gag antibodies.

serum (Table 1), only a small percentage (less than 6%) of the bcr-abl foci could grow in 0.5% serum (Table 1). This result suggested that the majority of bcr-abl-transfected cells required growth factors to proliferate. The inability of Bcr-Abl to stimulate DNA synthesis was demonstrated by another assay. P-3T3 cells were quantitatively infected with v-abl or bcr-abl virus and then assayed for DNA synthesis in 0.5% serum (Table 2). Infection with A-MuLV caused P-3T3 cells to synthesize DNA in reduced serum (47) (Table 2). However, infection with the bcr-abl virus did not

TABLE 2. DNA synthesis rates in A-MuLV- or bcr-abl virus-infected P-3T3 cells Virusa

Mock v-abl bcr-abl

[3H]thymidine incorporation (103 cpm; avg 6 SE)b Medium 1 10% CSc

Medium 1 0.5% CSd

68.8 6 4.7 73.6 6 7.1 66.9 6 6.5

3.7 6 0.5 60.7 6 5.9 7.0 6 2.3

a Cells were either mock infected or infected with v-abl at a multiplicity of infection of 5 or bcr-abl at a multiplicity of infection of 3. b During a 1-h pulse-labeling using 1 mCi/ml. c Fresh medium containing 10% calf serum (CS) was given 12 h prior to pulse-labeling. d Cells were placed in medium containing 0.5% calf serum 48 h prior to pulse-labeling.

have this effect (Table 2). This finding raised the question as to the origin of the few serum-independent foci found in bcr-abltransfected cells (Table 1). Daley et al. reported that a recombinant of Bcr-Abl with the retroviral Gag protein could transform 3T3 cells (9). Since helper viral DNA was included in our transfections, we examined whether the serum-independent foci expressed Gag-Bcr-Abl. As shown in Fig. 1B, normal-size Bcr-Abl was detected in a clone selected by neomycin resistance (lane 3), but a higher-molecular-weight form of Bcr-Abl was detected in a clone selected by serum independence (lane 4). The larger Bcr-Abl protein could be immunoprecipitated with anti-Gag antibodies, as was the Gag–v-Abl protein (lane 2). The normal Bcr-Abl protein did not react with anti-Gag (lane 3). The Gag-Bcr-Abl recombinant was detected in all of the serum-independent clones tested. The formation of serumindependent foci, therefore, appeared to be induced by GagBcr-Abl but not Bcr-Abl. We and others have previously shown that Bcr-Abl is associated with actin filaments (11, 38, 39). The Gag–v-Abl and Gag-Bcr-Abl proteins, however, show diffuse cytoplasmic staining and could associate with the membrane through a myristoylation site in Gag (9, 11, 39, 54). The subcellular locations of Bcr-Abl, Gag–v-Abl, and Gag-Bcr-Abl were examined in the transformed P-3T3 cells and found to be consistent with the previous reports (data not shown). Taken together, these results showed that Bcr-Abl induced anchorage-independent growth when bound to F-actin but could induce serumindependent growth when brought to the membrane. Bcr-Abl does not activate serum-responsive enhancer elements. The mitogenic signal transduction pathway is known to stimulate the c-fos and c-jun promoters in NIH 3T3 cells (24, 49). The key element in the c-fos promoter is the serum response element (SRE), which is activated by a mitogen-activated protein kinase-mediated stimulation of the p62elk transcription factor (34). The key element in the c-jun promoter is the tetradecanyl phorbol acetate response element (TRE), which is the binding site of Jun/Fos heterodimer (1, 26, 41). The activation of both the SRE and the TRE by serum can be demonstrated in transient assays using the appropriate reporter plasmids (Fig. 2). In the P-3T3 cells, cotransfection with v-abl led to 7- and 12-fold activation of the SRE and TRE, respectively (Fig. 2). Cotransfection with bcr-abl did not activate either the SRE or the TRE in the P-3T3 cells; however, these reporters remained responsive to serum stimulation (Fig. 2). The inability of bcr-abl to stimulate the SRE and TRE was consistent with the fact that bcr-abl could not stimulate DNA synthesis in these cells (Table 2). Bcr-Abl alleviates the anchorage requirement for TRE activation by serum. Fibroblastic cells, such as NIH 3T3 cells, are anchorage dependent for growth. When these cells are not properly anchored, they cannot respond to growth factors (4, 43). Since bcr-abl can abrogate anchorage dependence, we tested whether this activity would be demonstrated in a transient transfection assay. First, we examined whether anchorage was required for serum to induce the SRE or the TRE. As summarized in Table 3, induction of the SRE by serum was not dependent on anchorage. The same level of activation was observed in attached cells and in cells that were trypsinized and kept in suspension prior to the addition of serum (see Materials and Methods). Since the activation of the SRE was intact, the trypsinization procedure must not have damaged the cell surface receptors. In the detached cells, however, serum could no longer activate the TRE (Table 3). The TRE was activated between six- and sevenfold by serum in attached cells, but virtually no activation was observed in cells either trypsinized

VOL. 15, 1995

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FIG. 2. Effects of v-Abl and Bcr-Abl on the activation of the SRE and TRE enhancers. An SRE-luciferase reporter, pFL3 (A), or a 2XTRE-luciferase reporter (B) was cotransfected into P-3T3 cells with the indicated pSLX expression plasmids. Transactivation was measured by assaying luciferase activity from cells which had been placed in 0.5% serum for 24 h (0.5% calf serum [CS]). A duplicate sample of these transfected cells were treated with serum for 4 h (1 10% CS) as a positive control. Values are fold induction over that of the vector-transfected cells and represent the averages of three separate experiments. Equal amounts of protein were used in all luciferase assays, and transfection efficiencies were normalized with the cotransfection of b-galactosidase (see Materials and Methods).

or scraped (Table 3). Activation of the c-jun promoter by serum was also dependent on cell attachment (Table 3). With the knowledge that serum induction of the TRE is dependent on anchorage, we then tested if bcr-abl could abrogate that requirement when cotransfected with the reporter plasmid. Although bcr-abl by itself had little effect on the TRE activity in either attached or detached cells, the bcr-abl-transfected cells were able to respond to serum even when they were trypsinized or scraped (Table 3). This result suggests that BcrAbl can abrogate the anchorage requirement for the serum induction of the TRE. It has previously been reported that Bcr-Abl contains a binding site for the adapter protein Grb2, and it was suggested that Bcr-Abl might activate the Ras-Raf pathway (45, 46). In both the P- and N-3T3 cells, we could confirm the association of Bcr-Abl and Gag–v-Abl with Grb2 by coimmunoprecipitation (Fig. 3). Thus, the binding to Grb2 did not determine the biological response because the N-3T3 cells are not transformed by either bcr-abl or v-abl (Table 1). To test whether Ras or Raf was involved in the ability of Bcr-Abl to abrogate the anchorage requirement, we cotransfected dominant negative ras or raf mutants with bcr-abl and assayed the serum induction of TRE in attached or detached

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FIG. 3. Grb2 associates with oncogenic Abl proteins. Duplicate plates of P-3T3 (P) or N-3T3 (N) cells were either mock infected (lanes 1 to 4) or infected with virus bearing v-abl (lanes 5 to 8), bcr-abl (lanes 9 to 12), or recombinant gag-bcr-abl (lanes 13 to 16) at a multiplicity of infection of greater than 3 so that virtually every cell was infected. Three days after infection, the total cell populations were harvested and lysed, and the lysates were immunoprecipitated with anti-Abl 8E9 (see Materials and Methods). The immunoprecipitates were separated on SDS–7.5 or 12% polyacrylamide gels and then immunoblotted with anti-Abl or anti-Grb2 antibodies, respectively.

cells (Fig. 4). As expected, the dominant ras and raf both inhibited induction of the TRE by serum in attached cells (Fig. 4A, vector). Interestingly, when bcr-abl was cotransfected with these two mutants, less inhibition was observed in attached cells (Fig. 4A, Bcr-Abl). This result indicated that Bcr-Abl could block the inhibitory effects of these ras and raf mutants. As described above, serum did not induce the TRE in detached cells (Fig. 4B, vector), but serum could activate the TRE in detached cells transfected with bcr-abl (Fig. 4B, Bcr-Abl). The ability of Bcr-Abl to replace cell attachment was not blocked by the cotransfection with dominant ras or raf mutants. These results indicated that the abrogation of anchorage requirements by Bcr-Abl is likely to be mediated by a Ras-Raf-independent mechanism. Functional domains of Bcr-Abl required for abrogation of the anchorage requirement. To determine which of the many domains of Bcr-Abl were required to substitute for cell attachment, we tested a series of Bcr-Abl mutants (Fig. 5) in the P-3T3-based assays (Table 4). The expression and tyrosine kinase activities of these mutants have previously been characterized (39). A Bcr-Abl-p210 cDNA prepared from the CML cell line K562 (19) gave identical results in the assays as our constructed Bcr-Abl fusion containing the first 509 aa of Bcr (Fig. 5 and Table 4). Thus, addition of Bcr aa 510 to 927 did not alter the biological function of Bcr-Abl in these assays. The first 63 aa of Bcr contain a coiled-coil tetramerization domain which is responsible for the activation of the Abl tyrosine kinase (37). This domain was required, since the fusion protein

TABLE 3. Serum induction of the TRE in detached cellsa Serum induction in indicated cells Reporter

SRE 2XTRE Jun

Medium

0.1% CS 110% CS 0.1% CS 110% CS 0.1% CS 110% CS

Attached

Trypsinized

Scraped

Vector

bcr-abl

Vector

bcr-abl

Vector

bcr-abl

1.0 8.0 6 1.2 1.0 6.7 6 2.7 1.0 4.8 6 1.3

1.0 6 0.1 7.5 6 2.5 1.1 6 0.2 6.4 6 1.6 1.0 6 0.1 4.9 6 0.5

1.5 6 0.5 5.0 6 1.0 1.0 6 0.2 1.3 6 0.4 1.0 6 0.2 1.0 6 0.2

1.5 6 0.5 6.5 6 0.5 1.0 6 0.1 5.8 6 1.0 0.9 6 0.1 4.8 6 0.8

1.0 1.0

1.0 6 0.1 5.0 6 0.5

a Data were obtained from transient transfections as detailed in Materials and Methods. Values represent the average fold serum induction of luciferase activity compared with uninduced cells 6 standard error. Cells were allowed to remain attached to the plate (attached) or were removed from the plate by using trypsin (trypsinized) and placed in suspension in liquid medium as detailed in Materials and Methods. As a control for possible digestion of receptors by trypsin, other cells were removed from the plate by scraping and then placed in suspension (scraped). Cells either remained in serum-deprived medium (0.1% calf serum [CS]) or were stimulated by the addition of calf serum (110% CS) for 4 h. Suspension cells were stimulated with serum after being suspended.

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ble 4). We have previously shown that mutation in the F-actin binding domain or the SH2 domain caused only a two- to fourfold reduction in the transformation of Rat-1/myc cells (39). The same mutations had a more severe effect in the P-3T3 transformation. The overexpression of exogenous myc in the Rat-1/myc cells might have caused the observed quantitative difference between the two assay systems (33, 39). Thus, localization of an active Bcr-Abl tyrosine kinase to the actin filaments was required for promoting the anchorageindependent growth of P-3T3 cells. DISCUSSION

FIG. 4. Dominant negative ras and raf mutants do not affect Bcr-Abl activity. P-3T3 cells were cotransfected with a TRE-luciferase reporter and the combination of Bcr-Abl and dominant negative Ras or Raf (D.N.Ras or D.N.Raf) plasmid (Bcr-Abl). As controls, the ras and raf mutants were cotransfected with vector alone (Vector). The transfected cells were left on the culture dish (A) or trypsinized (B) at 36 h posttransfection. At 12 h posttransfection, cells were made quiescent (Q) by replacing the regular growth medium with that containing only 0.5% serum. The attached or detached cells were stimulated with 10% serum (S) for 4 h. The induction of TRE activity was measured as described in Materials and Methods. Each value represents the average of two independent experiments.

Bcr/63-509-Abl was unable to induce foci or soft agar colonies and did not allow serum to activate the TRE in detached cells (Table 4). The Bcr tetramerization domain, although necessary, was not sufficient to abrogate the anchorage requirement. This was indicated by the failure of Bcr/1-175-Abl to function in P-3T3 cells (Table 4). However, the inclusion of only 16 additional aa, as in Bcr/1-191-Abl, restored the biological activity (Table 4). Bcr aa 192 to 242 and 298 to 413, previously shown to bind the Abl SH2 domain (44), are not necessary for abrogation of the anchorage requirement. A hybrid construct containing Bcr aa 1 to 63 and 176 to 509 (Bcr/1-631176-509Abl) was also active in the P-3T3 assays (Table 4). These results demonstrated that at least two regions of Bcr, the Nterminal tetramerization domain and aa 176 to 191, are required for the promotion of anchorage-independent growth in P-3T3 cells. On the Abl side of the fusion, inactivation of the tyrosine kinase (Bcr-Abl/KD) completely abolished the anchorage-abrogating function (Table 4 and Fig. 5). Deletion of the DNA binding domain (Bcr-Abl/DXS) had only a small effect on the transforming activity (Table 4). Mutation of the actin binding domain (Bcr-Abl/isTth or Bcr-Abl/DSal) or the SH2 domain (Bcr-Abl/DSH2) caused a 30-fold reduction in the activity (Ta-

The Bcr-Abl tyrosine kinase is shown to abrogate the anchorage but not the growth factor requirement of the P subtype of NIH 3T3 cells (Tables 1 and 2). Bcr-Abl does not activate the SRE or TRE enhancer in these cells (Fig. 2). However, Bcr-Abl can restore the serum induction of the TRE in detached cells which normally cannot respond to mitogenic stimulation (Table 3). The ability of Bcr-Abl to abrogate the anchorage requirement requires two regions of Bcr, the Nterminal coiled-coil tetramerization domain and aa 176 to 191, which contain the binding site for the adapter protein Grb2 (Fig. 5 and Table 4). The putative SH2 binding regions of Bcr (44), however, are dispensable. The SH2, tyrosine kinase, and F-actin binding functions of Abl are also required for the induction of anchorage independence. It appears that the ability of the oncogenic Abl tyrosine kinase to activate the mitogenic pathway is determined by its subcellular localization. Bcr-Abl is mostly associated with the actin stress fibers, and it cannot activate the mitogenic pathway. A recombinant Gag-Bcr-Abl protein, however, can induce growth factor independence in the same cells, presumably as a result of the relocalization of Bcr-Abl to the membrane (reference 11 and data not shown). Membrane translocation of signal transducers has been shown to be critical for the activation of the mitogenic pathway. This is illustrated by the construction of a membrane-localized Raf (31, 55) or SOS (2). Under normal circumstances, activation of the Raf tyrosine kinase requires the activation of Ras (62). The activation of Ras requires the membrane translocation of the Grb2/SOS complex (42). With a membrane-localized Raf, however, the requirement for Grb2/SOS and Ras can be bypassed (31, 55). Our results with the Bcr-Abl protein are consistent with these findings. Because the phosphorylated tyrosine 177 of Bcr can bind the SH2 domain of Grb2, it has been proposed that Bcr-Abl signals through the Ras pathway (45). Although mutation of Y-177 impaired the ability of bcr-abl to induce soft agar colonies in Rat-1 cells, there was no evidence that Bcr-Abl could substitute for growth factors in those cells (45). With the transient transfection assay of TRE activation in detached cells, we have found that this activity of Bcr-Abl is not blocked by a dominant negative mutant of Ras or Raf (Fig. 4). Taken together, our results indicate that the Ras-Raf pathway may not be directly involved in the Bcr-Abl-mediated signaling. It is interesting to find that cell attachment is necessary for serum to induce the TRE but not the SRE. In human monocytes, adherence alone is sufficient to activate the immediateearly response indicated by the induction of c-fos and c-jun expression (52). Interestingly, the activation of c-jun but not c-fos is blocked by cytochalasin D, indicating that cytoskeleton rearrangement is important for the induction of c-jun but not c-fos. Cell attachment is known to activate protein tyrosine kinases, the best example being the focal adhesion kinase (3, 29, 61). Lack of adhesion also suppresses the synthesis of phosphatidylinositol biphosphate and impairs signal transduc-

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FIG. 5. Summary of Bcr, c-Abl, and the different Bcr-Abl fusion constructs. (A) The human Bcr contains 1,271 aa. Within the first 63 aa is a coiled-coil domain that mediates the formation of homotetramers. The tyrosine at position 177 (Y-177) is a binding site for Grb2. The Bcr-Abl of ALL contains the first 426 aa of Bcr. The region containing homology to the human Dbl oncoprotein and the budding yeast CDC24 is depicted (aa 501 to 755). While deleted in ALL, this region is present in the Bcr-Abl of CML, which contains the first 927 aa of Bcr. The Rac GTPase-activating protein (GAP) homology is at the C terminus of Bcr (aa 1197 to 1226). Two isoforms of c-Abl (murine types I and IV, human types 1a and 1b) are generated by alternative splicing of the first exon. Amino acids are numbered beginning with the first common amino acid of c-Abl. A kinase regulatory region contains the Src homology domains: SH3 and SH2 (located between aa 42 and 101 and between 101 and 214, respectively). The c-Abl protein contains a catalytic domain with tyrosine kinase activity (Tyr K; aa 214 to 474). A large C-terminal region that is unique to the Abl subfamily contains a nuclear translocation signal (NTS; aa 569 to 583). Also located within the C terminus is a DNA binding domain (DB; aa 836 to 935) and an F-actin binding domain (AB; aa 935 to 1097). (B) Diagram depicting the different Bcr-Abl constructs. Unless otherwise specified, the Bcr-Abl fusion contains the first 509 aa of Bcr. The Bcr-Abl-p210 clone contains 927 aa of Bcr. Bcr-Abl/isTth contains an insertion of 3 aa (RSV) at the Tth111I site in the c-abl portion disrupting the F-actin binding function. Bcr-Abl/DXS has an internal XhoI-SalI deletion which removes the DNA binding region of c-Abl. Bcr-Abl/DSH2 has no SH2 domain. Bcr-Abl/KD is a kinase-defective mutant in which Lys-295 in the ATP binding sites is substituted with His. Bcr/1-191-Abl has only the first 191 aa of Bcr, and Bcr/1-175-Abl has only the first 175 aa lacking Y-177. Bcr/63-509-Abl does not contain the coiled-coil domain, and Bcr/1-631176-509-Abl lacks aa 64 to 175 of Bcr.

tion through phospholipase C-g (51). The Bcr-Abl protein may constitutively activate an adhesion-mediated signal transduction pathway to allow serum induction of the TRE in detached cells. However, it is unlikely that the activation of the TRE in detached cells alone can account for the induction of anchorage-independent growth. Our finding that Bcr-Abl does not activate the mitogenic pathway is in keeping with the observation that proliferation of CML cells is dependent upon growth factors (5). The development of CML is due to the expression of Bcr-Abl in primitive hematopoietic progenitor cells (8). Proliferation of hematopoietic progenitor cells is regulated by positive and negative signals from the bone marrow stromal cells (16, 17). Direct contact with the stromal cells is not required for proliferation, indicating that proliferation is stimulated by diffusible factors (58). However, excess myeloid proliferation was observed in the absence of stromal contact, indicating that the negative regulation of proliferation involves cell-cell interaction (58). The progenitor cells isolated from CML patients are deficient in the ability to bind the marrow-derived stromal cells, and they are insensitive to the negative regulatory signals produced by the stromal layer (22). Treatment of these cells with alpha

interferon restores the expression of the adhesion molecule LFA-3 (56) and increases the binding of CML cells to the stromal layer (15). Restoration of normal attachment to the stromal layer might explain the success of alpha interferon treatment of CML (25). Our finding that Bcr-Abl binds to actin filaments and abrogates the anchorage requirement for growth is consistent with these clinical manifestations of CML. Fibroblasts are not the targets of Bcr-Abl in vivo, and this is consistent with our finding that Bcr-Abl does not transform the majority of NIH 3T3 cells, i.e., the N-3T3 cells. The context found with the P-3T3 cells may be present normally in the myeloid progenitors that are the natural targets of Bcr-Abl. With a specific murine cell line, BAF3, Bcr-Abl can replace interleukin-3 for the growth of a small percentage of transfected clones (7). This finding suggests that Bcr-Abl may generate a mitogenic signal in a certain cell context. The chronic phase of CML inevitably ends in an acute phase in which a Ph1-positive clone becomes malignant and loses the ability to differentiate (18, 60). This blast crisis is thought to be triggered by secondary mutations which may complement Bcr-Abl to induce factor-independent proliferation. Understanding of the

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MOL. CELL. BIOL. TABLE 4. Activities of various Bcr-Abl constructs in P-3T3 cells No. of foci (avg 6 SE) inb:

Plasmid construct

No. of Neor colonies (avg 6 SE)a

10% CS

Bcr-Abl-p210 Bcr-Abl Bcr-Abl/isTth Bcr-Abl/DSal Bcr-Abl/DSH2 Bcr-Abl/DXS Bcr-Abl/KD Bcr/1-191-Abl Bcr/1-175-Abl Bcr/63-509-Abl Bcr/1-631 176-509-Abl

1,290 6 250 1,635 6 145 1,660 6 210 1,630 6 200 1,550 6 230 1,180 6 60 1,695 6 135 1,220 6 70 1,320 6 90 1,480 6 110 1,390 6 140

610 6 10 765 6 95 0 0 0 375 6 55 0 570 6 50 0 0 590 6 70

0.5% CS

No. of colonies selected in soft agar (avg 6 SE)

Fold TRE activation in detached cells (avg 6 SE)c

20 6 10 60 6 20 0 0 0 10 6 10 0 10 6 10 0 0 20 610

630 6 120 850 6 150 30 6 10 25 6 15 20 6 10 365 6 40 0 540 6 60 0 0 530 6 70

6.5 6 0.5 5.8 6 1.0 1.0 6 0.2 1.0 6 0.1 ND 5.5 6 0.6 ND 5.0 6 0.5 1.1 6 0.2 ND ND

a

Per microgram of plasmid DNA selected in G418. Average number of foci per microgram selected for in Dulbecco’s modified Eagle’s medium plus 10 or 0.5% calf serum (CS). Average fold stimulation of the 2XTRE-luciferase reporter by serum, for cells in suspension, compared with unstimulated P-3T3 cells, using the transient cotransfection assay as described in Materials and Methods. ND, not determined. b c

cprimary biological effect of Bcr-Abl should facilitate the identification of events that mediate the transition into blast crisis.

16.

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