RESUMEN
Mechanisms underlying the effects of low-dose ionizing radiation (IR) exposure (10-100 mGy) remain unknown. Here we present a comparative study of early (less than 24h) and delayed (up to 11 post-irradiation passages) radiation effects caused by low (80 mGy) vs intermediate (1000 mGy) dose X-ray exposure in cultured human bone marrow mesenchymal stem cells (MSCs). We show that γÐ2ÐÐ¥ foci induced by an intermediate dose returned back to the control value by 24 h post-irradiation. In contrast, low-dose irradiation resulted in residual γÐ2ÐÐ¥ foci still present at 24 h. Notably, these low dose induced residual γÐ2ÐÐ¥ foci were not co-localized with ÑÐТРfoci and were observed predominantly in the proliferating Ði67 positive (Ði67+) cells. The number of γÐ2ÐÐ¥ foci and the fraction of nonproliferating (Ði67-) and senescent (SA-ß-gal+) cells measured at passage 11 were increased in cultures exposed to an intermediate dose compared to unirradiated controls. These delayed effects were not seen in the progeny of cells that were irradiated with low-dose X-rays, although such exposure resulted in residual γÐ2ÐÐ¥ foci in directly irradiated cells. Taken together, our results support the hypothesis that the low-dose IR induced residual γH2AÐ¥ foci do not play a role in delayed irradiation consequences, associated with cellular senescence in cultured MSCs.
Asunto(s)
Células de la Médula Ósea/efectos de la radiación , Proliferación Celular/efectos de la radiación , Senescencia Celular/efectos de la radiación , Histonas/metabolismo , Células Madre Mesenquimatosas/efectos de la radiación , Adulto , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Humanos , Antígeno Ki-67/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Transducción de Señal/efectos de la radiación , Factores de Tiempo , Rayos X , beta-Galactosidasa/metabolismoRESUMEN
Expansion of mesenchymal stromal/stem cells (MSCs) used in clinical practices may be associated with accumulation of genetic instability. Understanding temporal and mechanistic aspects of this process is important for improving stem cell therapy protocols. We used γH2AX foci as a marker of a genetic instability event and quantified it in MSCs that undergone various numbers of passage (3-22). We found that γH2AX foci numbers increased in cells of late passages, with a sharp increase at passage 16-18. By measuring in parallel foci of ATM phosphorylated at Ser-1981 and their co-localization with γaH2AX foci, along with differentiating cells into proliferating and resting by using a Ki67 marker, we conclude that the sharp increase in γH2AX foci numbers was ATM-independent and happened predominantly in proliferating cells. At the same time, gradual and moderate increase in γH2AX foci with passage number seen in both resting and proliferating cells may represent a slow, DNA double-strand break related component of the accumulation of genetic instability in MSCs. Our results provide important information on selecting appropriate passage numbers exceeding which would be associated with substantial risks to a patient-recipient, both with respect to therapeutic efficiency and side-effects related to potential neoplastic transformations due to genetic instability acquired by MSCs during expansion.