Study On Cell Signaling Pathways During Ultraviolet Irradiation-Induced Apoptosis

UV irradiation leads to DNA damage, which activates ataxia telangiectasia mutatedprotein(ATM). ATM can change the phosphorylation levels of p53 protein resulting incell cycle arrest and apoptosis, p53 stimulates a wide network of signals that act throughtwo major apoptotic pathways: transcription dependent and transcription independent.But the detail cellular signaling mechanisms mediating UV-induced apoptosis and howthe transcription-independent functions of p53 co-operate with its transcription-dependentfunctions in the induction of apoptosis are not fully understood. In this dissertation, theeffect of UV irradiation on human lung adenocarcinoma cells (ASTC-a-1) was analyzedusing Cell Counting Kit-8 and Hoechst 33258; the molecular mechanisms of the effectwere investigated using laser confocal scanning microscope, fluorescence resonanceenergy transfer (FRET), fluorescence spectrum, acceptor photobleaching, siRNA (smallinterfering RNA) and Western blotting techniques. Our studies help to clarify themechanisms of UV irradiation biological effects and guide the clinic therapy for p53.First in prolegomenon, the paper introduced the mechanisms of UV irradiation-inducedapoptosis, the research status quo of cell signaling pathways of UV irradiation. At thesame time, the paper generalized the study work in this dissertation and explained the aimand meaning of the study.The experimental results of this paper were divided into three parts:In the first experimental study, using the FRET technique based on YFP-Bid-CFP, weobserved the dynamics of Bid activation during UV-induced apoptosis. Furthermore, Bidactivation in response to UV irradiation was confirmed by Western blotting andfluorescence spectrum techniques. Meanwhile, we observed a collapse of themitochondrial membrane potential using Rhodamine123 fluorescence probes. The resultsshowed that Bid activation was initiated at 9±1 h after UV irradiation, and the averageduration of the activation was 75±10 min. Bid activation coincided with a collapse of themitochondrial membrane potential with an average duration of 50±10min. We also foundthat Bid activation was a caspase-8 dependent event.In the second experimental study, using FRET (fluorescence resonance energy transfer), acceptor photobleaching, Western blotting and siRNA (small interfering RNA)techniques, we investigated the relationship between Bid and Bax during UV-inducedapoptosis. The results showed that Bax translocation by UV irradiation was aBid-independent event and there was no interaction between Bax and Bid in both healthyand apoptotic cells, repression of Bid protein with siRNA (small interfering RNA) did notinhibit cell death by UV irradiation. Bax translocation, caspase-3 activation and cell deathby UV irradiation were delayed by Pifithrin-a (p53 inhibitor), overexpression of Bcl-x_L incells susceptible to UV-induced apoptosis prevented Bax translocation and cell death.In the third experimental study, using laser confocal scanning microscope (LCSM) andWestern blotting techniques, we investigated the biological effects by UV irradiation inthe presence or absence of Pifithrin-a or cycloheximide (CHX). The results indicated thatp53-dependent and -independent pathways cooperate to promote UV-induced apoptosis.