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  • Title: CHANGES OF CYCLIN D1-DEPENDENT REGULATION OF CELL CYCLE IN PERIPHERAL BLOOD LYMPHOCYTES OF CHORNOBYL CLEAN-UP WORKERS AT A REMOTE PERIOD AFTER RADIATION EXPOSURE.
    Author: Golyarnik NA, Ilyenko IM, Zvarych LM, Bazyka DA.
    Journal: Probl Radiac Med Radiobiol; 2020 Dec; 25():430-442. PubMed ID: 33361852.
    Abstract:
    OBJECTIVE: To study proliferative potential of peripheral blood lymphocytes of Chornobyl clean-up workers by levelof expression of cyclin D1 and quantitative parameters of cell cycle at a remote period after radiation exposure. MATERIALS AND METHODS: The research subject was the peripheral blood lymphocytes (PB) of Chornobyl clean-upworkers 30-33 years after radiation exposure. A total of 207 men were surveyed, 164 of them were clean-up workers exposed in the dose range 10.43-3623.31 mSv and 43 persons of the control group. Analysis of proliferationpotential (cell cycle initiation) and cyclin D1 expression in PB lymphocytes were performed in vitro by a micro methodof whole blood leukocytes culture with phytohemagglutinine-P (PHA). Sample preparation was performed by a standard immunofluorescent assay for intracellular proteins using the FITC labelled Mouse Anti-Human Cyclin D1Antibody Set. Cell distribution by cell cycle phases studied by propidium iodide DNA staining and analysis onFACSCalibur laser flow cytometer in histogram mode with separation of G0/G1-, S- and G2/M-regions and Sub-G0/G1-region (apoptotic cells). RESULTS AND CONCLUSIONS: An increase in the level of spontaneous сyclin D1 expression and disturbance of сyclinD1-dependent regulation of cell cycle of PB lymphocytes after mitogen activation were determined in a remote period after radiation exposure. An increase in the level of cyclin D1 expression was accompanied by increase in pool ofcells in the S- and G2/M-phases of cell cycle which characterizes the high proliferative potential of PB lymphocytes.Mitogen-induced delay of cell cycle of lymphocytes in G1/S check point and reduction of S-phase was revealed.These changes are a manifestation of genomic instability caused by the effect of radiation and depend on the radiation dose. The results confirm the hypothesis about the significance of levels of cyclin D1 expression, as a criterion for manifestations of genome instability and risks of oncogenesis in a remote period after irradiation. Meta: vyznachyty osoblyvosti proliferatyvnogo potentsialu limfotsytiv peryferychnoï krovi uchasnykiv likvidatsiï naslidkiv avariï na ChAES za rivnem ekspresiï tsyklinu D1 ta kil'kisnymy pokaznykamy klitynnogo tsyklu uviddalenomu periodi pislia oprominennia.Materialy i metody. Ob’iektom doslidzhennia buly limfotsyty peryferychnoï krovi (PK) uchasnykiv likvidatsiïnaslidkiv avariï (LNA) na ChAES u viddalenomu periodi pislia oprominennia (> 30 rokiv). Obstezheno 207 osib cholovichoï stati, iz nykh 164 uchasnykiv LNA na ChAES, oprominenykh u diapazoni doz 10,43–3623,31 mZv ta 43 osobykontrol'noï grupy. Doslidzhennia proliferatyvnogo potentsialu (initsiatsiia klitynnogo tsyklu) ta rivniv ekspresiïtsyklinu D1 u limfotsytakh PK provodyly in vitro z vykorystanniam mikrometodu kul'tyvuvannia leĭkotsytiv tsil'noïkrovi z fitogemagliutyninom-R (FGA). Probopidgotovku vykonuvaly za standartnoiu metodykoiu imunofluorestsentnogo zabarvlennia vnutrishn'oklitynnykh bilkiv z vykorystanniam reagentiv FITC Mouse Anti-Human Cyclin D1Antibody Set (BD, SShA). Parametry klitynnogo tsyklu doslidzhuvaly shliakhom zabarvlennia iader klityn propidiiuĭodydom (Sigma, SShA) u rezhymi DNK-gistogram z vidokremlenniam G0/G1-, S- ta G2/M-regioniv (fazy klitynnogotsyklu) ta Sub-G0/G1-regionu (iadra klityn u stani apoptozu). Analiz provodyly na protochnomu tsytometriFACSCalibur (BD, SShA).Rezul'taty ta vysnovky. Provedeno kil'kisnu otsinku proliferatyvnogo potentsialu limfotsytiv PK uchasnykivLNA na ChAES za pokaznykamy klitynnogo tsyklu i rivnem ekspresiï bilka tsyklin D1 spontanno ta u korotkochasniĭkul'turi limfotsytiv in vitro. U viddalenomu periodi pislia oprominennia vyznacheno pidvyshchennia rivnia spontannoïekspresiï tsyklinu D1 i porushennia tsyklin D1-zalezhnoï reguliatsiï klitynnogo tsyklu limfotsytiv PK pislia aktyvatsiïmitogenom. Pidvyshchennia rivnia ekspresiï tsyklinu D1 suprovodzhuvalos' zbil'shenniam pulu klityn u S- ta G2/M-fazakh klitynnogo tsyklu, shcho kharakteryzuie vysokyĭ proliferatyvnyĭ potentsial limfotsytiv PK. Vyiavleno mitogenindukovanu zatrymku klitynnogo tsyklu limfotsytiv u G1/S-kontrol'niĭ tochtsi, skorochennia S-fazy. Dani zminy ieproiavom nestabil'nosti genomu, zumovleni diieiu radiatsiï ta zalezhat' vid dozy oprominennia. Otrymani rezul'taty pidtverdzhuiut' gipotezu, shchodo znachennia rivniv ekspresiï tsyklinu D1, iak kryteriïv proiaviv nestabil'nosti genomu ta ryzykiv onkogenezu u viddalenomu periodi pislia oprominennia.
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