Bone and soft tissue sarcomas, tumors of the skinBone and soft tissue sarcomas, tumors of the skinСаркомы костей, мягких тканей и опухоли кожи2219-46142782-3687Publishing House ABV Press530EXPERIMENTAL ONCOLOGYЭКСПЕРИМЕНТАЛЬНАЯ ОНКОЛОГИЯEffect of B-RAF600 activating gene mutations on the ability of melanoma cells to autophagyВлияние активирующих мутаций V600 гена B-RAF на способность клеток меланомы к аутофагииRyabayaO. O.РябаяО. О.
Russian Federation
TsyganovaI. V.ЦыгановаИ. В.
Russian Federation
SidorovaT. A.СидороваТ. А.
Russian Federation
BurovaO. S.БуроваО. С.
Russian Federation
StepanovaE. V.СтепановаЕ. В.
Russian Federation
oxa2601@yandex.ruFSBI N.N. Blokhin Russian Cancer Research Center Of The Russian Academy Of Medical sciencesФГБУ «Российский онкологический научный центр им. И.И. Блохина» РАМН0308201353687203022022Copyright ©; 2013, Ryabaya O.O., Tsyganova I.V., Sidorova T.A., Burova O.S., Stepanova E.V.Copyright ©; 2013, Рябая О.О., Цыганова И.В., Сидорова Т.А., Бурова О.С., Степанова Е.В.2013Ryabaya O.O., Tsyganova I.V., Sidorova T.A., Burova O.S., Stepanova E.V.Рябая О.О., Цыганова И.В., Сидорова Т.А., Бурова О.С., Степанова Е.В.https://creativecommons.org/licenses/by/4.0https://sarbon.abvpress.ru/jour/article/view/530

Background. We investigate the interaction of the B-RAFgene mutations status and levels of autophagy induction in melanoma cell lines in vitro. Methods. In this report 12 human melanoma cell lines were investigated. B-RAF gene mutations were determined by PCR, expression of Beclin1 mRNA-by RT-PCR. Analysis of the LC3B protein expression and accumulation, specifically associated with the membranes of autophagosomes, was performed by immunocytochemical assay. Results. Among melanoma cell lines 7 had activating mutations in B-RAF gene. Basal expression level of Beclin1 mRNA was identified in all melanoma cell lines. The basal expression ranged from 0,15 to 1,16 r.u. Cell lines starvation resulted in increased expression of mRNA Beclin1 level in cells B-RAFwt, but B-RAFV600 cell lines, it remained almost unchanged. Autophagy induction by temozolomide has shown a significant increase in the amount of autophagosome punctuate in B-RAf and B-RAF600 melanoma cells. Conclusion. B-RAF mutations may affect the melanoma autophagy level in vitro. Investigation of the mechanisms of this process will enable the development of new predictive factors of drug resistance in melanoma and identify the original target for anticancer therapy.

Цель работы. исследована взаимосвязь статуса гена B-RAFсо степенью активации аутофагии в клеточных линиях меланомы человека in vitro. Материалы и методы. В работе были использованы 12 клеточных линий меланом человека. Наличие мутаций гена B-RAF в клетках определяли методом ПЦР, уровень экспрессии мРНк гена Beclin1 - методом оТ-ПЦР. Анализ экспрессии и аккумуляции белка LC3B, специфически ассоциированного с мембранами аутофагосом, проводили иммуноцитохимическим методом. Результаты. Среди исследованных клеточных линий 7 имели активирующие мутации в гене B-RAF. Базальный уровень экспрессии мРНк гена Beclin1 регистрировали в клетках всех исследованных линиях меланомы. Его величина варьировала в широком диапазоне от 0,15 до 1,16 о.е. Голодание клеточных линий приводило к увеличению экспрессии мРНк Beclin1 в клетках B-RAF\ а в B-RAF600 клеточных линиях он практически не менялся. однако противоопухолевый препарат темозоломид увеличивал количество аутофагосом в клетках меланомы человека с разным статусом B-RAF. Выводы. Активирующие мутации в гене B-RAFмогут влиять на уровень аутофагии в клетках меланомы in vitro. изучение механизмов этого процесса позволит разработать новые факторы предсказания лекарственной устойчивости меланомы и выявить оригинальные мишени для противоопухолевой терапии.

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