Cycline dependent kinase 4 decreased level in the population of Ki-67-negative melanoma cells

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Introduction. A transition of proliferating cancer cells to a G0 phase of a cell cycle considered to be implicated in cancer cell drug resistance.

The aim of the study. A separation of G0-positive melanoma B16 cells fraction that correspond to resting followed by cyclin dependent kinase 4 and cyclin dependent kinase inhibitor B1 expression estimation in these cells was the aim of the study.

Methods. Melanoma B16 cells were subjected to incubation with alkylating agent dacarbazine to induce a transition of cancer cells in a G0 phase of a cell cycle. Immunocytochemical assay was applied to visualize Ki-67-negative, Go-positive melanoma cells. Fluorescent sorting was used to separate an isolated population resting cells with subsequent cyclin dependent kinase 4 and cyclin dependent kinase inhibitor B1 expression evaluation.

Results. Dacarbazine induced the percentage of Ki-67-negative melanoma B16 cells that correspond to G0-positive cells. Fluorescent sorting application revealed 26% of resting cells among all melanoma cells population. Resting melanoma cells exhibited diminished levels of cyclin dependent kinase 4.

Conclusion. Alkylating agent dacarbazine induces the percentage of resting cancer cells that may be an additional mechanism of cancer cell drug resistance. Cell sorting allowas to separate of resting cancer cells population. Further study for these cell phenotype characterization is important for developing new strategies of targeting quiescent, G0-positive cancer cells.

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作者简介

Ekaterina Lapkina

Prof. V.F. Voino–Yasenetsky Krasnoyarsk State Medical University

编辑信件的主要联系方式.
Email: e.z.lapkina@mail.ru
ORCID iD: 0000-0002-7226-9565

associated professor of the Department of pharmacy. Candidate of biological sciences

俄罗斯联邦, Partizan Zheleznyak, 1, Krasnoyarsk, Krasnoyarsk Territory, 660022

Alexandra Esimbekova

Prof. V.F. Voino–Yasenetsky Krasnoyarsk State Medical University

Email: aleksandra.esimbekova.96@mail.ru
ORCID iD: 0000-0001-6363-5941

PhD student of the Department of pathological physiology

俄罗斯联邦, Partizan Zheleznyak, 1, Krasnoyarsk, Krasnoyarsk Territory, 660022

Ivan Zinchenko

Prof. V.F. Voino–Yasenetsky Krasnoyarsk State Medical University

Email: Zinchenko.Ivan.003@gmail.com
ORCID iD: 0000-0001-7085-6304

laboratory assistant of the Department of pathological physiology

俄罗斯联邦, Partizan Zheleznyak, 1, Krasnoyarsk, Krasnoyarsk Territory, 660022

Tatiana Ruksha

Prof. V.F. Voino–Yasenetsky Krasnoyarsk State Medical University

Email: tatyana_ruksha@mail.ru
ORCID iD: 0000-0001-8142-4283

head of pathophysiology department. Doctor of medical sciences, Professor

俄罗斯联邦, Partizan Zheleznyak, 1, Krasnoyarsk, Krasnoyarsk Territory, 660022

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2. Fig. 1. Melanoma B16 cells Ki-67 immunostaining after dacarbazine treatment at concentration 1,86 mM/l. a – control; b – dacarbazine-treated cells. Ki-67-positive cells stained green, alive cell nuclei stained dark blue (DAPI)

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3. Fig. 2. Flow cytometry plots (FSC-A – forward scatter, SSC-A – side scatter) representing melanoma B16 cells set subjected to fluorescent sorting due Ki-67 expression after dacarbazine treatment

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4. Fig. 3. Relative levels of CDK4 and CDKN1B expression in FACS populations of B16 melanoma cells after 72-hour exposure to dacarbazine

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