ИСТИНА

Microtubules (MT) are involved in diverse cellular processes such as cell division and cell migration. MT-targeted drugs applied at nanomolar concentrations inhibit dynamic instability of MTs affecting dynamicity, growth and shortening rate. The aim of this study was to assess the effects of concentrations range of MT inhibitors with different interaction mode with tubulin, using nocodazole, taxol and vinorelbine in an attempt to determine threshold dose for each drug. Cell culture of NIH-3T3 fibroblasts stably expressing EB3-RFP was developed. Cells images were recorded on AxioObserver microscope with a temperature control unit (Carl Zeiss) with Hamamatsu ORCAFLASH 2 camera using x63/1.4 PlanApo oil immersion objective with 2 seconds intervals. 16 bit images were processed for analysis using ImageJ software. From the qualitative analysis of MT dynamics we imposed upper and lower limits of drugs. The effects of different concentrations of drugs on MTs growth were categorized into four groups: no alteration; minimal alteration; obvious alterations; number of plus end comets reduces significantly. Minimal concentrations when alterations were visible appeared to be the follows: for nocodazole – 30 nM; for taxol – 100 nM and for vinorelbine – 10 nM. Concentrations when comets became rare and unmoved were for nocodazole – 1000 nM; for taxol – 1600 nM and for vinorelbine – 300 nM. To further quantify MT growth we addressed the question on variance of growth rates in individual cells. In the 11 cell groups had been analyzed independently from the same culture MT growth rate varied from 13.3±2.2 to 25.1±2.2 µm/min and even within a group of cells on the same coverslip the variance was in the range of 2.5 times. However, for a given cell the difference obtained in the repeated measurement was less than 2%. Thus using measurement in the same cells before and after treatment it was possible to quantify minor changes in microtubule behavior. In the range of concentrations between lower and upper limits MTs growth rate decreased gradually. Quantitative analysis demonstrated that at the threshold level MT growth rate decreased to 97% for taxol, and 90% for vinorelbine and nocodazole (insignificant from control). Significant slowdown of MT growth was observed for nocodazole treatment at a concentration of 100 nM, for taxol – at 300 nM and for vinorelbine – at 30 nM. We conclude that (1) there is rather narrow range of concentrations of drugs when MT growth is impeded, but not stopped, and (2) quantitative analysis of MT dynamics alterations under the action of different drugs requires repeated observations of the same cells before and after the treatment.