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Keywords:

  • BCR/ABL;
  • chronic myeloid leukaemia;
  • progenitor cells;
  • drug resistance;
  • actin binding domain

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Philadelphia chromosome-positive, chronic myeloid leukaemia (CML) stem and progenitor cells have a survival and growth advantage compared with their normal counterparts. The mechanisms through which the BCR/ABL protein tyrosine kinase (PTK) induces these effects and the important domains within this protein are not fully defined. The F- and G-actin binding region of the BCR/ABL C-terminus may be important in BCR/ABL-mediated events, and we have investigated this by expressing a C-terminus deletion mutant of the temperature-sensitive BCR/ABL PTK, in a haemopoietic progenitor cell line, which models the chronic phase of CML. The truncated BCR/ABL PTK displayed similar levels of PTK activity when compared with wild type and activation of second messenger formation (in the form of sn-1,2-diacylglycerol) remains intact. On fibronectin substrata, localisation of the protein to the periphery of the cell was, however, dependent on the C-terminus of BCR/ABL PTK. Deletion of the C-terminus reversed both BCR/ABL-mediated apoptotic suppression and drug resistance although the progenitor cells did retain a proliferative advantage at low concentrations of growth factor. These results demonstrated that the C-terminal actin-binding domain of BCR/ABL is important for some of BCR/ABL PTK-mediated leukaemogenic effects.

Chronic myeloid leukaemia (CML) is a neoplastic disease arising in the bone marrow and can be described as a ‘clonal, malignant myeloproliferative disorder’ which originates from malignant transformation of a single haemopoietic stem cell (Fialkow et al, 1967; Goldman, 1997). Over 90% of CML patients carry the Philadelphia chromosome (Ph+) (Kurzrock et al, 1988). This is a shortened chromosome 22 that has lost part of the long-arm due to the reciprocal translocation t(9;22)(q34;q11) (Rowley, 1973). This event generates the BCR-ABL fusion gene containing exons of the BCR gene linked to the second exon of the c-ABL gene. The resulting p210 BCR/ABL protein has constitutively activated protein tyrosine kinase (PTK) activity (Konopka et al, 1984; Kloetzer et al, 1985; Daley et al, 1990), which is central to the development and progression of CML.

The clinical course of CML is divided into three phases, chronic phase, accelerated phase and blast crisis. During the chronic phase, there is a marked expansion of the late myeloid progenitor cell population. These cells not only retain their dependence on growth factors or stromal cell layers for self-renewal, survival and proliferation, but also retain the capacity for multilineage differentiation into functionally mature cells. However, CML Ph+ progenitor cells possess a slight survival and growth advantage over normal progenitor cells (Bedi et al, 1994, 1995; Eaves et al, 1986; McGahon et al, 1994; Cortez et al, 1995).

The chimaeric BCR/ABL PTK has many protein domains and the molecular interactions between these domains and other proteins involved in mediating the downstream effects of BCR/ABL PTK, including activation of signal transduction cascades, are not well defined. At least two or three of these domains, e.g. the SH2 domain, the tyrosine kinase domain and the actin-binding domain, appear essential for the transforming activity of BCR/ABL PTK in fibroblasts and some differentiated haemopoietic cell lines (McLaughlin et al, 1987; McWhirter & Wang, 1991, 1993; Mayer et al, 1992; Muller et al, 1991; Tauchi et al, 1997). However, data from two mouse models provides conflicting evidence concerning the role of the C-terminal domain in leukaemogenesis (Heisterkamp et al, 2000; Wertheim et al, 2003). This current study is focussed on the role of the C-terminal actin-binding region of BCR/ABL PTK in the survival, enhanced proliferative potential and drug resistance observed in haemopoietic progenitor cells expressing this oncogene.

We have developed a model for the chronic phase of CML by transfecting the multipotent haemopoietic stem cell line, FDCP-mix, with a temperature sensitive mutant of BCR/ABL PTK (tsBCR/ABL) (Pierce et al, 1998). The expressed BCR/ABL PTK has constitutively activated tyrosine kinase activity at 32°C but is inactive at 39°C (Pierce et al, 1998) (Kabarowski et al, 1994). The cell biological characteristics of this cell line mimic those of CML stem cells in that cells remain factor dependent when grown at the permissive temperature for BCR/ABL PTK activity, but display enhanced survival in the absence of growth factor and enhanced proliferation in the presence of low concentrations of growth factor (interleukin 3; IL-3) (McLaughlin et al, 1987; Young & Witte, 1988; Scherle et al, 1990; Daley & Ben-Neriah, 1991; Gishizky & Witte, 1992; Pierce et al, 1998). In our current studies, the FDCP-mix cell line has been transfected with a mutant form of temperature sensitive BCR/ABL PTK, with the C-terminal actin-binding domain deleted, to investigate of the role of this domain in these haemopoietic progenitor cells.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell culture, generation of p210 tsBCR/ABL FDCP-mix cell models and differentiation assays

Multipotent FDCP-mix A4 cells were cultured in Fishers medium (FSS) supplemented with 5% (v/v) murine (m) IL-3 and 20% (v/v) horse serum. Cells were incubated with 5% CO2 in air at 37°C. Under these conditions the cells maintained a primitive phenotype (Spooncer et al, 1986; Heyworth et al, 1990).

The tsBCR-ABL constructs used in this investigation contained the b2a2 breakpoint. The tsBCR-ABLΔ construct was derived from the p210 tsBCR-ABL cDNA by site-directed mutagenesis. A stop codon was introduced using the Chameleon ds mutagenesis kit (Stratagene, La Jolla, CA, USA) to shorten the C-terminus by 167 amino acids. The mutation was confirmed by DNA sequence analysis. Derivation of the tsBCR/ABL FDCP-mix clone 5.2 cell line (tsBCR/ABL cell line) and tsBCR/ABLΔ clones 1 and 2 was carried out using retroviral-mediated gene transfer as previously described using pM5 Neo vectors (Pierce et al, 1998). pM5 constructs were transfected into the GP + env Am12 packaging cell lines using a lipofection procedure. The generated GP + env Am12 packaging cells were overlaid with FDCP-mix cells, in the logarithmic phase of growth in complete medium containing polybrene (6 μg/ml). After 4 h at 39°C (restrictive temperature for Bcr-Abl kinase activity), the FDCP-mix cells were harvested and washed to remove the polybrene. Cells were resuspended to a final concentration of 2 × 105 cells/ml in complete medium, supplemented with G418 (1 mg/ml). The cells were incubated at 39°C until the selection procedure was completed and cells were cloned and tested for expression of the relevant protein.

Cell differentiation analysis was performed as described previously (Pierce et al, 1998). Briefly, tsBcr-Abl, tsBcr-AblΔ and FDCP-mix control cells were washed twice in Fischer's medium, resuspended at 3 × 105 cells/ml in Iscoves Modified Dulbeccos Medium supplemented with preselected fetal calf serum (20% v/v), recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, 300 U/ml; Amgen, Thousand Oaks, CA, USA), recombinant human granulocyte colony-stimulating factor (G-CSF, 5000 U/ml; Amgen) and recombinant murine IL-3 (0·01 ng/ml; Calbiochem, Nottingham, UK). Cells were incubated at either 39° or 32°C for the times indicated, cytospins were prepared, stained with May–Grunwald–Giemsa solution and differential morphology scored for greater than 100 cells per slide.

Polymerase chain reaction and Western blot analysis of p210 tsBCR/ABL clones

Following selection and cloning at 39°C, incorporation of the tsBCR/ABLΔ gene into the FDCP-mix cell lines (tsBCR/ABLΔ1 and Δ2 cell lines) was confirmed by polymerase chain reaction (PCR) analysis using BCR and ABL-specific primers (Cross et al, 1994). Vector DNA carrying either the wild-type tsBCR-ABL gene (p210) or the truncated tsBCR-ABL gene (p210E) was used for positive control. Amplification of the region spanning this breakpoint was expected to generate a 297 base pair product. Gene incorporation was confirmed in each of the tsBCR/ABLΔ clones tested. FDCP-mix control cells were negative for the amplified product. Protein expression [using anti-Abl, Ab-2 (Oncogene Research, Nottingham, UK)] and PTK activity of tsBCR/ABL PTK was confirmed by Western blotting of total cell lysates as previously described (Owen et al, 1993; Owen-Lynch et al, 1995). Cells were lysed in ice-cold nonidet P-40 (NP-40) lysis buffer [50 mmol/l Tris acetate, 1 mmol/l EDTA, 1 mmol/l EGTA, 120 mmol/l NaCl, 50 mmol/l NaF, 1 mmol/l phenylmethylsulphonyl fluoride, 1% (v/v) NP-40 (pH 8), 1 mmol/l Na3VO4 and 10  μg/ml each of pepstatin, leupeptin, benzamidine, antipain, trypsin inhi-bitors and aprotinin] at 15 μl per 106 cells. Equivalent protein loading was achieved through measurement of protein concentration within each sample using Bio-Rad protein assay kit as per manufacturers instructions (Bio-Rad, Hemel Hempstead, UK) and loading of 30 μg/lane in all cases. This was also monitored through Ponceau-S staining of the blots after transfer. This method of equivalent loading was also verified through actin controls and an example of this is shown in Fig 1C. Densitometry was performed using a Typhoon 9410 to analyse the total intensity in the lanes on anti-phosphotyrosine blots.

image

Figure 1. Confirmation of the incorporation of the C-terminus truncated tsBCR/ABL gene into FDCP-mix and analysis of its temperature-dependent protein tyrosine kinase activity. (A) FDCP-mix tsBCR/ABL or tsBCR/ABLΔ cells from steady state normal culture conditions, after cloning and maintenance of cells for at least 1 month, were washed free of growth factor and serum and lysed. Expression of BCR/ABL was analysed by Western blotting as described. (B and C) tsBCR/ABLΔ cells were preincubated at 39°C for 18 h, washed to remove serum and growth factors, starved for 3 h and transferred to 32°C for the times shown. Levels of tyrosine phosphorylation of intracellular proteins [or β-actin expression (C)] were analysed by Western blotting as described. Results shown are from a representative of four separate experiments.

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Immunofluorescence

Cells were spun down, resuspended in phosphate-buffered saline (PBS) at approximately 5 × 105 cells/ml and cytospins prepared using a Shandon cytospin 2 (500 r.p.m. for 3 min). Alternatively, cells were adhered to fibronectin coated slides for 1 h. Following fixation in 4% paraformaldehyde for 15 min, slides were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min, incubated with 0·5 μg/ml polyclonal rabbit c-Abl IgG (Santacruz Biotechnology. Inc., Santa Cruz, CA, USA) and visualised with secondary anti-rabbit Alexa Fluor 488 or 546 (Molecular Probes, Paisley, UK), with or without phalloidin coupled to Alexa Fluor 488 as per manufacturers instructions. If required, cell nuclei were counter-stained with propidium iodide (0·05% in PBS) for 1–2 min and slides mounted using Vectashield mounting medium (Vector Laboratories, Peterborough, UK). Slides were analysed by confocal microscopy and Laserpix software.

Measurement of growth characteristics

To assess the ability of tsBcr-Abl and tsBcr-Abl transfected clones to survive in the absence of growth factor (IL-3), cells were maintained at 39°C for 18 h prior to experiments and washed in FSS three times to remove serum and growth factors. Cells were then resuspended in 96-well plates at 2 × 105 cells/ml in FSS containing 20% horse serum and incubated at either the restrictive (39°C) or permissive (32°C) temperature for tsBcr-Abl PTK activity. Aliquots of cells were removed from each well, at 24-h intervals, over 4 d and cell viability was determined by trypan blue exclusion (Owen-Lynch et al, 1995). Results are expressed as % viable cells in the culture [i.e. viable cell count/(viable + dead cell count) × 100%]. DNA synthesis was determined using the [3H]-thymidine uptake assay as described previously (Whetton et al, 1986). Cells were plated as above with increasing concentration of IL-3 as shown. After 24 h, [3H]-thymidine was added at 37 kBq/well for a further 4 h. The [3H]-thymidine incorporation into cellular DNA was determined by cell harvesting and liquid scintillation counting.

Measurement of drug resistance

Experiments examining the effect of cytotoxic drugs on cellular viability were carried out as previously described (Chapman et al, 1994, 1995). Cells were prepared as for the viability assay, but plated in the presence of 20% horse serum and 5% mIL-3, with and without the cytotoxic drug. The drugs used were cytosine arabinofuranoside (Ara-C) at 1 mg/ml and hydroxyurea (HU) at 10 mmol/l. Duplicate plates were prepared and incubated at either the restrictive (39°C) or the permissive (32°C) temperature for PTK activity. After 24, 48 and 72 h, aliquots of cells were removed and viability determined by trypan blue exclusion.

Measurement of diacylglycerol

Cells at a density of 7·5 × 105 cells/ml were labelled with 74 kBq/ml [3H]-palmitic acid (ICN Radiochemicals, Irvine, CA, USA) for 24 h at 39°C. Cells were labelled to equilibrium after approximately 18–20 h incubation, as judged when no further increase in label incorporation was observed. Labelled cells were washed free of growth factor and unincorporated label and maintained at 39°C for 4 h before switching to 32°C for 0, 1, 2, 4, 6 and 18 h. Lipids were extracted and separated by thin layer chromatography as described previously (Owen et al, 1993).

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

tsBCR/ABLΔ FDCP-mix clones display temperature-sensitive protein tyrosine kinase activity

To ensure that proviral integration of the tsBCR-ABLΔ gene, encoding the truncated form of Bcr-Abl PTK with 167 amino acids deleted from the C-terminus, had been achieved, DNA was isolated from control cells and several tsBcr-AblΔ clones and analysed by PCR using BCR and ABL-specific primers. Gene incorporation was confirmed in each of the tsBcr-AblΔ clones tested (data not shown). Confirmation of the presence, and expression levels, of tsBCR/ABL and tsBCR/ABLΔ proteins within the clones was obtained by immunoblotting experiments (Fig 1A). To confirm that, in common with the full-length version of the tsBCR/ABL PTK (Pierce et al, 1998), the truncated BCR/ABL protein retained temperature-dependent tyrosine kinase activity, the level of tyrosine phosphorylation of total cellular protein was analysed using antiphosphotyrosine antibody immunoblot. Switching the FDCP-mix tsBCR/ABLΔ clones 1 and 2 (Fig 1B) from the restrictive to the permissive temperature resulted in an increase in the level of tyrosine phosphorylation of a number of cellular proteins over a wide range of molecular weights within 2 h of temperature switch. The extent of tyrosine phosphorylation observed in the tsBCR/ABLΔ clones was not significantly different from that in the cells expressing full length BCR/ABL. At the permissive temperature, the total phosphorylation in the tsBCR/ABLΔ cells, as measured by densitometric analysis of the Western blots was 90 ± 17% of that observed in the cells expressing full length BCR/ABL [mean ± standard error of the mean (SEM) of six measurements]. We obtained further confirmation that signalling through the PTK was not compromised by truncation of the protein by assessing the effects of the mutation on the BCR/ABL-induced increases in the levels of the second messenger sn-1,2-diacylglycerol (DAG) and other neutral lipid species (Fig 2). After 6 h, full length BCR/ABL PTK induced a 29 ± 5% (mean ± SEM, n = 4) rise in DAG levels (Fig 2B). DAG levels also increased, with similar kinetics, in the tsBCR/ABLΔ clones after switching to the permissive temperature, with a maximal response of 25 ± 2% increase (mean ± SEM, n = 4) after 6 h. As expected, monoacyl- and triacylglycerol species were not affected by activation of BCR/ABL PTK (Fig 2A and C).

image

Figure 2. The effect of activation of BCR/ABL PTK on the levels of neutral lipid species. FDCP-mix cells control cells or those expressing full-length or the C-terminal deletion mutant of BCR/ABL were labelled to equilibrium with 3H-palmitic acid and maintained at 39°C for 18 h, washed to remove serum and growth factors, starved for a further 4 h and transferred to 32°C for the specified time points. Cellular lipids were isolated and separated as described in the methods. Lipid levels were calculated as a fraction of the radioactivity in the total cellular lipid and expressed as a percentage of the level at time 0. Results shown are mean values ± SEM of four separate experiments each performed in triplicate.

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Intracellular localisation of tsBCR/ABL and tsBCR/ABL Δ proteins

One of the key considerations when assessing the function of BCR/ABL is its location within the cell. To ensure that deletion of the C-terminus of the BCR/ABL protein did not result in a qualitative redistribution of the protein from cytoplasm to nucleus, we examined the intracellular localisation of BCR/ABL using confocal microscopy. Paraformaldehyde-fixed cells were probed with rabbit polyclonal c-Abl IgG (which recognises both c-Abl and BCR/ABL) and staining was visualised using anti-rabbit fluorescein isothiocyante (FITC) or Alexa Fluor 546. Murine c-Abl is primarily nuclear (Van Etten et al, 1989; Wetzler et al, 1993) whilst FDCP-mix transfected with full-length tsBcr/Abl exhibited increased, predominantly cytoplasmic, staining (Fig 3B and E). The staining in these cells was represented by bright dots and patches in the cytoplasmic and perinuclear regions indicating a correlation with the ability of Bcr/Abl to bind to components of the cytoskeleton (McWhirter & Wang, 1991). Nuclear staining was still present in these cells due to the continued expression of c-Abl (Fig 3A). FDCP-mix cells expressing tsBCR/ABLΔ (Clone Δ1) (Fig 3C and D) displayed similar general patterns to those observed in cells expressing full length BCR/ABL in cells prepared from suspension cultures, i.e. localisation of the tsBCR/ABLΔ to the cytoplasm, however, the staining was diffuse and not punctuate.

image

Figure 3. Intracellular localisation of BCR/ABL and BCR/ABLΔ proteins and F-actin in transfected FDCP-mix cells. Intracellular localisation of BCR/ABL proteins (false coloured green A-C, red D and E) and F-actin (green D and E) and the nucleus (false coloured red A–C) in parental control FDCP-mix (A), FDCP-mix tsBCR/ABL (B and E) or tsBCR/ABLΔ (C and D), was examined by immunofluorescence as described in the methods. Results shown are from a representative of three separate experiments.

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It was expected that the absence of the C-terminal actin binding domain would lead to a failure of tsBCR/ABLΔ protein to locate to specific areas within the cell, in particular to cytoskeletal actin filaments. Further analysis of the localisation of BCR/ABL and F-actin within the cell was carried out in dual staining experiments on cells adhered to fibronectin. In suspension cells there was no visible phalloidin staining, i.e. no F-actin ring, as this only developed as the cells adhered. Co-localisation analyses of cellular F-actin and BCR/ABL proteins, in cells adhered to fibronectin, are displayed in Fig 3D and E. Cells expressing full length BCR/ABL had a proportion of this BCR/ABL co-localised with the F-actin in the cell (seen as yellow staining in Fig 3E). This co-localisation of the staining was seen in over 95% of the cells analysed after 60 min of adherence to fibronectin (n = 1000 cells across three separate experiments). In contrast, in cells expressing the C-terminal truncation mutant of BCR/ABL no such co-localisation of the immunofluorescent signals was evident (Fig 3D).

Differentiation of FDCP-mix tsBCR/ABL and tsBCR/ABLΔ clones

Transfecting FDCP-mix with full-length tsBCR/ABL PTK does not block the differentiation of these cells when placed in conditions that induce myeloid differentiation (Pierce et al, 1998). Figure 4 confirms that this was the case with all clones used in this investigation. Quantitative morphological assessment of each cell line showed that, over the 10-d period, the proportion of blast cells was reduced whilst the proportion of early/late granulocytes and macrophages increased (Fig 4A and B). Thus, these cells retain their developmental potential in the presence of full length or truncated BCR/ABL and mimic CML progenitor cells, which, in the chronic phase of the disease, retain the ability to differentiate into mature myeloid cells.

image

Figure 4. Differentiation of tsBCR/ABL and tsBCR/ABLΔ FDCP-mix Clones. Control, tsBCR/ABL and tsBCR/ABLΔ FDCP-mix cells were plated in media designed to support myeloid differentiation and incubated at 39°C (A) or 32°C (B) over a 10-d period. Cytospins were prepared at the times shown, stained with May–Grunwald–Giemsa and differential morphology scored for greater than 100 cells/slide. Data shown represents mean values from two separate experiments (SEM is <10% for all data points shown).

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BCR/ABL PTK-mediated suppression of apoptosis in the absence of growth factor is lost in tsBCR/ABLΔ FDCP-mix clones

The parental FDCP-mix cells are growth factor (IL-3) dependent. Similarly, at the restrictive temperature, in the absence of growth factor all cell lines died after 3–4 d (Fig 5A). At 32°C (Fig 4B) only FDCP-mix tsBCR/ABL cells showed significant viability after 3 d (approximately 40% viability). tsBCR/ABLΔ cells did not survive over this time period although they did display a small survival advantage after 24 h. These results indicate that loss of the C-terminus from BCR/ABL reverses the prolonged, enhanced survival mediated by full-length BCR/ABL PTK.

image

Figure 5. Loss of the actin-binding domain reverses BCR/ABL-mediated enhanced cell survival in the absence of IL-3. FDCP-mix control, tsBCR/ABL and tsBCR/ABLΔ clones were washed free of growth factor and plated at 2 × 105 cells/ml in the absence of IL-3. Following incubation at 39°C (A) or 32°C (B) cell viability was assessed at 24-h intervals by trypan blue exclusion. Results shown are mean ± SEM values of eight experiments (***P < 0·001 compared with control cells at the same time point as analysed by Student's t-test).

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C-terminus-deleted tsBCR/ABL FDCP-mix clones retain the proliferation advantage mediated by BCR/ABL PTK at low doses of interleukin-3

Under normal culture conditions, at maximal doses of the growth factor IL-3 there was no difference in the doubling times for the cell lines. The doubling times were for FDCP-mix; 33 ± 4 h, tsBCR/ABL; 36 ± 7 h, tsBCR/ABLΔ clone 1; 29 ± 4 h, tsBCR/ABLΔ clone 2; 30 ± 3 h (all are mean ± SEM of four experiments). At the permissive temperature (32°C) for BCR/ABL PTK activity, tsBCR/ABL transfected FDCP-mix cells displayed a slight proliferation advantage over a 3-d period at low concentrations of IL-3 (around 0·1 ng/ml) compared with parental cells (Fig 6A and B). This effect was also observed in tsBCR/ABLΔ clones (Fig 6C and D). Compared with control cells and cells incubated at 39°C, tsBCR/ABL and tsBCR/ABLΔ clones 1 and 2 displayed higher levels of [3H]-thymidine incorporation at low concentrations (0·01–3·0 ng/ml) of IL-3 at the permissive temperature of 32°C. Thus, the cells have an enhanced ability to proliferate in the presence of active BCR/ABL PTK and the loss of the actin-binding domain has not affected this enhanced proliferative potential.

image

Figure 6. FDCP-mix transfected with tsBCR/ABLΔ retain proliferation advantage at low concentrations of IL-3. FDCP-mix control (A), tsBCR/ABL (B) and tsBCR/ABLΔ clones (C and D) were washed free of growth factor and plated at 2 × 105 cells/ml with increasing concentrations of IL-3 and incubated at 39°C or 32°C for 2 d. Proliferation was assessed by [3H]-thymidine incorporation. Results are expressed as a percentage of maximal [3H]-thymidine incorporation at 10 ng/ml IL-3 and show mean of five experiments.

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BCR/ABL-mediated resistance to cytotoxic drugs is lost in FDCP-mix tsBCR/ABLΔ clones

FDCP-mix cells expressing tsBCR/ABL displayed short-term resistance to the cytotoxic drugs, e.g. Ara-C and HU, at the permissive temperature for BCR/ABL PTK activity (Fig 7A and B). In contrast, truncation of the BCR/ABL actin-binding domain abrogated BCR/ABL-mediated drug resistance (Fig 7A and B). At 39°C there was minimal cell survival after 48 h for all cell lines, however, at 32°C only the cells expressing full-length tsBCR/ABL remained viable during the 48 h of drug treatment (Fig 7A and B). Thus, in common with resistance to apoptosis in the absence of added growth factor, resistance to drug-induced apoptosis requires the C-terminus of BCR/ABL.

image

Figure 7. Loss of the C-terminus actin-binding domain reverses BCR/ABL-mediated short-term drug resistance. FDCP-mix control, tsBCR/ABL clone 5.2 and tsBCR/ABL clones Δ1 and Δ2 were washed and plated in complete medium at 4 × 105 cells/ml in the presence of Ara-C (1 mg/ml) or HU (10 mmol/l). Following incubation at 39°C or 32°C, cell viability was assessed after 24 (A) and 48 (B) hours. Results shown are mean ± SEM values of six experiments (*P < 0·05 compared with controls as analysed by Student's t-test).

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Activation of BCR/ABL PTK in CML mediates several downstream biological responses ultimately leading to the leukaemic phenotype observed in the haemopoietic progenitor cells expressing this protein. For example, CML cells have a survival advantage compared with normal haemopoietic progenitor cells but do not proliferate in the absence of growth factors (Bedi et al, 1994). CML progenitor cells also retain their developmental potential but exhibit defective adhesion to fibronectin and stroma (Gordon et al, 1987; McLaughlin et al, 1987; Young & Witte, 1988; Scherle et al, 1990; Gishizky & Witte, 1992; Verfaillie et al, 1992; Eaves et al, 1993; Renshaw et al, 1995).

BCR/ABL PTK has several functional domains leading to activation of a wide range of signalling pathways and protein–protein interactions, thus complicating identification of the important domains and pathways. Using selective deletion of the C-terminal actin-binding domain of BCR/ABL we have focused on the role of this domain in BCR/ABL-mediated leukaemogenesis.

Deletion of the actin-binding domain of BCR/ABL abrogates prolonged cell survival in the absence of growth factor and short-term drug resistance. Both prolonged cell survival in the absence of growth factors and drug resistance are effectively a result of BCR/ABL-induced suppression of apoptosis (Chapman et al, 1994, 1995). Thus, the actin-binding domain is required for this BCR/ABL-mediated effect. In contrast, the enhanced proliferation associated with low doses of the growth factor IL-3 does not appear to require the C-terminal domain. Other studies on the role of the actin-binding domain of BCR/ABL have also suggested that it is required for the full manifestation of the leukaemogenic effects of the protein. For example, in Baf-3 cells, expression of BCR/ABL lacking the actin-binding domain results in a delay in the emergence of factor independent clones (McWhirter & Wang, 1993) and is required for enhanced proliferation in CD34+ progenitor cells (Ramaraj et al, 2004). Data from two mouse models of CML provides conflicting evidence concerning whether the C-terminal domain is required for leukaemogenesis. In a transgenic mouse model, p190 BCR/ABL F-actin-binding domain mutants increased the latency of development of leukaemia compared to full length protein, i.e. the F-actin-binding domain enhanced the leukaemogenic properties of BCR/ABL (Heisterkamp et al, 2000). In contrast, in an alterative murine model, the myeloproliferative disorder induced by the delta actin mutant was indistinguishable from that induced by the full length protein (Wertheim et al, 2003). These discrepancies may result from variation in the sequences of Bcr or the extent of the deletion. In the latter model, disease was induced through injection of retrovirally transfected bone marrow so the effects of the stem/progenitor cells in this extract may be masked by the excess of transfected lineage committed cells present in the population. In this study, we have directly analysed the effects of deletion of the C-terminal actin-binding domain on haemopoietic progenitor cells. In these cells, some aspects of the leukaemogenic activity of the BCR/ABL PTK were dependent on the presence of the C-terminal domain.

The effect of the loss of the C-terminus of BCR/ABL on cell survival may be a feature of the subcellular localisation of the protein. Full length BCR/ABL is located in the cytoplasm and, in agreement with others, staining showed a highly punctuate pattern indicative of association with the cellular cytoskeleton (Wetzler et al, 1993; Skourides et al, 1999). Further analysis here using confocal microscopy shows that, in adhered cells, full-length BCR/ABL is co-localised with F-actin filaments in common with other observations in BCR/ABL-transfected NIH3T3 and 32D cells and in crystallography studies (Wertheim et al, 2002; Hantschel et al, 2005). Truncation of the C-terminus abolishes this ability of BCR/ABL to localise with F-actin. The subcellular localisation of Bcr-Abl has been shown to be critical for other effects of this oncogene. For example, drug-induced trapping of Bcr-Abl in the nucleus induced apoptosis whilst, in response to DNA damaging agents, Bcr-Abl moves into the nucleus leading to a disruption of normal repair mechanisms (Vigneri & Wang, 2001; Dierov et al, 2004).

The results from the present study indicate that the actin-binding domain of BCR/ABL is important for some aspects of BCR/ABL PTK-mediated leukaemogenesis but not others. It is required for the suppression of apoptosis induced by growth factor withdrawal or drug treatment and for altered migration but appears to be superfluous to the altered proliferative properties of the cells.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We would like to thank Ms Sue Slack for her technical assistance and Mr John Dent for help with the confocal microscopy. This work was supported by the Leukaemia Research Fund UK and the Biotechnology and Biological Sciences Research Council (BBSRC).

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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