Background. Activating mutations in the BRAF gene leads to a constitutive activation of the MAPK signaling. The highly selective BRAFV600E inhibitor, vemurafenib, improves the overall survival of BRAF-mutant melanoma patients. However, despite the excellent results of response rate, the average duration of the response was short and acquired resistance develops in most BRAF mutated melanoma patients within a few months. Objective: to derive melanoma cell lines from surgical species of patients with BRAF mutant melanomas resistant to vemurafenib and to elucidate the mechanisms involved in acquired drug resistance. Materials and methods. Mel Ki and Mel F1702 melanoma cells were obtained from metastases of disseminated melanoma patients with BRAFV600E mutation. 2D tumor cell culture, MTT test, immunicytochemistry, flow cytometry, real-time polimerase chain reaction and osteogenic and adipocytic differentiation were used in the study. Results. We have derived two melanoma cell lines Mel Ki and Mel F1702 from tumor samples of patients with BRAFV600E mutation resistant to vemurafenib. These cells were homogenous and had fibroblastic morphology. The IC50 values for Mel Ki and Mel F1702 were 4.7 and 6.3 μM, respectively. The expression of cancer-testis antigens was not detected in both types of cells suggesting the stemness of Mel Ki and Mel F1702 melanoma cells. The immunophenotypic profile of the vemurafenib resistsant melanoma cells showed the expression of typical mesenchymal stem cells markers such as CD90, CD105 and CD44. In addition, we found that the melanoma cell lines derived from tumor resistant to vemurafenib differentiated into osteoblastand adipocyte-like cells. Conclusion. In this study we are offering an experimental evidence of the phenotypic transition of the vemurafenib-resistant melanoma cells into mesenchymal stem-like cells.

The free radical theory of aging was proposed in 1956. Although it does not fully describe the mechanisms of aging, it is generally accepted that reactive oxygen species (ROS) are one of the pathogenetic factors in aging and, in particular, in the development of pathologies associated with aging. The main source of ROS in the cell is mitochondria. Antioxidants directed to mitochondria have a positive effect, but have low efficiency. The problem is that increased amounts of antioxidants disrupt normal cellular redox reactions, and a low amount of antioxidants is not able to seriously affect the processes. Protection against ROS may be more effective if the rate of ROS formation is reduced. There is a natural mitochondrial uncoupling process that significantly reduces ROS production. The weak uncoupler dinitrophenol (DNP) prolongs the life span of mice, reduces traumatic brain damage, and inhibits the development of a number of neurodegenerative diseases. Unfortunately, DNP has a number of disadvantages that hinder its practical use. Uncoupling of oxidative phosphorylation by free fatty acids is a natural mechanism, the activation of which can be used in medicine. The third (after antioxidants and uncouplers), but so far little studied, method of reducing ROS is telomerase, which, under conditions of oxidative stress, is transported into the mitochondria and improves cell survival by reducing ROS production.

Background. Activating mutations in the BRAF gene leads to a constitutive activation of the MAPK signaling. The highly selective BRAFV600E inhibitor, vemurafenib, improves the overall survival of BRAF-mutant melanoma patients. However, despite the excellent results of response rate, the average duration of the response was short and acquired resistance develops in most BRAF mutated melanoma patients within a few months. Objective: to derive melanoma cell lines from surgical species of patients with BRAF mutant melanomas resistant to vemurafenib and to elucidate the mechanisms involved in acquired drug resistance.Materials and methods. Mel Ki and Mel F1702 melanoma cells were obtained from metastases of disseminated melanoma patients with BRAFV600E mutation. 2D tumor cell culture, MTT test, immunicytochemistry, flow cytometry, real-time polimerase chain reaction and osteogenic and adipocytic differentiation were used in the study.Results. We have derived two melanoma cell lines Mel Ki and Mel F1702 from tumor samples of patients with BRAFV600E mutation resistant to vemurafenib. These cells were homogenous and had fibroblastic morphology. The IC50 values for Mel Ki and Mel F1702 were 4.7 and 6.3 μM, respectively. The expression of cancer-testis antigens was not detected in both types of cells suggesting the stemness of Mel Ki and Mel F1702 melanoma cells. The immunophenotypic profile of the vemurafenib resistsant melanoma cells showed the expression of typical mesenchymal stem cells markers such as CD90, CD105 and CD44. In addition, we found that the melanoma cell lines derived from tumor resistant to vemurafenib differentiated into osteoblastand adipocyte-like cells. Conclusion. In this study we are offering an experimental evidence of the phenotypic transition of the vemurafenib-resistant melanoma cells into mesenchymal stem-like cells.

In order to expand the informational capabilities of molecular genetic research, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.

Concentration of endogenous dipeptide carnosine in human muscle tissue reaches tens of millimoles. For more than 100 years of research, a lot of data concerning carnosine functions were accumulated, among which anti-aging effects are regarded most important. Heire, effect of carnosine in cell cultures was studied. It has been found that apart from the known action--an increase of the Hayflick limit and morphological rejuvenation--carnosine stimulates cell division in colony-forming assays and in the course of transition of cells to the quiescent state. The analysis of the transcriptome showed that carnosine-induced changes are mainly related to positive regulation of the cell cycle at all levels, from the onset of the DNA synthesis to chromosome condensation. One can suppose that the revealed stimulation of the cell cycle account for the carnosine-induced rejuvenation processes and a high concentration ofcarnosine in muscle tissue is required for the muscle recovery (regeneration) after excess loads.

Концентрация эндогенного дипептида карнозина в мышечной ткани человека достигает десятков миллимолей. За более чем 100 лет исследований накоплен огромный материал о самых разнообразных функциях карнозина, из которых основными считают действия, направленные против процессов старения. При исследовании действия карнозина на культуры клеток нами обнаружено, что помимо уже известных активностей: увеличения предела Хейфлика и морфологического омоложения клеток – карнозин стимулирует клеточные деления в тесте колониеобразования и тормозит процесс перехода клеток в состояние покоя. При анализе изменений в транскриптоме, происходящих под действием карнозина, выявлено, что основные сдвиги связаны с позитивной регуляцией клеточного цикла на всех уровнях – от начала синтеза ДНК до конденсации хромосом. Можно предположить, что именно способность карнозина стимулировать клеточный цикл лежит в основе его омолаживающего действия, а высокие концентрации этого дипептида в мышечной ткани играют роль не только физиологического буфера, но и необходимы для восстановления (регенерации) мышц после чрезмерных нагрузок.

We studied the properties of human skin fibroblast in filamentous polyglycolic microtransplant. Fibroblast adhesion to the microtransplant filaments is followed by the formation of a network cross-linked with fibroblasts. The cells rapidly proliferate during the first few days; after transfer of the microtransplant to the standard culture flask, the cells migrate to the plastic and continue proliferation. The cells are uniform and exhibit high colony-formation capacity. The bundles of microtransplant filaments persist in the culture for several days and through the cells completely leave them, the area around these filaments remains the most populated for 40 days. Mitotic cells are seen in the immediate proximity to the degrading filaments of the transplant. The effect of cell “rejuvenation” in the microtransplant can be explained by selection of cells by their adhesion to relatively thin (about 15 μ) filaments, which excludes large old cells.

The expression of human telomerase catalytic subunit in HL-60 and HT-1080 malignant transformed cells and telomerized fibroblasts was studied by quantitative PCR. It was found that the number of transcripts of human telomerase catalytic subunit per cell in telomerized fibroblasts could be hundreds of times higher than in HL-60 and HT-1080 cells. Telomerized fibroblast cultures are suggested as experimental systems for selection of basal compounds for creation of anticancer drug prototypes, the molecular target of which is human telomerase catalytic subunit. The effects of human telomerase catalytic subunit expression on the fibroblast proteome are analyzed.

It is known that most somatic cells do not possess telomerase activity. It led to end ternunae underreplication of chromosomes and, as the result, to proliferative senescence of cells. We investigated the consequences of introduction of gene of catalytic subunit of human telomerase (hTERT) into normal dermal fibroblasts of adult person. We showed that hTERT expression led to the appearance of telomerase activity, telomere lengthening, and immortalization. Proliferation of one of the fibroblast clones (1608tel7) was investigated up to 200 population doublings level (PDL), another clone - 1608tel2 - up to 120 PDL, After Hayflick limit, transition the cells did not change significantly, they preserved constant rate of proliferation, and morphologically they resembled young human diploid fibroblasts. Original cells and cells after transfection with empty vector can achieve not more than 68PDL, their proliferation decreased, and they acquired senescent-like morphology, Telomeres of hTERT-transfected cells became longer up to 10-20 kb. Original cells possess 8 kb-long telomeres. Long term blocking of telomerase function with azidothymidine induced telomere shortening and significant decrease of proliferation rate, but after approximately three weeks nondivided cells resumed proliferation with the previous rate. Long term nondivided cells became bigger, they preserved viability and morphologically resembled senescent cells. Thus, human cells immortalized via introduction of hTERT gene, in contrast to human tumour cells, preserve mechanisms of proliferative senescence that can be induced by telomere shortening.

The long-term action of azidothymidine, reverse transcriptase inhibitor, on cultivated U-937 (human promyelocyte leukemia) and MeWo (human melanoma) cells led to the concentration-dependent decrease in the length of telomeric chromosomal repeats. Telomere shortening was accompanied by temporary retardation of cell proliferation. Combined with the data obtained previously, these results suggest that azidothymidine inhibits telomerase functioning in cultivated cells.

It was shown what microinjection of polyclonal antibodies to the myc protein specifically inhibits DNA synthesis in serum-stimulated 3T3 Swiss cells during the first 6 h of the prereplicative period. The effect depends on the concentration of antibodies. Microinjections of polyclonal antibodies against the whole protein were more effective when microinjections of antibodies against parts of the protein. Microinjections of five kinds of monoclonal antibodies and their mixture were in effective. It was also shown what induction of expression of the antisense myc sequence in 3T3 Swiss cells leads to potent inhibition of DNA synthesis during the first 6 h of the prereplicative period. Thus it is clear what the myc protein participates in the early stages of preparation to replication, i.e., transition of cells from G0 to G1.