The Plk1 inhibitor BI 2536 temporarily arrests primary cardiac fibroblasts in mitosis and generates aneuploidy in vitro

Abstract

BI 2536 is a new anti-mitotic drug that targets polo-like kinase 1 (Plk1) and is currently under clinical development for cancer therapy. The effect of this drug on cancer cells has been extensively investigated, but information about the effects on primary dividing cells and differentiated non-dividing cells is scarce. We have investigated the effects of this drug on primary neonatal rat cardiac fibroblasts and on differentiated cardiomyocytes and explored the possibility to use this drug to enrich differentiated cell populations in vitro. BI 2536 had a profound effect on cardiac fibroblast proliferation in vitro and arrested these cells in mitosis with an IC50 of about 43 nM. Similar results were observed with primary human cells (HUVEC, IC50 = 30 nM), whereas the cancer cell line HeLa was more sensitive (IC50 of 9 nM). Further analysis revealed that prolonged mitotic arrest resulted in cell death for about 40% of cardiac fibroblasts. The remaining cells showed an interphase morphology with mostly multi- and micro-nucleated nuclei. This indicates that a significant number of primary fibroblasts are able to escape BI 2536 induced mitotic arrest and apparently become aneuploid. No effects were observed on cardiomyocytes and hypertrophic response (growth) upon endothelin-1 and phenylephrine stimulation was normal in the presence of BI 2536. This indicates that BI 2536 has no adverse effects on terminally differentiated cells and still allows proliferation independent growth induction in these cells. In conclusion, cardiomyocytes could be enriched using BI 2536, but the formation of aneuploidy in proliferating cells most likely limits this in vitro application and does not allow its use in putative cell based therapies.

Figure 1. Effect of BI 2536 on neonatal rat cardiomyocytes.

Neonatal rat cardiomyocytes were cultured for three days before 100 nM BI 2536, or DMSO as control, was added. A. Light microscopy pictures of control and 24 hours BI 2536 treated cultures. B. Immunofluorescence microscopy pictures of BI 2536 treated cells stained with anti-troponinT (red) and anti-α-tubulin (green). In the middle a mitotic cell is shown with monopolar spindle devoid of troponin T staining. Scale bar is 10 µM. C. FACS analysis of 24 h treated and untreated cells stained with anti-α-actinin and anti-Phospho-Histone H3. Four regions are selected and marked as follow: C-I =  cardiomyocytes in interphase, C-M  =  cardiomyocytes in mitosis, F-I =  fibroblasts in interphase, F-M  =  fibroblasts in mitosis. D. Quantification of FACS profiles of control and 24 h BI 2536 (BI) treated cells stained like in C. Dark grey bars indicate the percentage interphase cells and light grey bars indicate the percentage mitotic cells. CM =  cardiomyocyte, Fibro =  fibroblast.

Figure 2. Effect of BI 2536 on hypertrophy induction in neonatal rat cardiomyocytes.

Cardiomyocyte cultures were grown as before with or without 100 nM BI 2536 and subsequently serum starved in the presence or absence of BI 2536. A. Effect of treatment with the hypertrophy inducing hormone endothelin-1 on cellular growth ([³H]-Leucine incorporation) in the presence or absence of BI. [³H]-Leucine incorporation into cellular proteins was determined by liquid scintillation counting. Values are relative to the respective non-treated cultures and error bars depict standard deviations (n = 3). B. Analysis of ANP gene expression by real-time PCR in cultures treated with or without ET-1 in the presence or absence of BI 2536. Levels are corrected for expression of the cyclophilin A housekeeping gene. Error bars depict standard deviations (n = 3).

Figure 3. BI 2536 reduces fibroblast numbers in cardiomyocyte cultures.

Cardiomyocyte cultures were grown as before with or without 100 nM BI 2536. A) The percentage cardiomyocytes and cardiac fibroblasts were determined by FACS analysis, using anti- α-actinin staining. Before harvest mitotic cells were removed by shake off. Light gray bars indicate fibroblasts, dark grey bars show the percentage cardiomyocytes. B After 24 hour treatment RNA was isolated from cells and real time PCR was performed with primers against cardiac troponin T to determine the expression level of this cardiomyocyte specific gene, providing an indirect measure for the number of cardiomyocytes. Error bars depict standard deviations (n = 3). C. Same as in B, but with primers against periostin, a marker for cardiac fibroblasts.

Figure 4. Effect of BI 2536 on rat cardiac fibroblasts.

Rat cardiac fibroblasts were cultured for two days before the indicated doses of BI 2536 or DMSO as a control were added. HeLa cells were cultured under identical conditions. A. Dose response curve of primary cardiac fibroblasts and HeLa cells. Cells were cultured for 24 hours with BI 2536 and subsequently fixed and stained with anti-Phospho-Histone H3, as a mitotic marker, and then quantified by FACS analysis. Closed circles, HeLa cells; open circles, cardiac fibroblasts. B. Light microcopy pictures of control and 24 h BI 2536 treated cardiac fibroblast cultures C. Immunofluorescence microscopy pictures of same cells as in B, but stained with anti-α-tubulin (green) and the DNA stain TOPRO-3 (blue). Scale bar is 10 µM.

Figure 5. BI 2536 temporarily arrests cardiac fibroblasts.

Rat cardiac fibroblasts were cultured for two days before 100 nM BI 2536 or DMSO as control were added. Cells were subsequently analysed at different time points. A. FACS analysis of cells stained with anti-Phospho-Histone H3 and propidium iodide. In the upper panel the control cells are shown and in the lower panel the BI2536 treated cells. The different regions are marked as follow: I is interphase, M is mitosis and D denotes dead cells. B. Quantification of FACS profiles, as shown in A (n = 3). Dark grey bar depict interphase cells, light grey bar depicts mitotic cells and white bar depicts dead cells. C. Immunofluorescence pictures of remaining interphase cells after 72 hours culture in the presence or absence of BI 2536. Cells were stained with anti-α-tubulin (green) and the DNA stain TOPRO-3 (blue). Control cells are shown in the upper panel. Scale bar is 10 µM.