Induction of Polyploidization in Leukemic Cell Lines and Primary Bone Marrow by Src Kinase Inhibitor SU6656.

Abstract

Megakaryocytes (MKs) undergo successive rounds of endomitosis during differentiation, resulting in polyploidy (typically, 16-64N). Previous studies have demonstrated that this occurs through an interruption of normal cell cycle progression during anaphase. However, the molecular mechanism(s) controlling this unique process is undefined. Our studies of Src family kinases demonstrate that Lyn and Fyn kinases are activated by thrombopoietin (TPO) and have an inhibitory effect on cellular proliferation. In fact, adding PP1 to murine bone marrow cells in culture resulted in a higher percentage of polyploid cells. In the present report, we examined the effect of SU6656 on TPO-induced growth and differentiation. SU6656 is reported to be more specific than previous inhibitors for the Src family of tyrosine kinases. Remarkably, when SU6656 (2.5 mM) was added to a megakaryocytic cell line, UT-7/TPO, the cells underwent dramatic increase in cell size and polyploidization. By 24 h, 8N cells were present. After 48 h a 16N population was identified by FACS, and continued growth in SU6656 resulted in higher ploidy states (32N and 64N). This was accompanied by cessation of cellular proliferation (i.e. cell number remained constant), increase in CD41 and CD61 expression, and was notable for the absence of apoptosis. Similarly, polyploidization was observed when SU6656 was added to expanded human bone marrow progenitors with partial MK differentiation. The ability to induce differentiation was also seen using bone marrow from two patients with myelodysplastic syndrome and thrombocytopenia, suggesting that SU6656 might be useful as a differentiation-inducing agent for MKs. Although SU6656 is clearly a potent inhibitor of the Src family kinases, we have undertaken studies to determine if an additional target can be identified. We found no affect on Jak2, STAT3, and STAT5 tyrosine phosphorylation. However, the activity of Aurora kinase B was inhibited in vitro by as little as 50 nM SU6656. We propose that SU6656 may be an important tool for understanding the molecular basis of MK endomitosis and may also have therapeutic potential for individuals with MDS, thrombocytopenia, or other disorders resulting from incomplete megakaryocyte maturation.

Nicotinamide (vitamin B3) increases the polyploidisation and proplatelet formation of cultured primary human megakaryocytes

“…It has proven difficult to obtain high‐ploidy human Mk cells in vitro . Cultured primary human Mk cells typically reach a maximum DNA content of only 16N (Guerriero et al , 2001; Lannutti et al , 2005; Ma et al , 2005; Raslova et al , 2006), with only a few reports of cultures containing Mk cells with a maximum DNA content of 32N (De Bruyn et al , 2005) or 64N (Mattia et al , 2002). Also, cultures with Tpo only typically contain 15–25% high‐ploidy (≥8N) Mk cells (De Bruyn et al , 2005; Ma et al , 2005).…”

“…Although most of these studies have been performed using primary murine Mk cells or Mk cell lines, four recent studies with primary human cells have also demonstrated enhanced Mk maturation. Treatment with the Src kinase inhibitor SU6656 increased Mk cell size and the number of high‐ploidy Mk cells in cultures of human bone‐marrow‐derived CD34 + cells and in cultures of bone marrow mononuclear cells from patients with myelodysplastic syndromes (Lannutti et al , 2005). In another study, addition of the chemokine stromal‐cell‐derived factor‐1 α (SDF‐1 α ) to cells cultured with Tpo increased proplatelet frequency twofold and approximately doubled the number of Mk cells with both four and eight nuclear lobes compared to those with Tpo only (Guerriero et al , 2001).…”

“…Human bone marrow Mk cells reach a maximum DNA content of 128N, with a modal ploidy of 16N (Tomer, 2004). In contrast, cultured human Mk cells typically reach a maximum DNA content of 16N (Guerriero et al , 2001; Lannutti et al , 2005; Ma et al , 2005; Raslova et al , 2006), with only a few reports of cultures containing a low frequency of cells with a ploidy of 32N or 64N (Mattia et al , 2002; De Bruyn et al , 2005). Culture conditions that yield high Mk ploidy are desirable because there is a direct correlation between Mk DNA content and platelet production (Mattia et al , 2002).…”

“…Some progress has been made to increase Mk ploidy using genetic modification or chemical modulation of signalling pathways. However, most studies have been conducted using Mk cell line models and murine Mk cells, with only a limited number of studies performed using primary human Mk cells (Guerriero et al , 2001, 2006; Lannutti et al , 2005; Ma et al , 2005).…”

Ex Vivo production of platelets from stem cells

“…Lastly, Src‐family kinases inhibit megakaryocyte proliferation and differentiation (Lannutti & Drachman, ). Accordingly, inhibition of Src‐family kinases increased megakaryocyte polyploidization and proplatelet formation in leukaemic cell lines through the Lyn/Fyn pathway and inhibition of actin polymerization (Lannutti et al , ; Kaminska et al , ). Each (or all) of these pathways could be manipulated to force ex vivo cultured megakaryocytes to undergo polyploidization.…”