The development of chronic myelogenous leukemia (CML) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequential expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequences in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of individual clones and cannot be applied to primary hematopoietic cells. To generate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-ABL p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet OP sequences. Retroviral particles were obtained by transfecting the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotyping. To determine the functionality of the retrovirus, the IL-3-dependent murine Ba/F3 cell line was retrovirally transduced and clones were grown in the absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones expressing BCR-ABL were obtained within 2–3 weeks. A single dose of doxycycline added to the medium (1 μg/ml), induced in different clones, a reduction of BCR-ABL protein levels by 60–90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was further reduced to become almost undetectable at 48 h. The doxycycline-regulated BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition in the medium. In in vivo experiments, subcutaneous injection of 2 × 106 Ba/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 μg/ml). To determine the functionality of the inducible BCR-ABL retrovirus in vivo, primary Lin− bone marrow cells were transduced with SIN-p210 and transplanted in lethally irradiated mice. All transplanted mice had successful hematopoietic reconstitution and BCR-ABL integration was found in the peripheral blood of seven out of 14 mice available for long-term analysis (>6 months). However, despite evidence of retrovirus-mediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter in primary hematopoietic cells. Thus, these results demonstrate for the first time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. Our data suggest that with further improvements of the retrovirus-mediated gene transfer technology, it might be possible to generate inducible leukemia models in mice by the use of single retroviral constructs.
BCR-ABL oncogene is the molecular counterpart of the Philadelphia chromosome (Ph1), the molecular marker of the human chronic myelogenous leukemia (CML). During the last few years, experimental models generated by transferring BCR-ABL into hematopoietic cells have allowed significant advances to be accomplished in the understanding of the pathogenesis of CML.1,2 The systems allowing the expression of BCR-ABL in heterologous cells include essentially retroviral vectors with control of BCR-ABL expression through the LTR-based promoters.3,4,5 During recent years it has also become possible to control gene expression by different strategies, including doxycycline-sensitive promoters,6,7,8 the ecdysone-sensitive promoters9 or the the rapamycin-based regulatory systems.10 In the tetracycline-sensitive promoter strategy, developed initially by Gossen and Bujard,6 the transactivator sequences (tTA) and the tet-OP sequences need to be separately expressed in a single cell. The selection of such a clone expressing non-toxic levels of tTA6 and able to respond to tetracycline is very cumbersome and time consuming, requiring several months of cell culture. This strategy has already been used to generate a tetracycline-inducible BCR-ABL expressing Ba/F3 cell line.11 Beside its labor intensiveness, this double vector strategy has the major disadvantage of being totally inadequate for the gene transfer into primary hematopoietic cells which are most efficiently transduced by the use of defective retroviruses. Mice models of CML are extremely important to understand the molecular events leading from chronic phase to acute phase, the occurrence of which conditions the prognosis.1 It is currently possible to generate a model of CML-like disease in mice using retrovirus-mediated gene transfer but in all cases this leads to rapid death by hyperleukocytosis, thus precluding long-term study of mice.4,5 The development of an inducible retrovirus would therefore be of great interest, allowing BCR-ABL to be expressed in primary murine hematopoietic cells in a regulated manner.
A retroviral gene strategy allowing the regulated expression of a transgene has already been developed and showed its efficiency by the regulated expression of β-gal gene transfer in fibroblasts.12 We now report the generation of a doxycycline-inducible BCR-ABL retrovirus based on this vector (Retro-TET-SIN-p210 vector) allowing an efficient, stable and regulated expression of BCR-ABL in hematopoietic cells.
Materials and methods
SIN-TET- BCR-ABL retroviral vector
SIN-TETp210 retroviral vector was constructed by ligating the 7.2-kb EcoRI fragment containing the BCR-ABL cDNA from MSCV 2.1 p21013 into SIN-Retro-TET vector12 in the 5′–3′ orientation. The resulting SIN-TET-BCR-ABL plasmid was amplified using Qiagen (Courtaboeuf, France) and the purity of the plasmid was verified using standard methods.
Generation of retrovirus producer cell lines
Transient SIN-TET-BCR-ABL supernant production was accomplished by transfecting the 293 EBNA cell line (Invitrogen, Cergy Pontoise, France) with the amphotropic host range in a fully equipped level III facility. To improve the stability of the retrovirus particles, we have chosen a vesicular stomatitis virus G (VSV-G) pseudotyping technique. Briefly, 0.5 μg of each SIN-TET-BCR-ABL, VSV-G and gag-pol expression plasmids were co-transfected using EXGEN reagent according to the manufacturer's recommendations (Euromedex, Mundolsheim, France). The supernatants were collected at 48 h, filtered and frozen at −80°C. The use of this strategy with control GFP vectors regularly gives a transfection efficiency of 50% and a viral titer of 106 CFU/ml using NIH-3T3 cell line as a target (data not shown).
Gene transfer and cell cloning
Exponentially growing Ba/F3 cell line was infected with the transient retroviral supernatants once, twice or three times by exposing the cells to fresh filtered viral supernatant, after 50% dilution with RPMI medium containing 10% heat-inactivated fetal bovine serum, 5% WEHI-conditioned medium as a source of mIL-3 and 4 μg/ml of polybrene (Sigma, St Louis, MO, USA). Forty-eight hours later, cells were washed twice in medium without mIL-3 and were distributed in 600 wells in a final volume of 500 μl in the absence of mIL-3. Half of the medium and cells were changed every 2 days and wells with mIL-3-independent growth were identified after 2 weeks. All mIL-3-independent pools were individually amplified and a survival test was performed by adding doxycycline (Sigma Chemicals, St Louis, MO, USA) at the concentration of 0.1 μg/ml for each of the pools divided into two fractions. The pools in which the doxycycline addition induced significant cell death were selected, amplified either in the presence of mIL-3 (5% WCM) and doxycycline (0.1 μg/ml) (to minimize the expression of BCR-ABL) or in the absence of mIL-3 and frozen. All candidate pools were then analyzed for BCR-ABL expression and for inducibility by Western blotting. The pools with significant inhibitory responses to mIL-3 were then individually cloned by cell sorting (FACS-VANTAGE).
Cell proliferation assays
Ba/F3 cells (two inducible clones, SIN1–4 and SIN3–2 as well as Ba/F3 p210 cells constitutively expressing BCR-ABL) growing in the absence of mIL-3 were plated in triplicate at the concentration of 105 cells in the presence or in the absence of doxycycline (1 μg/ml). Cells were counted daily using trypan blue exclusion for 4 days and the cumulative cell numbers were counted in each condition.
Cells 0.4–1 × 106 were lysed in Laemmli lysis buffer, 30 μg of total protein were run on a 7.5% SDS-PAGE and transferred to a Hybond-C membrane (Amersham, Les Ulis, France). After saturation with 5% dry milk and 0.1% NP40, the membrane was incubated for 1 h at room temperature in × 1 PBS containing 0.1% Tween 20, 5% dry milk and the c-ABL antibody (Ab-3; Oncogene Science). After washing, bound antibodies were revealed by using peroxidase-conjugated sheep anti-mouse antibody and the ECL chemoluminescent detection kit (Amersham).
BaF3 SIN 1–4 cells (2 × 106) were injected subcutaneously in nude mice previously irradiated at the dose of 2.5 Gy. Mice were monitored for the appearance of tumors which were measured at indicated times. Doxycycline was dissolved in the drinking water to a final concentration of 200 μg/ml with 5% sucrose.
Retrovirus-mediated gene transfer in primary hematopoietic cells
Bone marrow cells were obtained by flushing femurs from thirty 6- to 8-week-old C57BL/6 mice treated with 5-FU (150 mg/kg) 5 days earlier. From this population, a Lin− fraction was obtained by the use of a negative selection procedure (lineage marker rat anti-mouse antibodies used were B220, GR-1, TER-199, CD4, CD8, and Mac1). Cell stained with a combination of the above antibodies were then treated with magnetic beads conjugated with anti-rat antibodies according to the manufacturer's recommendations (Dynabeads M450, Oslo, Norway). After elimination of magnetic beads, 5 × 106 Lin− cells were then co-cultured in the presence of the ecotropic Phoenix cell line that was transfected by SIN-p210 retrovirus 2 days earlier. After a co-culture period of 48 h in the presence of mIL3, Tpo, l-Flt3, IL6 and SCF, non-adherent cells were collected and were used to transplant 20 syngeneic, lethally irradiated (9.5 Gy) C57BL/6 mice (250 000 cells/mouse) in the presence of appropriate irradiation controls. After transplantation, mice were divided into two groups. Group 1 consisted of six mice with doxycycline in their drinking water and group 2 included 14 mice without doxycycline. Peripheral blood was collected every 2 to 3 weeks to perform blood counts and to detect BCR-ABL by PCR analysis at the DNA level to detect BCR-ABL integration in the peripheral blood cells.
Results and discussion
Figure 1 shows the backbone of the retro-TET-SIN vector which has been used to generate the inducible BCR-ABL retrovirus. The characteristics of this vector have been previously described.12 Briefly, in this autoregulatory cassette, the tet operator (Tet-OP) sequences and the cytomegalovirus (CMV) minimal promoter allow expression of a bicistronic mRNA, leading to transcription of the gene of interest (BCR-ABL) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). The vector contains an inactivating mutation in its 3′ long-terminal repeat (LTR) sequences, leading to the inhibition of transcription from the 5′ LTR after infection of target cells. This strategy allows the tet-regulated CMV minimal promoter to be active in the target cell, without the interference of the strong retroviral LTR enhancer and promoter elements. In the absence of doxycycline, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of doxycycline, gene expression is prevented. We have initially transfected the retro-TET-SIN p210 vector into the ecotropic Phoenix cell line to generate transient retroviral supernatants, which were tested by their efficiencies to induce growth factor independence in the IL-3-dependent murine Ba/F3 cell line. In several series of experiments, we have not been able to generate infectious retroviral particles using this supernatant strategy (data not shown). One of the possible reasons for this failure could be the size of the retro-TET-SIN-p210 vector (12 kb) precluding successful packaging or the reduced stability of the virus, leading eventually to low viral titers. We then decided to perform a VSV-G pseudotyping strategy using 293-EBNA cells.14,15 This cell line was simultaneously transfected with the retro-SIN-TETp210 plasmid as well as with the gag-pol, and the VSV-G plasmids. The transient supernatants collected from the cultures at day +2 were used to transduce Ba/F3 cells in the exponential phase of their growth. In order to increase the copy numbers of the retrovirus in the target cells, one, two or three rounds of infections were performed in the presence of IL-3 (5% WEHI-conditioned medium) and polybrene (4 μg/ml). Two days later, cells were washed and were distributed in microplates in the absence of IL-3 and doxycycline, at the concentration of 5000 cells/well. A polyclonal stock was also generated in the same manner. During regular medium changes performed during 2 weeks, the majority of the cells in the wells died but growth factor-independent Ba/F3 cells could be rapidly identified at the end of this period. Out of 600 wells screened, IL-3-independent Ba/F3 growth was obtained in 480/600 wells, whereas in the same period of time, no growth was obtained in the parental Ba/F3 cells upon IL-3 deprivation. The contents of each well were amplified in the presence of mIL-3 without doxycycline and protein extracts were prepared. BCR-ABL expression was determined by Western blotting in the conditions where BCR-ABL was expected to be present (in the absence of doxycycline). In all polyclonal Ba/F3 cells as well as growth factor-independent pools obtained from individual wells in these conditions, there was a strong BCR-ABL expression. Upon addition of doxycycline in the culture medium, (0.1 μg/ml or 1 μg/ml) BCR-ABL expression was reduced by 60–90% in the majority, but not all, of the clones tested (data not shown). The pools that were estimated to give the best inhibitory response were individually cloned by cell sorting and amplified. Using this strategy, several individual clones able to respond to doxycycline were generated within weeks. Figure 2 shows the kinetics of the BCR-ABL down-regulation upon a single addition of doxycycline in the culture medium at day 0 in the clone SIN-1–4. Cells were cultured in the presence of IL-3 and doxycycline and extracts were performed at 24 h and at 48 h, followed by Western blot analysis. As can be seen in Figure 2, the expression of BCR-ABL was dramatically reduced (90% reduction) 24 h after a single dose of doxycycline was added to the medium (1 μg/ml). In the extracts obtained at 48 h, there was a further reduction of BCR-ABL expression that was hardly detectable at the end of this period (Figure 2, lane 5). In order to determine the functionality of the retrovirus, two cell lines (SIN 1–4 and SIN 3–2) were cultured in the presence or in the absence of doxycycline after IL-3 deprivation. As can be seen in Figure 3, cell growth was partially inhibited at 48 h (SIN 1–4: 50% inhibition; SIN 3–2: 70% inhibition) whereas at 96 h, no viable cells were present in either cultures (Figure 3). As expected, Ba/F3 cells transfected with a constitutionally active, non-inducible BCR-ABL vector continued to grow in the presence of doxycycline.
In order to determine if the tumorigenicity of the cell lines that we generated could be controlled in vivo, we have injected Ba/F3-SIN 1–4 clone subcutaneously into nude mice (Figure 4). In earlier experiments we determined the optimal tumorigenic dose to be of 2 × 106 cells per mouse, with the appearance of macroscopic tumors 2 weeks after injection in these conditions. Thirty mice were then injected with 2 × 106 SIN 1–4 cells. One group of 10 mice were immediately placed on doxycycline (Dox) administered through their drinking water at the concentration of 200 μg/ml. The second group of 20 mice were left without treatment. At day +12 of the evaluation, none of the 10 mice treated with doxycycline were found to have developed a tumor whereas all mice that did not receive doxycycline presented with tumors whose size varied between 0.11 cm3 and 0.13 cm3. Half of the mice with tumors at day +12 were then divided into two groups of 10 and one group was treated with doxycycline in their drinking water. Nine days later, all 10 mice treated with doxycycline were free of tumors whereas the tumors in the second group continued to grow (Figure 4). After 2 weeks, small tumors appeared at the injection site of the mice initially treated with doxycycline, confirming the presence of leakage in the regulatory system, already demonstrated in Western blots.
To determine if the SIN BCR-ABL retrovirus could be used to transduce murine bone marrow cells, we have performed a co-culture of Lin− bone marrow cells along with the ecotropic Phoenix packaging cell line transfected with the SIN-p210 plasmid. The efficiency of the transfection procedure was difficult to control due to the absence of selectable gene marker. Out of 20 mice transplanted with transduced cells, 14 were available for long-term analysis over 6 months. During this period, 2/4 mice maintained on doxycycline (group 1) and 5/10 mice maintained without doxycycline (group 2) had evidence of BCR-ABL DNA integration by PCR analysis (data not shown). However, there was no evidence of overt leukemia in any mice and deprivation of doxycycline did not induce any increase of blood cell counts in the group placed on doxycycline (group 1).
The availability of an inducible BCR-ABL retrovirus is of major interest to study the biology of CML. In all mice models studied to date, the rapid death of mice transplanted with retrovirally infected cells, precludes the study of the behavior of the hematopoietic clones in vivo. A single study has been able to generate an inducible BCR-ABL-associated leukemia in mice with the use of two transgenic mice strains, allowing the tTA and tet-OP-BCR-ABL sequences to be expressed in their progeny.16 This study is the first demonstrating the reversibility of a BCR-ABL-associated leukemia upon inhibition of BCR-ABL expression. However, all mice in this study developed pre-B leukemia with no evidence of myeloid involvement. Moreover, the transgenic mice strategy is costly and labor intensive.
We believe the strategy that we report to be useful in many experimental settings as viral particles of sufficient titers could be obtained by the use of a VSV-G pseudotyping strategy in the 293-EBNA cell line. It has previously been shown that pseudotyping with the vesicular stomatitis virus G protein in this cell line considerably increases retroviral titers.17,18,19 Our results show the possibility of generating a functional, doxycycline-inducible BCR-ABL retrovirus by using this simple transfection strategy. The regulation of the BCR-ABL was of several orders of magnitude in all clones, with evidence of a dramatic decrease in several of them. The simplicity of the methodology could certainly be counterbalanced by the fact that some leakage of the expression could eventually lead to the emergence of doxycycline-unresponsive clones. However, the magnitude of the decrease of BCR-ABL expression (down to 95% upon doxycycline addition) and the stability of the doxycycline responsiveness that we have observed over 8 months in several clones, indicate that this methodology could be applied to several in vitro studies by the use of other genes.
Our results demonstrate, however, that the SIN-p210 retrovirus was not efficient to induce a myeloproliferative disease in vivo, by the transduction strategy that we have used, despite evidence of BCR-ABL integration in circulating hematopoietic cells. This might be due to the low efficiency of BCR-ABL gene transfer due to the large size of the vector (Figure 1) or to the low efficiency of the minimal CMV promoter in primary cells. Studies are now underway to improve the gene transfer strategy in order to generate an inducible CML model by the use of the retro-SIN-TET-p210 retrovirus.
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This work was supported by grants from the French Ministry of Research to AD and from the Association pour la Recherche contre le Cancer, Ligue Nationale contre le Cancer and Comite de Recherche Clinique of the Institut Gustave-Roussy to AGT. We also thank Dr HM Blau (Stanford University, CA, USA) for providing the retro-TET SIN vector.
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Dugray, A., Geay, J., Foudi, A. et al. Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette. Leukemia 15, 1658–1662 (2001). https://doi.org/10.1038/sj.leu.2402225
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