To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK-) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK- cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D0 for PIC-20 cells was 7.4 +/- 0.2 Gy, which is three times higher than that for parental cells (2.5 +/- 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK- cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK- cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.

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The possibility of using VEGF-R1 receptor for targeted therapy in oncology was investigated. Using the approach to measuring the protein content in intact nuclei of cells, which was developed by us, we showed the presence of this receptor in the nuclei of tumor, but not normal cells. A direct correlation between the level of VEGF-R1 expression in the nucleus and the degree of malignancy of tumor cells, indicating the prognostic value of this parameter, was found. The mechanisms of the functioning of this receptor and the pathways of inhibiting its activity are discussed, and the validity of the selection of VEGF-R1 as a molecular target for anticancer therapy is conformed.

Background. Identification of cellular proteins can be performed by indirect immunofluorescence assay using flow cytometry. However, this method allows to detect cellular proteins that are either on the cell surface or inside the cell and cannot demonstrate a protein distribution within cellular compartments, particularly in nucleus. Meanwhile, the nuclear localization of the protein of interest in many respects gives an indication of the mechanisms of its action. Therefore, the development of the protocol of extraction of nuclei suitable for analysis on a flow cytometer is able to complement the information about the localization and functional significance of many nucleus-associated proteins. Objective. The aim of this study was to develope a method for extraction, purification and stabilization of intact nuclei suitable for flow cytofluorimetry analysis. Materials and methods. In this work we studied the nuclear localization of the receptor type 1 for vascular endothelial growth factor (VEGF-R1). The A431 human cancer cell line was used as an object of the study. The quality of extracted nuclei was assessed by microscopic examination of stained smears of nuclear suspension. Expression of the receptor was determined by indirect immunofluorescence assay using flow cytometry. Results. New method was successfully applied to obtain the suspension of intact cellular nuclei that is crucial to perform further flow cytometry. Applied method revealed the presence of the receptor type 1 for vascular endothelial growth factor at the external nuclear membrane and inside of the nucleus. Interesting to note that the presence of the receptor type 1 for vascular endothelial growth factor inside ofthe nucleus was 3,8 times as much as its surface location (17,9 ± 1,04 % and 4,8 + 0,26 % respectively). Conclusions. The new method of extraction, purification and stabilization ofthe nuclei is applicable for proteins identification by flow cytometry. In combination with other methods (ICC, Western blotting, etc.) flow cytometry of intact nuclei is able to complement the information about the properties of nucleus-associated proteins.