Induction of polyploidization in leukemic cell lines and primary bone marrow by Src kinase inhibitor SU6656

Thrombopoiesis is a complex process of megakaryocyte (MK) differentiation and fragmentation that can be divided into 4 distinct stages: (1) commitment of pluripotent stem cell to the MK lineage; (2) proliferation of MK progenitors (ie, cell division without differentiation); (3) terminal differentiation of MKs, characterized by endomitosis and cellular expansion; and (4) platelet shedding through fragmentation (reviewed in Vainchenker et al¹  and Italiano and Shivdasani² ). Among hematopoietic cells, polyploidization through endomitosis is unique to MKs. However, it is also seen in other cell types, including salivary glands, trophoblast, and urinary bladder epithelium.^3,4 

The progression of cell cycle through chromosome duplication, assembly of nuclear spindles, dissolution of the nuclear envelope, and partial separation of homologous chromosomes has been well documented in MKs.^5,6  However, the process is interrupted prior to cell division, resulting in cells with twice the number of chromosomes. Our prior studies demonstrate that activation of the Src family kinases (SFKs) Lyn and Fyn may partially block MK development.^7,8  We have shown that SFK inhibitors PP1 and PP2 as well as a dominant-negative form of Lyn lead to increased proliferation, higher ploidy classes, and increased extracellular signal-related kinase 1/2 (Erk1/2) activity.⁸  These data led us to hypothesize that SU6656, a small molecule reported to be a more specific SFK inhibitor, might be useful in evaluating MK cell cycle control during endoreduplication.⁹ 

The results presented in this paper add to the growing application of protein kinase inhibitors as tools in treating cancer and inflammatory, neurodegenerative, and cardiovascular diseases.^10-13  Recently, it was demonstrated that Src family kinase (SFK) inhibitors are capable of blocking growth of leukemic cells, suggesting that targeted inhibition of SFKs may have a therapeutic role in human disease.¹⁰  Currently, a number of studies are using SU6656 to examine molecular aspects of signal transduction pathways.^11,14^,15  In this study we demonstrate that SU6656 may also induce polyploidization and maturation of human leukemic cell lines and primary human bone marrow progenitors.

Myelodysplastic bone marrow cells and cadaveric organ donor marrow were used after obtaining approval from the University of Washington institutional review board. No personal identifying data were provided to the investigators, and bone marrow cells from living individuals were leftover diagnostic specimens. K562 and HEL cell lines were cultured in Iscoves modified Dulbecco medium (IMDM; Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD), 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine (BioWhittaker). UT-7/thromobopoietin (TPO; kindly provided by N. Komatsu¹⁶ ) was maintained in IMDM with 10% fetal calf serum and 5 ng/mL human TPO (PeproTech, Rocky Hill, NJ). SU6656 (CalBiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide (DMSO) and added to cells at a final concentration of 2.5 μM and 0.1% DMSO (dosage was determined based on titration vs phenotypic effect). Primary human CD34⁺/CD38^lo were isolated and maintained as previously described.⁷  After 10 days in culture, cytokines were removed by washing the cells 3 times, and the pellet was resuspended in serum-free media containing recombinant human TPO (rhTPO, 35 ng/mL).

Cells were labeled with propidium iodide and nuclear ploidy was determined by flow cytometry as previously described.⁸  Flow cytometric analysis after immunostaining for surface expression of CD41, CD61, or an isotype-matched control antibody was performed as previously described.⁷  Analysis was performed using a FACScan analyzer using CELLQuest software (Becton Dickinson, San Diego, CA).

Cells were observed by inverted light microscopy; aliquots were stained with trypan blue and counted for total viable cells by a hemocytometer. Each cell count was performed 4 times, and results were counted in triplicate. Cytospins were prepared and stained as previously described.⁷ 

Whole cell lysates were analyzed on 10% polyacrylamide gels. Transfer to nitrocellulose, blocking, probing with antibodies, and chemiluminescence were performed as previously described.⁸  Kinase reactions were performed at 30°C for 10 minutes in kinase buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, pH 7.6], 5 mM EGTA [ethylene glycol tetraacetic acid], 1 mM dithiothreitol, 25 mM β-glycerophosphate, 7.5 mM magnesium chloride, 200 μM adenosine triphosphate (ATP), 1 μg Histone H3 [Upstate Biotechnology, Lake Placid, NY]) and 1 μg active Aurora B kinases (Upstate Biotechnology) in a total volume of 15 μL. Kinase reactions were terminated by the addition of 7 μL 3x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, separated by SDS-PAGE, blotted to nitrocellulose, and probed with anti-phosphohistone H3 (Upstate Biotechnology). Immunocomplex kinase assays using anti-Aurora B (Cell Signaling Technology, Beverly, MA) were performed as previously described.⁸ 

UT-7/TPO is a human cell line, derived from the leukemic UT-7 line, that has characteristics of both megakaryocyte and erythroid cells. These cells proliferate but do not differentiate significantly in response to exogenous rhTPO.¹⁶  We have found that the addition of the SFK inhibitor, SU6656, at a concentration of 2.5 μM, results in rapid terminal differentiation of UT-7/TPO cells. In as early as 12 hours, a dramatic shift in cell cycle was apparent, with the majority of the cells shifting to 4N. After 24 hours 8N cells appear, and by 48 hours, 16N are readily seen (Figure 1A). Continued growth in Src kinase inhibitor yields higher ploidy states (32 and 64N; data not shown). In contrast, cells grown in the presence of 0.1% DMSO showed no change in ploidy (Figure 1A). We found a similar effect on polyploidization when other cells with megakaryocytic potential (ie, HEL and K562) were cultured in the presence of SU6656 (Figure 1B). Of note, HEL and K562 cells do not require exogenous cytokines for proliferation.

In addition to polyploidization, we recognized morphologic changes indicative of megakaryocytic maturation after prolonged exposure to TPO and the SFK inhibitor SU6656. Cells cultured longer than 48 hours displayed cellular enlargement and the appearance of proplatelet processes and platelet-sized particles (Figure 1C-D, and data not shown). Interestingly, cell division ceased in SU6656, but there was no significant change in the apoptotic rate as measured by annexin V staining (Figure 1E; data not shown). The expression of MK-specific markers was then evaluated using flow cytometry. The percentage of cells expressing glycoprotein IIb (CD41) and glycoprotein IIIa (CD61) increased significantly after 48 hours in the presence of SU6656 (Figure 1F). In contrast, there was no change in the expression of erythroid antigen glycophorin A (data not shown). These results suggest that SU6656 induces MK differentiation, as assessed by cell morphology and expression of specific differentiation markers. Furthermore, this represents a powerful in vitro model system to study polyploidization and cell cycle regulation.

To test the effects of SU6656 on primary cells, human undifferentiated hematopoietic progenitors (CD34⁺/CD38^lo) were isolated by flow cytometry from whole marrow. Purified CD34⁺/CD38^lo cells were cultured in serum-free media supplemented with interleukin-3 (IL-3; 50 pg/mL), IL-6 (10 ng/mL), stem cell factor (SCF, 10 ng/mL), and TPO (50 ng/mL) (PeproTech) to expand MKs as previously described.⁷  After 10 days in culture, cytokines were removed by washing the cells 3 times, and cells were then resuspended in serum-free media. Half of the cells received TPO (35 ng/mL) + 0.1% DMSO, and half were treated with TPO (35 ng/mL) + SU6656 (2.5 μM). Similar to our previous studies using cell lines, primary cells demonstrated an increase in the number of higher ploidy megakaryocytes as well as an increase in cell size when SU6656 was present (Figure 2A).

MDS is a relatively common cause of acquired thrombocytopenia and increases in incidence with advanced age. In many cases, thrombocytopenia results from ineffective thrombopoiesis and abnormal (ie, dysplastic) MKs. Therefore, we tested the hypothesis that SU6656 could improve MK differentiation. Bone marrow cells were obtained from 2 individuals with confirmed MDS and were cultured under serum-free conditions with rhTPO (35 ng/mL) ± SU6656 (2.5 μM). After 72 hours of incubation, polyploid cells (8N and 16N) and cells with increased size and MK morphology were substantially increased in the presence of inhibitor (Figure 2B-C). Of interest, cultured MDS cells demonstrated constitutive activation of Lyn kinase, which was substantially decreased in the presence of SU6656 (Figure 2D). Finally, there were fewer small cells in the presence of SU6656, suggesting that the inhibitor might diminish the growth rate of clonal malignant cells.

In each of the above experiments (Figure 2B-C), the level of Lyn kinase activation was markedly decreased in the presence of SU6656 (data not shown). A similar decrease in intracellular tyrosine phosphorylation was observed in cells grown in the presence of SU6656, compared with our previous results using the Src inhibitor PP2 (data not shown).⁸  Although SU6656 is clearly a potent inhibitor of the Src family kinases, we have undertaken studies to determine if additional targets can be identified. We found no affect on Janus kinase 2 (Jak2), signal transducer and activator of transcription 3 (STAT3), and STAT5 tyrosine phosphorylation (data not shown). However, the activity of Aurora kinase B was inhibited in vitro by as little as 50 nM SU6656 (Figure 2E). To further extend these observations, we examined the activity of Aurora B in the presence of SU6656 in UT-7/TPO cells. Cells were incubated with SU6656 for 45 minutes before TPO stimulation. Aurora B was immunoprecipitated from the lysates and kinase activity was measured using in vitro phosphorylation assay. As shown in Figure 2F, Aurora B activity was inhibited by SU6656 in the context of intact cells. Previous studies have shown that Aurora B kinase (AIM-1) is down-regulated in human MK and erythromegakaryocytic cell lines during endomitosis and that ectopic expression of AIM-1 blocks phorbol ester-induced polyploidization.^17-19  In contrast, a recent observation has shown that Aurora B is expressed and active in polyploidy MKs.²⁰  Until additional studies are carried out, we cannot exclude the possibility that SU6656 inhibits enzymes in addition to Lyn and Aurora kinases, and we are currently studying its effect on other families of kinases. These results demonstrate that SU6656 is an important tool for understanding the molecular basis of MK endomitosis and may also have therapeutic potential.

We wish to thank Jennifer Minear for manuscript preparation.