Mitochondrial DNA-dependent Signaling Pathway Is Involved In The Process Of α Particles-induced Malignant Transformation In Vitro

Objective：To explore the role of mitochondrial DNA （mtDNA） in the process ofmalignant transformation induced by α-particles, and in the mechanisms of disabledapoptosis.Methods:（1） Human bronchial epithelial cells （HBE） were cultured in mediumcontaining50ng/mL ethidium bromide （EtBr） for9days and consecutively with12.5ng/mL EtBr for other30days to establish mtDNA-depleted HBE （HBE ρ^-）. The mtDNAcopy numbers were determined by real-time PCR. The EtBr-treated HBE were thenscored for mtDNA depletion by live cell imaging after staining with Picogreen andMitoTracker Red. Also, HBE ρ^-was cultured in “ρ0test medium” to verify itsauxotrophic characteristic for uridine and pyruvate. We assessed the growth kinetics ofHBE ρ⁺and HBE ρ^-by recording cells growth curve. Cytochrome c oxidase （COX）activity was measured with biochemical kit. Flow cytometry （FCM） was used to detectmitochondrial membrane potential （ΔΨm） and intracellular reactive oxygen species（ROS）.（2） HBE ρ⁺and ρ^-cells were exposed to different doses （0、0.3、0.6Gy） ofα-particles, and were sub-cultured to passage20and40. Anchorage-independent cellproliferation was determined by the soft agar assay and wound-healing assay wasperformed to assess invasiveness/metastatic potential. The plating efficiency assay wasperformed to detect the surviving fraction. Spontaneous apoptosis was measured withthe use of JC-1, Annexin V-FITC/PI or PI-staining flow cytometry separately.Calculation of the doubling times was based on cells growth curve. Distribution of thecell cycle was analyzed by flow cytometry after being stained with PI.（3） Theexpression of apoptosis-related molecules Bax, Bcl-2, cyt-c, caspase-9, caspase-8, caspase-3in irradiated ρ⁺and ρ^-at passage20and40was detected by western blottinganalysis. ROS production was determined by fluorescent staining with H₂DCFDA andmtDNA content was analyzed by real-time PCR.Results:（1） Real-time PCR and mtDNA stainning showed that the content ofmtDNA in EtBr-treated HBE cells was24%of the wide-type HBE. Moreover, theEtBr-treated cells couldn’t survive in “ρ0test medium”. These indicated that, EtBr canbe used to delete mtDNA of HBE. Compared with parent HBE, HBE ρ^-showed reducedCOX activity, slower growth rate, decreased ΔΨm and ROS.（2）At passage40afterirradiation, the plating efficiency，the colony forming efficiency in soft agar andmigration index of irradiated HBE ρ^-were higher than irradiated HBE ρ⁺. Comparingapoptosis of irradiated HBE ρ^-with ρ⁺at passage40, we found that early apoptosispercentage, postapoptosis/necrotic percentage and DNA fragmentation debrispercentage of irradiated HBE ρ^-was significantly lower than irradiated HBE ρ⁺, notably,the progressive rate of disabled death in irradiated HBE ρ^-was faster than irradiatedHBE ρ⁺. Cells growth curve showed that, compared to mitochondrial control at P40,irradiated HBE ρ^-possessed higher saturation density and shorter doubling times.Cell-cycle distribution analysis indicated that, compared to irradiated HBE ρ⁺, thepercentage of G1and G2/M phase decreased but S phase increased in irradiated HBE ρ^-.（3） The expression of apoptosis-related molecules was detected by western blottinganalysis. Bax expression of irradiated HBE ρ^-at passage20was higher than irradiatedHBE ρ⁺, but there was no difference at passage40. Reversely, Bcl-2expression ofirradiated HBE ρ^-at passage20was lower than irradiated HBE ρ⁺, but at passage40,irradiated HBE ρ^-’ was higher than irradiated HBE ρ⁺’. The expression of cyt-c inirradiated HBE ρ^-was higher than irradiated HBE ρ⁺at passage40. Contrarily, theexpressions of caspase-3and caspase-8in irradiated HBE ρ^-were lower than irradiatedHBE ρ⁺at passage20and40. The expression of caspase-9in irradiated HBE ρ^-waslower than irradiated HBE ρ⁺at passage40. Real-time PCR showed progressivedecrease in mtDNA copy numbers of irradiated HBE ρ⁺and ρ^-. The levels ofintracellular ROS in irradiated HBE ρ⁺and ρ^-were both maintained at high levels. Conclusions: Mitochondrial DNA knock down and deficient respiratory chainpromote the process of α particles-induced malignant transformation in vitro, includingaccelerated proliferation, disabled apoptosis, invasiveness potential and cell cyclearresting.