Background:Cancer stem cells (CSCs) paradigm suggests that CSCs might have important clinical implications in cancer therapy. Previously, we reported that accumulation efficiency of CSCs is different post low- and high-LET irradiation in 48 h.Methods:Cancer stem cells and non-stem cancer cells (NSCCs) were sorted and functionally identified through a variety of assays such as antigen profiles and sphere formation. Inter-conversion between CSCs and NSCCs were in situ visualised. Cancer stem cells proportions were assayed over multiple generations under normal and irradiation surroundings. Supplement and inhibition of TGF-β1, as well as immunofluorescence assay of E-cadherin and Vimentin, were performed.Results:Surface antigen markers of CSCs and NSCCs exist in an intrinsic homoeostasis state with spontaneous and in situ visualisable inter-conversions, irrespective of prior radiations. Supplement with TGF-β1 accelerates the equilibrium, whereas inhibition of TGF-β signalling disturbs the equilibrium and significantly decreases CSC proportion. Epithelial mesenchymal transition (EMT) might be activated during the process.Conclusion:Our results indicate that the intrinsic inter-conversion and dynamic equilibrium between CSCs and NSCCs exist under normal and irradiation surroundings, and TGF-β might have important roles in the equilibrium through activating EMT.

Bystander effects induced by cytoplasmic irradiation have been reported recently. However, the mechanism(s) underlying, such as the functional role of mitochondria, is not clear. In the present study, we used either mtDNA-depleted (r 0 ) A L or normal (r þ ) A L cells as irradiated donor cells and normal human skin fibroblasts as receptor cells in a series of medium transfer experiments to investigate the mitochondria-related signal process. Our results indicated that mtDNA-depleted cells or normal A L cells treated with mitochondrial respiratory chain function inhibitors had an attenuated g-H2AX induction, which indicates that mitochondria play a functional role in bystander effects. Moreover, it was found that treatment of normal A L donor cells with specific inhibitors of NOS, or inhibitor of mitochondrial calcium uptake (ruthenium red) significantly decreased g-H2AX induction and that radiation could stimulate cellular NO and O 2 KÀ production in irradiated r þ A L cells, but not in r 0 A L cells. These observations, together with the findings that ruthenium red treatment significantly reduced the NO and O 2 KÀ levels in irradiated r þ A L cells, suggest that radiationinduced NO derived from mitochondria might be an intracellular bystander factor and calcium-dependent mitochondrial NOS might play an essential role in the process.

The initiation and propagation of the early processes of bystander signaling induced by low-dose a-particle irradiation are very important for understanding the underlying mechanism of the bystander process. Our previous investigation showed that the medium collected from cell culture exposed to low-dose a-particle rapidly induced phosphorylated form of H2AX protein foci formation among the non-irradiated medium receptor cells in a timedependent manner. Using N G -methyl-L-arginine, 4-amino-5-methylamino-2 0 ,7 0 -difluorofluorescein diacetate and N xnitro-L-arginine (L-NNA) treatment before exposure to 1 cGy a-particle, we showed in the present study that nitric oxide (NO ) produced in the irradiated cells was important and necessary for the DNA double strand break inducing activity (DIA) of conditioned medium and the generation of NO in irradiated confluent AG1522 cells is in a time-dependent manner and that almost all NO was generated within 15 min post-irradiation. Concurrently, the kinetics of NO production in the medium of irradiated cells after irradiation was rapid and in a timedependent manner as well, with a maximum yield observed at 10 min after irradiation with electron spin resonance analysis. Furthermore, our results that 7-Nitroindazole and L-NNA, but not aminoguanidine hemisulfate, treatment before exposure to 1 cGy a-particle significantly decrease the DIA of the conditioned medium suggested that constitutive NO from the irradiated cells possibly acted as an intercellular signaling molecule to initiate and activate the early process (p30 min) of bystander response after low-dose irradiation.