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[Cancer Research 61, 138-144, January 1, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Expression of a Truncated First Exon BCR Sequence in Chronic Myelogenous Leukemia Cells Blocks Cell Growth and Induces Cell Death1

Yan Wang, Jiaxin Liu, Yun Wu, Weiping Luo, Sue-Hwa Lin, Hui Lin, Natalyn Hawk, Tong Sun, Jie Qiang Guo, Zeev Estrov, Moshe Talpaz, Richard Champlin and Ralph B. Arlinghaus2

Departments of Molecular Pathology [Y. Wa., J. L., Y. Wu., W. L., S-H. L., H. L., N. H., T. S., J. Q. G., R. B. A.], Bioimmunotherapy [Z. E., M. T.], and Bone Marrow Transplantation [R. C.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown that a deletion mutant form of Bcr [Bcr(64-413)] is a strong inhibitor of the tyrosine kinase of Bcr-Abl in vitro and also inhibits its oncogenic growth effects (Liu et al., Cancer Res., 56: 5120–5124, 1996). To determine the effects of this Bcr-Abl kinase inhibitor on chronic myelogenous leukemia (CML) cells, we cloned BCR(64-413) into a recombinant, replication-defective adenovirus to express useful quantities of Bcr(64-413) in a wide variety of cells in culture. Infection of Cos1 cells with plaque-purified virus at a multiplicity of infection of 20–40 induced high expression of Bcr(64-413) as detected by Western blotting. Infection of hematopoietic cells at modest multiplicities of infection (20–40) required special conditions involving shifting cycling cells to a nongrowing condition involving serum starvation and cell crowding. Under these conditions, both Bcr-Abl-positive and -negative hematopoietic cells can be efficiently infected by adenovirus, as demonstrated by 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining of cells infected by ß-galactosidase (ß-GAL) adenovirus. We found that expression of Bcr(64-413) in Bcr-Abl-positive K562 and BV-173 cells, but not Bcr-Abl-negative SMS-SB cells, increased cell-cell clumping and inhibited cell growth. In contrast to the effects of the Bcr(64-413) adenovirus, the ß-GAL adenovirus, despite infecting both types of cells, did not block growth or increase cell-cell clumping of Bcr-Abl-positive and -negative hematopoietic cells. Expression of Bcr(64-413) protein in primary cultures of cells from CML patients with active disease interfered with cell growth, induced apoptosis (as measured by annexin staining), and increased cell-cell clumping, whereas the ß-GAL adenovirus and mock-infected cells lacked these effects. In contrast, normal marrow cells did not exhibit these effects on infection with Bcr(64-413) adenovirus. We conclude from these findings that Bcr(64-413) interferes with the oncogenic effects of Bcr-Abl and therefore has the potential for use in therapy of CML.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Bcr-Abl oncoprotein induces myeloid and lymphoid leukemias. The principal driving force responsible for causing these leukemias is the activated tyrosine protein kinase of Bcr-Abl. The Bcr-Abl oncoprotein phosphorylates a number of protein targets including Jak2, Stat5, Shc, Bcr itself, and a number of cytoskeletal proteins including paxilin and Cbl, among others (1) . The oncogenic activity of Bcr-Abl is due in large part to the induction of proliferation and prolonged survival of precursor stem cells, which in CML3 leads to a large accumulation of granulocytes. The importance of the kinase function in causing these leukemias was confirmed by the finding that a tyrosine kinase inhibitor, ST-571, inhibits the Bcr-Abl tyrosine kinase (2) and induces remission in CML patients undergoing therapy as part of a clinical trial (3) .

Our findings have demonstrated mutual cross-regulation of the Bcr-Abl tyrosine kinase and the Bcr serine/threonine kinase. Tyrosine phosphorylation of Bcr by Bcr-Abl inhibits the serine/threonine kinase of Bcr (4) . The mechanism for this inhibition involves phosphorylation of two tyrosine residues (Tyr328 and Tyr360) located within the kinase domain of Bcr (5) . Of interest, however, Bcr can inhibit the Abl tyrosine kinase not by phosphorylating Abl but by a mechanism involving a phosphoserine form of Bcr (6 , 7) . The region of Bcr responsible for this Abl kinase inhibition contains two serine-rich boxes (termed A and B) that bind to the Abl SH2 in a phosphotyrosine-independent manner (8) . We have pinpointed one Abl inhibitory region to the serine-rich B box surrounding serine 354. A 17-amino acid peptide (350–366) in the phosphoserine form inhibits the Bcr-Abl and the activated form of c-Abl kinase (6) .

In this report, we show that a replication-defective recombinant adenovirus encoding Bcr(64-413) can readily infect hematopoietic cells and induce expression of Bcr(64-413). Expression of Bcr(64-413) in Bcr-Abl-positive cell lines caused growth inhibition and induction of apoptosis. Moreover, the cell lines undergo morphological changes as manifested by cell-cell clumping. Similar findings were found with blood cells from CML patients with active disease. In contrast, a hematopoietic cell line lacking Bcr-Abl expression and cells from marrow of normal individuals lacked these effects.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Antibodies.
K562 cells (9) and BV-173 cells (10) , both of which express Bcr-Abl, and SMS-SB cells (11) , which lack Bcr-Abl, were grown in RPMI 1640 in 10% FBS. Peripheral blood cells from CML patients with active disease were fractionated by Ficoll-Hypaque to select low-density cells (which lack granulocytes). Cells from patient 1 were fractionated to isolate buffy coat cells (total WBCs). Fractionated cells were maintained in RPMI 1640/10% FBS supplemented with GM-CSF (250 units/ml), G-CSF (200 ng/ml), and SCF (50 ng/ml). Patients 1, 3, 4, and 5 were treated with pegylated IFN; patient 2 was undergoing treatment with STI-571; and patient 6 received busulfan.

Adenovirus BCR(64-413) Construction.
The BCR(64-413) gene was inserted into an adenovirus 5 shuttle vector containing a CMV promoter (12) , and the construct was transfected into Cos1 cells to confirm protein expression by Western blotting with anti-Bcr(181-194). After verifying the expression of Bcr(64-413), the shuttle vector was transfected into 293 cells along with the adenovirus 5 vector pJM17 using methods described previously (13) . After plaque purification, a number of plaques were tested by infection of 293 cells, and extracts were screened by Western blotting with anti-Bcr(181-194) (Fig. 1B)Citation . One of the recombinant viruses [BCR(64-413) adenovirus (Ad-{Delta}BCR)] had high expression of Bcr(64-413) and was used to mass infect 293 cells to produce viral stocks. The ß-GAL adenovirus [Ad-ßGAL (5 x 109)] and C-CAM adenovirus (Ad-C-CAM) (14) were supplied by the laboratory of S-H. L. (14) . In most experiments, virus was purified by cesium chloride centrifugation (twice banding) by ourselves, by a commercial company (Quantum Biotechnologies, Inc., Montreal, Canada), or by our Virus Production core facility (University of Texas M. D. Anderson Cancer Center, Houston, TX) and the virus titer measured by plaque assay. Virus titers ranged from 109 to 1010 infectious particles/ml. Plaque assays were performed on human 293 cells by counting plaques formed at 10–14 days after infection (15) .



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  		Fig. 1. Construction of adenovirus 5 encoding Bcr(64-413). A, Bcr(64-413) expression in adenovirus shuttle vector. Lysates of either Cos1 cells or 293 cells transfected with either the vector only or the Bcr(64-413) vector were Western blotted with anti-Bcr(181-194). Lane 1, pSG5 {Delta}Bcr was transiently transfected into Cos1 cells; Lane 2, the pSG5 vector only; Lane 3, Cos1 cells only; Lane 4, XCMV {Delta}BCR was transfected into 293 cells; Lane 5, XCMV vector only was transfected into 293 cells. B, plaque purification of adenovirus 5 Bcr(64-413). Several plaque-purified Bcr(64-413) recombinant adenovirus preparations were expanded by infection of 293 cells. Cells were extracted with SDS/mercaptoethanol containing sample buffer before analysis on a SDS-polyacrylamide gel. After transferring the blot to a filter, the blot was analyzed by anti-Bcr(181-194) using enhanced chemiluminescence reagents.

 
Adenovirus Infection.
For cell lines, cells were concentrated to 1–2 x 107 cells/ml in fresh growth medium containing 10% FBS for up to 16 h before switching to serum-free medium and adding adenovirus at a MOI of 20–40 infectious particles/cell. Infection was done at 1–2 x 106 cells/ml. After an additional 16 h, cell cultures were adjusted to 10% FBS to allow growth to continue. Complete growth medium with serum was added to the culture to maintain active growth over the course of the experiment. For primary blood cells from CML patients or normal marrow cells, about 108 WBCs (Ficoll purified, low-density fraction) from CML patients with active disease were maintained in 50 ml of RPMI 1640 culture medium with 10% FBS and the three growth factors mentioned above in a T-75 flask for 3 days. Cells from 50 ml of medium were pelleted and placed in 5 ml of the above-mentioned (conditioned) culture medium for 16 h in the presence of adenovirus at a MOI of 20–40. Sufficient medium was then added to stimulate cell growth in 6-well plates at about 107 cells/ml. Virus infection was done only once. Viable cells were counted in a hemocytometer after trypan blue staining. Marrow cells from healthy donors were used as the source of normal marrow cells. They were fractionated as described above.

Actively growing Cos1 cells were infected directly without the need for serum starvation and concentration. Adenovirus was added as described above to a MOI of 40. To assess the effect of the of Ad-{Delta}BCR on the phosphotyrosine content of Bcr-Abl, cells were first transfected with the pSG5 plasmid containing the gene for P210 BCR-ABL as described previously (4) . After 1 day, cells were infected with either Ad-ßGAL or Ad-{Delta}BCR at similar MOIs. Cells were harvested after a total of 3.5 days.

Western Blotting.
Western blotting was performed as described previously (16) .

X-Gal Staining.
About 106 cells were processed for X-Gal staining. After pelleting the cells, the cells were suspended and fixed in 200 µl of PBS containing 1% formaldehyde/0.2% glutaraldehyde at 37°C for 15 min. Cells were pelleted and washed twice in 1 ml of PBS at room temperature. The cell pellet was resuspended in 250 µl of staining solution (4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl2, and 0.4 mg/ml X-Gal reagent substrate). After 1 h at 37°C, the cell suspension was distributed in wells of a 24-well plate for photography using a Nikon camera/microscope. Typically, Ad-ßGAL infection induced staining of about 27–65% of patient CML cells and normal marrow cells.

Cos1 Transfection.
Cells were transiently transfected as described previously (6) . For these experiments, BCR(64-413) was inserted into a Cos expression vector (pSG5; Ref. 5 ).

Quantitative PCR.
WBCs from patients were processed by RNA extraction with Trizol (Life Technologies, Inc., Gaithersburg, MD), and cDNA was prepared by use of reverse transcriptase as described by Lin et al. (17) . The amount of cDNA from the sample was estimated by competitive PCR using an internal competitor composed of the b3a2 BCR-ABL junction containing an insert (17) .

Annexin/PI Staining.
We used the procedure described in the manufacturer’s protocol (Annexin V-FITC; Clontech, Palo Alto, CA); cell sorting was done by the Division of Pathology and Laboratory Medicine core facility (University of Texas M. D. Anderson Cancer Center, Houston, TX).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction and Purification of Adenovirus BCR(64-413).
The BCR(64-413) gene was inserted into an adenovirus shuttle vector controlled by a CMV promoter. Transient transfection experiments were done to confirm the presence of the BCR(64-413) gene. As a positive control, transient transfection with the pSG5 expression vector (Fig. 1ACitation , Lane 2) and pSG5 containing BCR(64-413) (Fig. 1Citation A, Lane 1) were performed in Cos1 cells. As is typical with this system, high expression of Bcr(64-413) was obtained as measured by Western blotting with anti-Bcr(181-194) (6) . Bcr(64-413) is always expressed as one major band of about Mr 43,000 with variable amounts of faster migrating bands (6) , which appear to be degradation products. The 293 cells transfected with the BCR(64-413) shuttle vector also expressed an intense band of Bcr(64-413) (Fig. 1Citation A, Lane 4), which was lacking in vector-transfected cells (Fig. 1Citation A, Lane 5).

The shuttle vector was transfected into 293 cells along with the adenovirus vector pJM17 to generate the recombinant adenovirus 5 virus encoding Bcr(64-413). The virus was plaque purified, and plaques were expanded for analysis by Western blotting (Fig. 1B)Citation . One of the high expressing clones was further expanded and the purified by cesium chloride banding (Ad-{Delta}BCR).

Ad-{Delta}BCR Infection Inhibits Tyrosine Phosphorylation of P210 Bcr-Abl.
To assess the effects of Ad-{Delta}BCR infection on the phosphotyrosine content of Bcr-Abl, we transfected Cos1 cells with BCR-ABL (P210) DNA to generate cells expressing P210 BCR-ABL. After 1 day, cells were infected with Ad-{Delta}BCR, Ad-ßGAL, or mock infected. Cells were harvested at 3.5 days after beginning the experiment for Western blotting with anti-Bcr(181-194) and anti-phosphotyrosine 4G10 (Fig. 2)Citation . Fig. 2ACitation shows that P210 BCR-ABL was produced in similar amounts in all three cultures as judged by anti-Bcr(181-194) blotting. Strong expression of Bcr(64-413) was detected in the Ad-{Delta}BCR-infected cells (Lane 3). The phosphotyrosine blot showed a significant decrease in the amount of phosphotyrosine in the P210 Bcr-Abl band. Volume densitometry analyses showed that the level of phosphotyrosine in P210 Bcr-Abl was reduced about 60% compared with mock-infected cultures or Ad-ßGAL-infected cells (Fig. 2B)Citation . These data indicate that Bcr(64-413) expression inhibits the tyrosine phosphorylation of P210 BCR-ABL within the cells and supports our earlier in vitro findings published previously (6) .



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  		Fig. 2. Bcr(64-413) expression as a result of Ad-{Delta}BCR infection inhibits tyrosine phosphorylation of P210 Bcr-Abl. Cos1 cells were transfected with pSG5 plasmid encoding P210 Bcr-Abl for 1 day, and cells were subsequently infected with Ad-{Delta}BCR, Ad-ßGAL, or mock infected. After 3.5 days, cells were harvested for Western blotting. A, Western blotting with anti-phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY) and anti-Bcr(181-194). B, quantitation of the phosphotyrosine content of P210 Bcr-Abl after expression of Bcr(64-413). Band intensities were determined with the Personal Densitometer (Molecular Dynamics, Sunnyvale, CA). The results show the average of two blots (see the error bars).

 
Infection of Hematopoietic Cells with Adenovirus.
Others have used adenovirus to infect hematopoietic cells with mixed results (18 , 19) . We had problems infecting cycling K562 cells because Ad-ßGAL was found to poorly infect actively growing cells. To optimize conditions, we performed experiments with an adenovirus 5 recombinant virus expressing the cell surface protein C-CAM (20) , which allowed rapid monitoring for virus infection by flow cytometry. C-CAM is a cell surface protein that can be detected by an antibody made against a cell surface epitope (20) . Various conditions were tested first by infecting K562 cells with various MOIs of the C-CAM virus (Ad-C-CAM) followed by flow cytometry analysis (data not shown). Optimal conditions involved infecting a 10-fold concentrated suspension of serum-starved K562 cells with MOIs of 20–40 for 16 h in serum-free medium. Sufficient culture medium containing 10% FBS was added to stimulate cell growth, and infection was allowed to proceed at a concentration of 2.5 x 106 (for K562/BV-173 cells) and 5.0 x 106 (for SMS-SB cells) cells/ml. Fig. 3Citation shows the results of Ad-ßGAL infection of hematopoietic K562 cells, which express Bcr-Abl. Similar results were found with BV-173 cells, which also express P210 Bcr-Abl, and with Bcr-Abl-negative SMS-SB cells (data not shown). Blue-stained cells were counted manually, indicating that between 38% and 77% of cells were infected (Table 1)Citation .



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  		Fig. 3. Ad-ßGAL infection of K562 hematopoietic cells. Cells were infected with a 20 MOI for 6 days and analyzed by X-Gal staining. The photo was taken at x10 magnification.

 

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  		Table 1 Quantitation of ß-Gal staining after Ad-ßGAL infection

 
Adenovirus Bcr(64-413) Infection of Bcr-Abl-positive K562/BV-173 Cells Induced Morphological Changes and Growth Inhibition.
We infected K562 cells, BV-173 cells, and Bcr-Abl-negative SMS-SB cells with Ad-{Delta}BCR and Ad-ßGAL. In addition, mock-infected cells were used as an additional negative control (data not shown). The results showed that Ad-{Delta}BCR infection induced Bcr(64-413) expression within 3 days after infection, as detected by Western blotting (Table 2)Citation . Morphological changes were observed later (5–6 days after infection) in K562 cells and BV-173 cells as viewed by cell-cell clumping. In contrast, Bcr-Abl-negative hematopoietic SMS-SB cells showed no significant level of cell-cell clumping. Detection of Bcr(64-413) expression preceded the induction of cell-cell clumping. Examples of clumping induced by the Bcr(64-413) adenovirus are shown in Fig. 4Citation . Uninfected K562 cells had little clumping after mock infection (Fig. 4A)Citation , but cells infected with Ad-{Delta}BCR had a considerable degree of clumping (Fig. 4C)Citation . Infection of Bcr-Abl-negative SMS-SB cells with Ad-{Delta}BCR had little effect on cell-cell clumping (Fig. 4D)Citation ; Ad-ßGAL also did not cause clumping of K562 cells (Fig. 4B)Citation . Further work is needed to determine whether cell-cell clumping is due to early stages of cell death processes or changes in cell adhesion. It is important to note that these clumps of cells are not stable to pelleting and resuspension; therefore, it is difficult to assess the viability condition of such cells.


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  		Table 2 Induction of cell-cell clumping in Bcr-Abl-expressing cells by Bcr(64-413) expression

Cells maintained in conditions of growth saturation were infected for 1–6 days with Ad-{Delta}BCR at a MO1 of 20 using purified virus. Aliquots were removed for Western blotting. Each day, cells were viewed in the microscope for changes in morphology, and representative photos were taken. Bcr-Abl-positive cells expressed Bcr(64-413) beginning at day 3; large clumps of cells formed in the culture beginning at day 5. Clumps contained 10–30 cells. Bcr-Abl-negative cells (SMS-SB) did not form significant numbers of clumps despite expressing Bcr(64-413). Increasing the number of clumps (cell-cell adhesion) was scored from + to +++. Bcr(64-413) expression was measured by Western blotting with anti-Bcr(181-194). Increasing band intensity is indicated by + to +++; +/- is a barely detectable signal.

 


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  		Fig. 4. Bcr(64-413) induces cell-cell adhesion in Bcr-Abl-positive K562 cells. Cells were infected for 6 days with a 20 MOI of virus. A, uninfected K562 cells; B, K562 cells infected with Ad-ßGAL; C, K562 cells infected with Ad-{Delta}BCR; D, SMS-SB cells (Bcr-Abl negative) infected with Ad-{Delta}BCR. The magnification was x10.

 
Effects of Bcr(64-413) expression on cell growth were also examined (Fig. 5, A and B)Citation . In these experiments, cells were concentrated 10-fold to facilitate infection and then diluted to conditions permissive for cell growth. Growth of Bcr-Abl-positive K562 cells was strongly inhibited by infection with Ad-{Delta}BCR but not with Ad-ßGAL (Fig. 5A)Citation . In contrast, infection of Bcr-Abl-negative SMS-SB cells had no effect on cell growth of these hematopoietic cells (Fig. 5B)Citation . ß-GAL staining of the Ad-ßGAL-infected cells indicated that about 75% of the K562 cells stained positive; the value for SMS-SB cells was about 50%. These observations indicate that Bcr(64-413) expression blocks the growth-stimulating effects of the Bcr-Abl oncoprotein in cell line experiments.



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  		Fig. 5. Infection of Bcr-Abl-positive K562 cells with Ad-{Delta}BCR inhibits cell growth. Cells were infected with 20 MOI of Ad-{Delta}BCR or Ad-ßGAL or mock infected. A, K562 cells; B, SMS-SB cells. In each case, the number of viable cells (cells lacking trypan blue staining) was determined in triplicate.

 
In a similar experiment, cell death was measured by annexin staining using flow cytometry to detect and quantitate the amount of apoptosis. These results showed that adenovirus Bcr(64-413) infection stimulated apoptosis in about 25% of K562 cells compared with 11% of mock-infected cells. In contrast, adenovirus infection had no detectable effects on Bcr-Abl-negative SMS-SB cells because mock-infected and Ad-{Delta}BCR-infected cells each had about 12% apoptosis using the same methods. Confirming the lack of effect of Ad-{Delta}BCR on Bcr-Abl-negative cells, primary cultures of marrow cells from normal individuals also showed no significant apoptotic effects after infection with Ad-{Delta}BCR compared with either mock infection or infection with Ad-ßGAL (see Table 4Citation ).


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  		Table 4 Expression of Bcr(64-413) by adenovirus infection of CML patient cells induces apoptosis

WBCs from peripheral blood of patients with active CML or normal marrow cells were harvested and cultured in RPMI-1640-10% FBS medium supplemented with GM-CSF (250 units/ml), G-CSF (200 ng/ml), and SCF (50 ng/ml). At day 3 of culture, cells were concentrated and infected with recombinant adenovirus containing Bcr(64-413) or ß-Gal with the same MOI or mock infected. Cells were harvested 2–9 days after infection and analyzed by annexin/PI staining to estimate late-stage apoptosis and by quantitative/competitive reverse transcription-PCR to determine the number of Bcr-Abl transcripts per µg of total cellular RNA. Data shown is for the maximum levels of apoptosis; samples were analyzed at least twice during the infection. Samples were also analyzed by Western blotting to determine whether the cells were expressing Bcr(64-413) using anti-Bcr(181-194). Cells were also stained for ß-Gal activity. Manual counting of blue cells indicated infection ranged from 27–65% of the cells (Table 1)Citation . Mock, Ad-ßGal, and Ad-{Delta}Bcr infection was performed at the same time on parallel batches of cells.

 
Bcr(64-413) Expression in Cells from CML Patients with Active Disease Inhibits Cell Growth and Induces Cell Death.
We performed experiments with Ad-{Delta}BCR and Ad-ßGAL using primary cultures of CML patients with active disease. In these experiments, low-density WBCs (which would contain CML blast cells) from the blood of active leukemia patients were selected for infection under the conditions described for hematopoietic cell lines. Viable cells were counted as infection progressed to compare the cell growth effects of Ad-{Delta}BCR with those of mock-infected cultures. In the first patient studied (who was off therapy for CML and not included in Table 4Citation ), Western blotting with anti-Bcr(181-194) detected a strong band of Bcr(64-413) at 3.5 days after infection (Fig. 6Citation , Lane 2). Live cells were reduced dramatically by Ad-{Delta}BCR infection as compared with mock infection. At this time point after infection, there was a strong signal of Bcr(64-413) expression, which correlated with strong growth inhibition (Table 3)Citation .



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  		Fig. 6. Expression of Bcr(64-413) in primary cultures of human WBCs after Ad-{Delta}BCR infection. A, infection of primary CML patient WBCs (low-density fraction) with Ad-{Delta}BCR. Ten million cells at various times after infection were lysed directly in SDS/mercaptoethanol sample buffer and analyzed by Western blotting using anti-Bcr(181-194). Lane 1, mock-infected cells; Lane 2, Bcr(64-413) adenovirus infection after 3.5 days; Lane 3, mock-infected Cos1 cells after 1 day; Lane 4, Bcr(64-413) adenovirus infection of Cos1 cells after 1 day. Bcr-Abl was detected in this patient by blotting with anti-Abl 8E9 in a separate gel. B, infection of primary bone marrow cells from a normal individual with adenovirus Bcr(64-413). The procedures described in A were used in this experiment. Lane 1, infection with Ad-{Delta}BCR for 1 day; Lane 2, infection for 2 days.

 

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  		Table 3 Induction of cell death in bone marrow cells from a Philadelphia chromosome-positive CML patient by infection with Ad-{Delta}BCR recombinant adenovirus

Low-density cells from a chronic phase Bcr-Abl-positive CML patient with active disease were incubated in growth factor containing maintenance medium for 3.5 days. One aliquot of cells was infected at day 0 with Ad-{Delta}BCR. Viable cells were counted after trypan blue staining. Ad-{Delta}BCR was twice banded in CsCl gradients and used at a MOI of 20. Philadelphia chromosome-positive cells infected with the Ad-{Delta}BCR showed a large amount of dead cells; mock-infected cultures showed no significant amount of cell death. The values are the average of triplicates with less than 10% divergence from the mean. Cos1 cells, either mock infected or infected with 20 MOI of Ad-{Delta}BCR, showed no significant differences in viable cell counts.

 
Expression of Bcr(64-413) by Adenovirus Infection of Leukemia Cells from CML Patients with Active Disease Induced Apoptosis.
For these experiments, we chose CML patients with active disease (Table 4)Citation . This was reflected in the level of Bcr-Abl transcripts, which ranged from 0.2 -3.6 x 105 transcripts/µg total RNA. We also measured Bcr(64-413) expression by Western blotting with anti-Bcr(181-194). Of six patient samples, five expressed Bcr(64-413) after infection with the adenovirus Bcr(64-413) (Table 4)Citation . Also, the cells were sorted for both annexin V-positive staining and PI staining. The latter detects cells that have become permeable to PI. The annexin V staining percentages in Table 4Citation show cells that are stained by both reagents and thus represent later stages of apoptosis. Of the five samples that expressed detectable levels of Bcr(64-413) after Ad-{Delta}BCR infection, all samples but one (sample 3) had increased levels of annexin V staining compared with Ad-ßGAL-infected cultures. In these cases, apoptosis increased dramatically (typically more than doubling the level of apoptotic cells) after infection with the Ad-{Delta}BCR compared with Ad-ßGAL. The one case (sample 3) that expressed Bcr(64-413) with little effect on the induction of cell death also had the lowest levels of Bcr-Abl transcripts. Importantly, expression of Bcr(64-413) in normal marrow cells had no significant effect on apoptosis (samples 7–10 of Table 4Citation ). This result is consistent with our studies with a Bcr-Abl-negative cell line (Fig. 5B)Citation . Together, these data indicate that normal hematopoietic cells are not affected by Bcr(64-413) expression, whereas, in contrast, Bcr-Abl-positive cells undergo apoptosis. Additional studies are needed to determine the effect of longer-term Bcr(64-413) expression.

Bcr(64-413) Expression Induces Morphological Changes.
Using cells from one CML patient, we examined cells for morphological changes (Fig. 7)Citation . Mock-infected cells were similar in appearance to Ad-ßGAL virus-infected cultures (compare top and middle panels of Fig. 7Citation ). In contrast, Bcr(64-413) virus-infected cultures had dramatic changes in morphology (large clumps of floating cells were seen) and showed evidence of crenated dead cells (apoptotic in nature; Fig. 7Citation , bottom panels). Whether these clumps of cells are the result of dying cells or cell adhesion changes (21 , 22) remains to be determined.



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  		Fig. 7. Infection of CML patient cells by adenovirus Bcr(64-413) causes cell-cell adhesion and cell death. Low-density cells from the blood of a CML patient with active leukemia (sample 6, Table 4Citation ) were cultured for 3 days in a T-75 flask with 50 ml of RPMI 1640–10% FBS containing the growth factors listed in Table 4Citation . Total cells (both adherent and nonadherent) were harvested and placed in 5 ml of the above-mentioned conditioned growth medium for 2 days. Cells were either mock infected, infected with Ad-ßGAL, or infected with Ad-{Delta}BCR for 2 days at a MOI of 40 infectious particles/cell in 6-well plates at a concentration of 107 cells/ml/well. Photographs were taken using a Nikon camera attached to a Nikon microscope. Both x40 and x10 fields were selected. The bottom panels show morphological changes induced specifically by Bcr(64-413) expression. Two types of changes were observed. In the x10 panel (bottom right), large clumps of cells (seen at very high frequency) were observed, and it was difficult to obtain a sharp picture of these cells because they were in suspension. The second change (seen in the bottom left, x40 magnification) visualizes a large number of dying cells and cell debris. These changes were specific because they were not observed in either the control (top panels) or the Ad-ßGAL-infected cells (middle panels).

 
The degree of adenovirus infection was quite high as judged by X-Gal staining of cells from patients with active CML after infection with Ad-ßGAL (Table 1)Citation . Positively stained cells ranged up to 65% of the culture, as determined by manual counting of blue-stained cells. Mock infection showed little staining (data not shown). Table 1Citation also showed that adenovirus readily infects normal marrow cells, as judged by X-Gal staining of Ad-ßGAL-infected cells (sample 8 of Table 4Citation ).

Buffy coat WBCs from patient 1 (Table 4)Citation with active disease also exhibited morphological changes and growth inhibition after expression of Bcr(64-413) (Fig. 8A)Citation . In contrast, mock infection and infection with Ad-ßGAL had no effects on cell morphology (Fig. 8, B and C)Citation .



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  		Fig. 8. Morphological changes of buffy coat WBCs from a patient with active CML. Cells from patient 1 (sample 1, Table 4Citation ) were infected with Ad-{Delta}BCR or Ad-ßGAL for 9 days. A, Ad-{Delta}BCR infection; B, Ad-ßGAL infection; C, mock-infected cells. The magnification was x40.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous studies indicated that Bcr(64-413) blocks the Bcr-Abl tyrosine kinase in vitro and severely inhibits the growth of Bcr-Abl-positive K562 cells in colony assays (6) . Unlike the intact Bcr protein, Bcr(64-413) is not a target for Bcr-Abl (6) , and because it is not tyrosine phosphorylated, it will retain its Ser/Thr protein kinase activity (4 , 5) . In fact, our recent results have shown that Bcr(64-413) is active as a Ser/Thr kinase both in vivo and in kinase assays (23) . Based on these studies, we decided to introduce Bcr(64-413) into normal and Bcr-Abl-positive hematopoietic cells (both cell lines and primary cultures from CML patients) for the purpose of testing the physiological effects of this form of Bcr on CML cells. We chose an adenovirus system to be able infect noncycling cells and to allow relatively high expression of the truncated Bcr protein. We had to overcome the difficulty experienced by others, namely, that hematopoietic cells are resistant to adenovirus infection. This problem was overcome by shifting cycling cells into a nongrowing condition during exposure to the virus. This resistance of hematopoietic cells did not pose a problem for infecting primary cultures of marrow cells when we used the same protocol used for hematopoietic cell lines. Of interest, the primary cultures were routinely more susceptible to adenovirus infection than cell lines, as measured by the level of Bcr(64-413) expression.

The expression of Bcr(64-413) in both Bcr-Abl-positive hematopoietic cell lines and primary blood cell cultures inhibited cell growth (Fig. 5Citation ; Table 3Citation ) and induced apoptosis strongly in primary cultures (Table 4)Citation . The effects were specific because four normal marrow cell samples were unaffected by expression of Bcr(64-413) (Table 4)Citation . Of importance, the inhibitory effects of Bcr(64-413) were seen even in cells that were stimulated with three cytokines (GM-CSF, G-CSF, and SCF), which are known to be involved in the growth of marrow stem cells. Thus, the blocking effects of Bcr(64-413) appear to be cytokine independent.

Some comment is appropriate with regard to the ability of Bcr(64-413) to inhibit the kinase of c-Abl when c-Abl is activated by overexpression (6) . Our findings show that Bcr(64-413) expression did not affect normal hematopoietic cell growth or induce apoptosis in short-term cultures (Fig. 5Citation ; Table 4Citation ). We believe that the lack of effects of Bcr(64-413) on cell cultures lacking Bcr-Abl expression is due to the fact that c-Abl is normally in a kinase-inhibited state in the cytoplasm of cycling cells. It is well known that c-Abl is normally not active in cells (except for a brief period in the nuclei of S-phase cells) unless the cells are treated with DNA-damaging agents or other forms of stress (24) . We have also tested the effects of Bcr(64-413) on HeLa cell foci formation and found that it had no significant effect.4

Our results indicate that Bcr(64-413) expression in primary cultures of CML cells specifically inhibits the oncogenic effects of Bcr-Abl. These inhibitory effects are the result of the inhibition of Bcr-Abl’s tyrosine kinase (6) , some other aspect of Bcr(64-413) action, or both. In this study, we showed that Bcr(64-413) expression as a result of Ad-{Delta}BCR infection reduced the phosphotyrosine content of P210 expressed in Cos1 cells by about 60% (Fig. 2)Citation . Similar reductions in phosphotyrosine Bcr-Abl were seen in Bcr-Abl-positive hematopoietic cells (K562 cells) infected with Ad-{Delta}BCR (data not shown). Therefore, these findings prompt additional studies to determine the therapeutic usefulness of introducing Bcr(64-413) into CML patients as a strategy to treat the leukemia.


   ACKNOWLEDGMENTS
 
We thank Tammy Trlicek for typing assistance on this manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant CA49369, Therapy of CML PO1 Grant from the NIH, Project 7 Molecular Inhibition of bcr-abl Tyrosine Kinase by bcr Sequences and Core C2 Minimal Disease Detection by Polymerase Chain Reaction, NIH CA16672 Cancer Center Support Grant, Project Synthetic Antigen Laboratory. Back

2 To whom requests for reprints should be addressed, at Department of Molecular Pathology, Box 89, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-8995; Fax: (713) 794-1395; E-mail: rarlingh{at}mdanderson.org Back

3 The abbreviations used are: CML, chronic myelogenous leukemia; MOI, multiplicity of infection; X-Gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; ß-GAL, ß-galactosidase; FBS, fetal bovine serum; GM-CSF, granulocyte macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; SCF, stem cell factor; PI, propidium iodide; CMV, cytomegalovirus. Back

4 Y. Wu and R. Arlinghaus, unpublished results. Back

Received 4/26/00. Accepted 10/31/00.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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