By employing a new semi-quantitative assay system that includes co-culturing leukemia cells with the mouse bone marrow-derived stromal cell line MS-5, we examined the suppressive effect of a selective inhibitor of ABL tyrosine kinase, STI571, on acute lymphoblastic leukemia (ALL) cells with BCR-ABL fusion. Leukemic blast cells from eight patients with B-precursor ALL, including three patients with BCR-ABL-positive ALL, were cultured on monolayers of MS-5 cells for 3 weeks with or without addition of variable amounts of STI571. In all cases, cobblestone areas (CAs) were formed, showing clear linear cell dose-dependent curves, allowing quantitative assessment of blast cell growth. The progenitor frequencies obtained by this direct CA-forming cell (CAFC) assay were equivalent to ALL progenitor frequencies assessed by the standard limiting dilution assay. The number of CAFCs ranged from 12.3 to 140.3/104 cells. In BCR-ABL-positive ALL patients, CA-containing cells were examined by FISH, and all contained BCR-ABL fusion genes. STI571 inhibited CA formation of BCR-ABL-positive ALL cells virtually 100% at 0.1–1.0 μmol/l. None of the five BCR-ABL-negative ALL patients showed this growth inhibition by STI571 at 0.1–1.0 μmol/l. Our results indicate that STI571 selectively inhibits in vitro growth of BCR-ABL-positive ALL cells.
Acute lymphoblastic leukemia (ALL) is quite heterogeneous in genetic phenotype and in therapeutic response. The Philadelphia (Ph) chromosome is seen in 25–30% of adult patients with ALL.123 Many studies have indicated that the Ph chromosome is the strongest risk factor. The Ph chromosome is characterized molecularly by the BCR-ABL fusion gene resulting in the creation of p190, p210 or p230 chimeric proteins. These BCR-ABL proteins show higher tyrosine phosphorylation activity on their substrates compared with normal p145 tyrosine kinase (TK).45 The BCR-ABL gene was proven to be capable of inducing either acute leukemia or chronic leukemia in mice, implicating these abnormal TK as the basis of Ph-positive leukemia.67
Since TK activity of BCR-ABL is considered essential for malignant transformation,8 the inhibition of TK activity represents a specific therapeutic strategy for Ph-positive leukemias.910 In 1996, Druker et al11 reported that a new ABL-specific synthetic inhibitor, STI571, exerted a potent inhibitory effect on in vitro growth of BCR-ABL-positive leukemia cells. The drug is a synthetic 2-phenylaminopyrimidine class compound that can inhibit proliferation of BCR-ABL-positive cells and induce apoptosis in these cells both in vitro and in vivo.1112131415 STI571 is also known to inhibit TK activity of c-Kit and platelet-derived growth factor (PDGF) receptor α and β.1116171819 Clinical trials of this drug on Ph-positive leukemias have indicated reasonably selective effects in inducing hematological remission and reducing Ph-positive rates in patients with Ph-positive CML who did not respond well to interferon α therapy.20 Substantial responses were also observed in Ph-positive acute leukemias, including acute crisis in CML and primary Ph-positive ALL.21 Adverse effects were reportedly minimal, such as tolerable nausea and mild myelosuppression. The drug can be given over several months without any apparent dose-limiting toxicity.
In CML, it is easy to assay Ph-positive progenitor cell growth by culturing CML cells in semi-solid media containing cytokines that preferentially stimulate myeloid progenitors. Such clonal cultures provide the most suitable assay system for observing the inhibitory effect of STI571 on Ph-positive clones. STI571 selectively inhibited colony formation of CML progenitors at 1.0 μM.1114 However, it is far more difficult to assay colony formation by ALL progenitor cells in the presence of some cytokines such as SCF and IL-7. Campana and colleagues developed an assay system by employing co-culture with human bone marrow-derived stromal cells, which provided reproducible growth of leukemic B-precursor cells from children with ALL.222324 Nishihara et al25 recently reported a similar but xenogenic co-culture system for normal pro-B cells with the mouse bone marrow-derived stroma cell line MS-5. We co-cultured ALL blasts with MS-5 to develop an assay system that allows easy quantitative assessment of ALL cell growth. We successfully developed a semi-quantitative assay system using this xenogenic co-culture system by directly counting cobblestone areas (CAs) formed by ALL progenitor cells and observed selective inhibitory effects of STI571 on BCR-ABL-positive ALL cells.
Materials and methods
Leukemic cell samples were taken from patients after obtaining their informed consent with the approval of our institutional ethical committee. The samples were taken from peripheral blood (PB) or bone marrow (BM) from eight patients with B-precursor ALL, five of whom were BCR-ABL negative and three of whom were positive as determined by reverse transcription-polymerase chain reaction (RT-PCR). Hematological profiles of these patients are summarized in Table 1. Mononuclear cells (MNC) were isolated from these samples by Ficoll (1.077 g/dl; Nycomed Pharma, Oslo, Norway) centrifugation at 1200 r.p.m. for 20 min. MNC were stored in media containing 10% dimethylsulfoxide and 40% fetal calf serum (FCS; Hyclone, Logan, UT, USA) at −180°C in liquid nitrogen and were thawed at 37°C before use.
STI571 (formerly CGP57148B)
STI571 was kindly provided by Novartis Phama (Basel, Switzerland). A stock solution of STI571 at a concentration of 10 mmol/l was prepared by dissolving the compound in sterile phosphate-buffered saline (PBS) and was diluted in tissue culture medium before use.
MS-5 cells26 were kindly provided by Kirin Brewery Co. Ltd (Gunma, Japan) The cells were maintained in Iscove's modified Dulbecco's medium (IMDM; Gibco BRL, Tokyo, Japan) with 10% FCS. For direct CA assay, six-well tissue culture plates (Falcon, Lincoln Park, NJ, USA) were inoculated with 5 × 104 trypsinized MS-5 cells in each well containing 1.0 ml IMDM with 10% FCS, and for the limiting dilution assay, 24-well plates (Falcon) with 1 × 104 cells in each well containing 0.5 ml IMDM with 10% FCS were used. MS-5 cells became confluent after 7 to 14 days of culture in 5% CO2 at 37°C, forming an even monolayer.
Direct cobblestone area-forming cell (CAFC) assay
Aliquots of 1 × 102 to 5 × 104 MNC from an ALL sample were inoculated on to confluent MS-5 feeder layers in triplicate wells of six-well plates and cultured in 1.0 ml IMDM containing 30% FCS at 37°C in 5% CO2. Cytokines were not added. CAFCs were directly counted under an inverted microscope with phase contrast every week of co-culture. CAs were defined as a group of more than five cells and visualized as dark-contrast cells closely contacting MS-5.27 After 2 weeks, culture media were completely replaced with fresh 30% FCS-containing IMDM. After 3 weeks, media were discarded from the wells, and the wells were washed thoroughly with PBS, air-dried and stained with May-Giemsa solution. CAFCs were counted on these permanent plates.
Fluorescence in situ hybridization (FISH)
Interphase FISH analysis was performed on the samples fixed on slides, as described above, to confirm that these CAFCs originated from BCR-ABL-positive leukemic clone. To detect all BCR-ABL translocations, a BCR-ABL dual-color translocation probe (Vysis, Fujisawa, Japan) was used, and the procedure was performed according to the manufacturer's instructions. The slides were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) and observed under an E-800 fluorescence microscope (Nikon, Tokyo, Japan). At least 100 interphase nuclei were analyzed for each experiment.
Limiting dilution assay
As a standard reference method28 to quantify plating efficiency or progenitor cell number of ALL samples in this co-culture system, we employed the limiting dilution assay. Nine concentrations from 5000 × 20 to 2−8 MNC from each ALL sample were inoculated on to MS-5 feeder layer in a well of 24-well plates, using 16 wells in total, and cultured at 37°C in 5% CO2. After 2 weeks of culture, media were replaced with completely fresh media, and after 3 weeks the wells were washed and stained as in the direct CAFC assay. Each well was tested for the presence of CAs (r is the number of wells without CAs). At each cell concentration, the proportion of wells without CA, r/16, was calculated to obtain progenitor cell frequency in the starting MNC sample.
Growth inhibition assay and cytotoxicity assay with STI571
Two types of experiments were designed to determine the direct inhibitory effect of STI571 on ALL progenitor cell growth (A) and cytotoxic effects of STI571 on pre-formed CA-composing cells (B). In experiment A, STI571 was added at final concentrations of 0.01 to 10 μmol/l to each well in triplicate six-well plates when starting the co-culture, and in experiment B STI571 was added when CAs were formed after 2 weeks of co-culture. In both experiments, wells were washed out, air-dried and stained after 3 weeks of co-culture.
Cobblestone areas (CAs)
CAs were formed in samples from all patients tested. CAs were identified as a group of dark-contrast cells as observed by phase contrast microscopy (Figure 1a). On May-Giemsa-stained permanent plates, CAs were observed as aggregations of small cells with dark purple-stained nuclei with a background of large MS-5 cells with large light purple nuclei (Figure 1b). Some CAs consisted of a few hundred small cells; some of them were pricked out from MS-5 layer, cyto-centrifuged on to glass slides and stained with May-Giemsa. The cells were clearly blastic in morphology with fine nuclear chromatin texture and clear nucleoli (Figure 2a). Blast cells from three cases of BCR-ABL-positive ALL were 100% positive for BCR-ABL fusion genes, as demonstrated by three signals, one of which stained yellow with FISH (Figure 2b). In patients 1, 2, 3 and 4, blast cells were immunocytochemically 100% positive for CD10.
Direct CAFC assay
CAs were observed from approximately day 14 to several weeks. Therefore, the number of CAFCs was counted on day 21. In some patient samples, CAs were followed-up for 4 weeks; some CAs disappeared rapidly but others continued to grow and reached huge sizes, containing more than 1000 cells after 6 weeks. Some of the latter CAs continued to grow over several months when maintained with bi-weekly medium changes and transferred on to newly formed MS-5 layers, suggesting establishment of stromal cell-dependent cell lines.
Figure 3a shows clear linear relationships between inoculated cell number and CAFCs in four cases, including two cases of BCR-ABL-positive ALL, indicating the clonal nature of our direct CAFC assay.
CAFC in eight patients ranged between 12.3 and 142.3 (median 60.5)/104 cells (Table 1).
Limiting dilution assay
Figure 3b shows the results of limiting dilution assay in patient 7. CAFC concentration based on the direct assay was 1/610, whereas the progenitor cell number was calculated as 1/570 by the limiting dilution assay, indicating that direct assay can be utilized as a semi-quantitative method for enumerating CAFCs of ALL blasts.
Effects of STI571 on CAFCs
In experiment A, which tested the effects of 0.1, 1.0 and 10 μmol/l STI571 on growth of CAFCs in eight cases, CAFC numbers were expressed as percentages of CAFC number without addition of STI571. As shown in Figure 4, in samples from patients 1 and 2, who had b3a2 BCR-ABL, and in the sample from patient 3, who had e1a2 BCR-ABL, almost 100% of CAFCs were inhibited at the low concentration of 0.1–1.0 μmol/l, whereas there was virtually no inhibition in samples from patients 4, 5, 6 or 8, who did not have BCR-ABL, at this concentration. In the sample from patient 7, who also did not have BCR-ABL, significant inhibition was observed at 1.0 μmol/l.
In experiment B, which tested the effects of 0.1, 1.0 and 10 μmol/l STI571 on pre-formed CAs, as shown in Figure 4, in samples from patients 1, 2 and 3, who had BCR-ABL, apparent inhibition of over 90% was observed at concentrations of 1.0 μmol/l or more. In samples from patients 4, 5, 6, 7 and 8, who did not have BCR-ABL, this inhibition was not observed.
Since the above two experiments demonstrated almost 100% inhibitory effects of STI571 at the lowest concentration of 0.1 μmol/l, we performed a fine STI571 dose adjustment ranging from 0.01 to 0.075 μmol/l to find out whether positive dose responses could actually be demonstrated in the inhibition. As shown in Figure 5, in samples from both patient 1, who had b3a2 BCR-ABL, and from patient 3, who had e1a2 BCR-ABL, a similar dose-dependent inhibitory effect of STI571 was demonstrated in both experiment A and experiment B.
By co-culturing ALL samples with the mouse bone marrow-derived stroma cell line MS-5, we observed consistent growth of ALL blasts as CAs in all eight cases of B-precursor cell ALL. Demonstration of fusion genes for BCR-ABL by FISH and CD10 by immunocytochemistry clearly indicated that the CAs were derived from leukemic lymphoblasts. Furthermore, the clear linear relationships between inoculated cells and CAFCs in some representative cases guaranteed the quantitative nature of our direct CAFC assay when we inoculated less than 104 MNCs to avoid overlapping of individual CAs. Comparison of progenitor cell frequency between the formal limiting dilution assay and the direct CAFC assay in patient 7 provided almost equal values, further proving the quantitative capacity of the latter assay, which has an apparent advantage in performing complicated experiments such as dose–response experiments; indeed, the limiting dilution assay is almost impossible to use for this type of experiment.
The first step of our experiments thus established a reliable and reproducible clonal assay for primary ALL blast. Although the precise growth signals generated by this co-culture with MS-5 cells remain to be elucidated, the apparent growth inhibition by specific ABL-inhibitor STI571 of BCR-ABL-positive lymphoblasts strongly suggested that the growth of BCR-ABL-positive blasts in contact with MS-5 cells actually was dependent on p210 or p190-dependent signal transduction.
The addition of STI571 from the beginning of co-culture (experiment A) apparently inhibited the ALL CAFCs in a dose-dependent manner. Although we could not determine how STI571 inhibited CA formation by ALL progenitors, the results of experiment B seemed to provide an indirect answer. In this culture, we added STI571 after we confirmed a definite CA formation in each case at 2 weeks of co-culture by expecting to observe a decrease in number or size of CAs due to the cytotoxic effect on CA-composing blasts. This phenomenon actually took place, proving cytotoxic effects of STI571 on surviving blasts in CAs. Dan et al15 previously examined the molecular mechanism of STI571 inhibition using cell lines derived from crisis cases of Ph-positive CML from the point of view of induction of apoptosis. They demonstrated that STI571 had not only antiproliferative but also apoptogenic effects on BCR-ABL-positive cells by blocking BCR-ABL-initiated signaling pathways.
There was a slight but consistent difference in the extent of inhibition between experiments A and B; at a concentration of 0.1 μmol/l, experiment A demonstrated virtually 100% inhibition in all three cases, but experiment B indicated that 10–20% of CAs still survived at this concentration. Whether such surviving CAs still possess clonogenic capacity must be tested in the future by washing out STI571 and refeeding the cells with fresh medium or transferring on to a new MS-5 monolayer. In some cases of ALL, including Ph-positive cases, leukenia cells continued to proliferate for several months, strongly suggesting a high clonogenic capacity of CAFCs in CAs. They may be called ALL stem cells. It will be interesting to test whether STI571 can completely inhibit these ALL stem cells with high self-renewal capacity. Such experiments will answer the question of whether STI571 can not only inhibit growth of ALL stem cells but also kill them.
In the sample from one patient, who did not have BCR-ABL, growth inhibition was apparently observed at a higher concentration of 1.0 μmol/l, indicating that STI571 exerts some inhibitory effect in a minority of ALL cases other than Ph-positive ALL. This may imply that some ALLs use TK(s) for their growth that are similar to that of BCR-ABL TK. It has been reported that STI571 can inhibit PDGF receptors TK and also c-kit TK.1116171819 In this respect, STI571 is not BCR-ABL TK-specific, but is highly selective.
Our co-culture system may be useful for testing the sensitivity to STI571 of leukemic cells from patients with BCR-ABL-positive ALL and also for developing new treatments consisting of STI571 and other drugs.
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We are grateful to Novartis Pharma, Inc. for providing STI571 and to Kirin Brewery Co. Ltd. for providing MS-5.
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Kawaguchi, Y., Jinnai, I., Nagai, K. et al. Effect of a selective Abl tyrosine kinase inhibitor, STI571, on in vitro growth of BCR-ABL-positive acute lymphoblastic leukemia cells. Leukemia 15, 590–594 (2001). https://doi.org/10.1038/sj.leu.2402068
- cobblestone area
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