Abstract
JNK與p38訊號傳遞分子在細胞遭受逆境時會被誘發，這些分子之訊號傳遞路徑往往與細胞的存活有關，然而它們是否傳遞與產生基因突變有關的訊號仍不為人知。本論文以衍生自人類肺癌細胞CL3的四個細胞株：CL3-JNK1DN、CL3-JNK2DN、CL3-p38DN以及CL3-pLNCX為研究對象，分別代表細胞含有表現JNK1、JNK2與p38優勢負效應的蛋白質以及含有載體pLNCX的對照組細胞，來探討當以及p38訊號傳遞路徑被抑制的狀況下，對烷基致癌物所誘發的細胞毒性以及致突變性的影響。這些細胞經烷基化合物methyl methanesulfonate (MMS; 0.5-2 mM)與N-methyl-N*-nitro-N-nitrosoguanine (MNNG; 2.5-10 mM)處理一小時後，以群落形成能力分析細胞毒性，並利用對6-thioguanine的抗性偵測hypoxanthine (guanine)phosphoribosyl transferase (hprt)基因突變被誘發的頻率。結果顯示MMS與MNNG誘發CL3-JNK1DN及CL3-JNK2DN的細胞毒性比其等CL3-pLNCX細胞株略為敏感，然而差異並不顯著。MMS與MNNG均能對CL3-JNK1DN細胞株誘發出比對照組細胞較高的突變頻率。MMS並不誘發CL3-JNK2DN產生突變，然而MNNG卻非常顯著地誘發CL3-JNK2DN細胞產生突變。MMS與MNNG對CL3-p38DN細胞的細胞毒性都較CL3-pLNCX細胞略低，而誘發CL3-p38DN細胞的突變頻率也都較CL3-pLNCX為低。抑制JNK之活化使細胞被烷基化合物誘發的突變率提高，推測JNK的活化會引發抑制細胞突變形成的機制。相反地，抑制p38之活化提高了細胞存活率並降低基因突變率，可能與p38的調控細胞凋亡以及傳遞一些與基因突變產生的訊號有關。JNK and p38 signal transduction molecules were activated whencell suffered environmental stresses. The signal transductionpathways containing these molecules usually associate with cellsurvival and death. However, little is known about therelationship between these signals and gene mutations . In thisthesis, we use CL3-JNK1DN, CL3-JNK2DN,CL3-p38DN and CL3-pLNCXcell-lines which derived from human lung adenocarcinoma cellline CL3, express dominant negative proteins JNK1, JNK2 and p38and vector pLNCX for control, respectively. Using these celllines, we investigate the effects of JNK or p38 on cytotoxictyand mutagenicity induced by alkylating mutagens. These cellswere treated with methyl methanesulfonate (MMS; 0.5-2 mM) or N-methyl-N*-nitro-N-nitrosoguanine (MNNG; 2.5-10 mM) for 1 hour.Aftertreatment, the cytotoxicity was assayed by colony formingability, and the induced hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene mutation frequency was detected by theirresistance to 6-thioguanine. Our results demonstrated that thecytotoxicities induced by MMS and MNNG in CL3-JNK1DN and CL3-JNK2DN cell lines are slightly higher than that observed in CL3-pLNCX cells. MMS didn*t induce hprt gene mutation in CL3-JNK2DNcells, however the mutation frequency are markedly induced byMNNG in CL3-JNK2DN cells. CL3-p38DN cells were less sensitive toMMS or MNNG when compared with that observed in CL3-pLNCX cells.Also, the mutation frequencies induced by MMS or MNNG in CL3-p38DN cells were lower than those induced in CL3-pLNCX cells. Theresults suggest that JNK activation may inhibit some mechanismsleading to mutation formation because the mutation frequenciesinduced by alkylating agents were enhanced when JNK activationwas inhibited. Conversely, the cell survival increased andmutation frequencies decreased in alkylating agents-treatedcells whose p38 activity was inhibited. This finding isconsistent with that p38 plays an important role in apoptosis,and also suggest a novel function of p38, i.e., enhancingmutagenesis occurred spontaneous and induced by alkylatingagents.