Imatinib mesylate (IM)-induced growth inhibition is associated with production of spliced osteocalcin—mRNA in cell lines
Introduction
IM (STI571, Glivec) selectively targets the Bcr-Abl protein tyrosine kinase that competes with ATP for its specific binding site in the kinase domain. In addition, it has activity against platelet-derived growth factor (PDGF) receptor alpha and beta [1], and c-KIT, the receptor for the stem cell factor (SCF). As the signalling pathways mediated by activation of the above mentioned cytokines may act as survival signals in some cancers including both, solid tumours and leukemias, an inhibition of these pathways may potentiate the activity of some cytotoxic drugs [2], [3].
Recent data indicate that IM-treatment influences bone remodelling as evidenced by clinical observations of hypophosphatemia and up-regulation [4] or down-regulation of osteocalcin-serum-levels [5].
In addition, IM potently induces osteoclast apoptosis and strongly suppresses the bone resorbing activity of osteoclasts [6]. Osteoclast survival is coordinated in part by proteins synthesised and presented by cells of osteoblast lineage [7]. Among these, macrophage colony stimulating factor (M-CSF) is known to play a critical role not only in osteoclast differentiation [8], but also as one of the most potent osteoclast survival factors [9], [10]. M-CSF triggers osteoclast survival by binding to its cognate receptor cFMS [11]. It has been demonstrated that IM can also target the cFMS of some non-malignant hematopoietic cells, including monocytes/macrophages [12], [13], [14]. Further data also demonstrated that IM decreases osteoclast formation from primary human peripheral blood monocytes [15], because osteoclasts are cells derived from progenitors of the monocyte/macrophage lineage whose differentiation is dependent on M-CSF.
We have shown that osteocalcin, an osteoblast specific protein and differentiation marker, is transcribed in AML and CML, in a normal spliced and an non-spliced variant in c-KIT positive cells [16].
Aim of the study was to find out whether IM has a direct effect on osteocalcin expression and whether osteocalcin is up- or down-regulated in osteoblasts to substantiate the in vivo data [4], [5].
The activity of IM on c-KIT signal transduction suggests that this drug could influence the splicing process of osteocalcin as well. A further topic of this work was to find out, if IM influences telomerase activity, because apart from the Runt domain transcription factors AML1 and AML3, telomerase has been discussed to influence OCN-transcription [17].
Thus, besides evaluating the effect of IM, this study provides some insight into the network of OCN-regulation in c-KIT positive cell lines from leukemic (HL-60) and osteoblastic origin such as MG-63 in comparison to the c-KIT negative U-2 OS osteosarcoma cell line as well as to osteoblastic cells MC3T3-E1.
Section snippets
Cell culture and treatment
HL-60, MG-63, U-2 OS and MC3T3-E1 cells were cultured in petri dishes (8 cm in diameter) in αMEM (Biochrom AG, Berlin, Germany), supplemented with 4.5 g/l glucose, 5% foetal calf serum (Sigma) and 30 μg/l gentamycin (Sigma) at 37 °C under 5% CO2 in humidified air. The adherent cell-lines were sub-cultured twice a week using 0.001% pronase E (Roche) and 0.02% EDTA in Ca2+ and Mg2+ free phosphate buffered saline (PBS). HL-60 cells were sub-cultured by centrifuging at 400 × g for 5 min. To prevent a
Attenuation of cell multiplication and telomerase activity (TA) by IM
Cell lines (HL-60, MG-63, U-2 OS, MC3T3-E1), were cultured with different concentrations of IM between 100 nM and 20 μM and compared to untreated and VD-treated cultures, respectively. For appropriate statistical coverage, each culture was kept as hexaplicate.
VD stimulated cell multiplication in MG-63 cells but inhibited it slightly but significantly in all other cell lines tested (Fig. 2). IM, however, irrespective of cell line, inhibited cell multiplication significantly with respect to the
Discussion
The results of this study confirm an antiproliferative effect of IM without induction of cell death [24], [25], irrespective of cell type and presence of the stem cell marker c-KIT. Inhibition of TA by IM was dose-dependent and associated with inhibition of cell multiplication. This confirms previous data concerning IM-associated down-regulation of cell proliferation [1] and the TA in various malignant cell lines [34].
Besides down-regulation of the TA activity IM attenuated expression of both,
Acknowledgements
We thank Helmut Mühlberger and Sylvia Spitzer for skilful technical assistance. This study was supported by the Jubiläumsfonds der Österreichischen Nationalbank (Project No. 11455).
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