Objective: Rat liver stem-like epithelial cells (WB-F344) that under certain conditions may differentiate into hepa- tocyte and biliary lineages were subjected to acute X-irradiation with the aim to examine cell cycle peculiarities dur- ing the course of survival.
Materials and methods: Suspensions of WB-F344 cells that grew as a monolayer and reached sub-confluence were irradiated with 1, 5, and 10 Gy of X-rays (2 Gy/min). As an intact control, sham-irradiated cells were used. After irra- diation, cells were plated into 25-cm2 tissue culture flasks to culture them for over several days without reaching contact inhibition. On days 1, 2, 3, and 5 post-irradiation, cells were harvested and examined for nuclear morpholo- gy and DNA ploidy by stoichiometric toluidine blue reaction and image cytometry. On days 7 and 9 post-irradiation, only heavily irradiated (10 Gy) cells were examined. Also, 10 Gy-irradiated cells were chosen for immunofluorescence staining to monitor persistence of DNA lesions (γ-H2AX), cell proliferation (Ki-67), and self-renewal factors charac- teristic for stem cells (OCT4 and NANOG).
Results: Radioresistance of WB-F344 cells was evidenced by the findings that they do not undergo rapid and mas- sive cell death that in fact was weakly manifested as apoptotic even in heavily irradiated cells. Instead, there was cell cycle progression delay accompanied by polyploidization (via Ki-67-positive mitotic slippage or via impaired cytokinesis) and micronucleation in a dose-dependent manner, although micronucleation to some extent went ahead of polyploidization. Polyploid cells amenable for recovering from DNA damage can mitotically depolyploidize. Many micronuclei contained γ-H2AX clusters, suggesting isolation of severely damaged DNA fragments. Both factors, OCT4 and NANOG, were expressed in the intact control, but became enhanced after irradiation.
Conclusions: Although the fact of micronucleation is indicative of genotoxic effect, WB-F344 cells can probably escape cell death via sorting of damaged DNA by micronuclei. Induction of polyploidy in these cells can be adaptive to promote cell survival and tissue regeneration with possible involvement of self-renewal mechanism.
Meta: vyvchennia osoblyvostey̆ klitynnogo tsyklu oprominenykh epitelial'nykh stovburovykh klityn pechinky shchuriv (klityny liniï WB-F344) u protsesi vyzhyvannia, otsiniuiuchy radiatsiy̆no-indukovani zminy iadernoï morfologiï ta ploïdnosti DNK iz zastosuvanniam analizu zobrazhen', otrymanykh za dopomogoiu optychnogo mikroskopa. Materialy i metody. WB-F344 klityny, iaki vyroshchuvalysia u monoshari bez dosiagnennia povnogo pokryttia, op- rominiuvaly u suspenziï v dozakh 1,0; 5,0 i 10,0 Gr rentgenivs'kymy promeniamy potuzhnosti 2,0 Gr/khv. Iak intaktnyy̆ kontrol' vykorystovuvaly neopromineni klityny. Pislia oprominennia klityny vysivaly u 25-sm2 flakony v kil'- kosti, shcho ne dozvoliaie kontaktne gal'muvannia protiagom kil'kokh dniv ïkh kul'tyvuvannia. Na 1, 2, 3 ta 5-y̆ den' pislia oprominennia doslidzhuvaly iadernu morfologiiu i ploïdnist' klityn, vykorystovuiuchy stekhiometrychnu reaktsiiu z toluïdynovym synim i tsytometriiu zobrazhen'. Na 7-y̆ ta 9-y̆ den' pislia oprominennia doslidzhuvaly til'ky klity- ny, opromineni u velykykh dozakh (10,0 Gr), v iakykh dodatkovo otsiniuvaly naiavnist' poshkodzhen' DNK (γ-H2AX), proliferatyvnyy̆ stan (Ki-67), a takozh naiavnist' faktoriv samoonovlennia, prytamannykh stovburovym klitynam (OCT4 i NANOG).Rezul'taty. Radiorezystentnist' WB-F344 klityn bula dovedena rezul'tatamy, iaki vkazuiut' na te, shcho tsi klityny ne piddaiut'sia shvydkiy̆ ta masyvniy̆ zagybeli, malo skhozhiy̆ na apoptoz navit' u syl'no oprominenykh klitynakh. Na- tomist' sposterigaly zatrymku klitynnogo tsyklu, iaka suprovodzhuvalasia poliploïdyzatsiieiu (cherez Ki-67-pozy- tyvne mitotychne proslyzannia abo porushenyy̆ tsytokinez) i mikronukleatsiieiu zalezhno vid dozy, prote mikronuk- leatsiia deiakoiu miroiu vyperedzhala poliploïdyzatsiiu. Poliploïdni klityny, iaki zdatni pozbavliatysia poshkodzhen' DNK, mozhut' depoliploïdyzuvatysia cherez mitoz. Bagato mikroiader mistyly γ-H2AX klastery, shcho peredbachaie vydilennia syl'no poshkodzhenykh fragmentiv DNK. Obydva faktory, OCT4 i NANOG, buly vyrazheni v intaktnomu kontroli, prote ïkh ekspresiia posyliuvalasia pislia oprominennia.Vysnovky. Khocha fakt mikronukleatsiï svidchyt' pro naiavnist' genotoksychnogo efektu, WB-F344 klityny, y̆movirno, mozhut' unykaty zagybeli shliakhom sortuvannia poshkodzhenoï DNK za dopomogoiu mikroiader. Induktsiia poliploïdiï v tsykh klitynakh mozhe buty adaptyvnoiu dlia spryiannia vyzhyvanniu klityn i regeneratsiï tkanyn z mozhlyvym zalu- chenniam mekhanizmu samoonovlennia.
Keywords: WB-F344 cells; X-irradiation; cell survival; micronucleation; polyploidization; self-renewal.
B. I. Gerashchenko, K. Salmina, J. Krigerts, J. Erenpreisa, A. M. Babsky.