本研究團隊之前對117位非小細胞肺癌病人及70位非肺癌病人組織中的MDM2mRNA表現情形進行分析，證實肺癌病人組織中的MDM2mRNA選擇性短小變異比例為26.4%(n=31)明顯高於非肺癌病人(8.5%,n=6)(p<0.001)；其中抽煙習慣及性別與MDM2mRNA的選擇性短小變異發生有高度的正相關性，而MDM2mRNA選擇性短小變異型態又以具有腫瘤惡質化特性的MDM2-657居多。本研究欲進一步探討是否香菸中的毒性物質可造成MDM2mRNA選擇性短小變異。將H1355細胞處理於香菸中多環芳香烴化合物；NestedRT-PCR分析發現：B[a]P與BPDE於1μM及0.5、2μM可誘發三種選擇性短小變異的MDM2mRNA表現。值得注意是，透過序列分析選擇性短小變異的MDM2mRNA發現缺失部位存有某些特定序列。另外，ActinomycinD預處理可有效抑制BPDE誘發MDM2mRNA發生選擇性短小變異。本研究結果證實香菸中所含的毒性物質-B[a]P及BPDE會造成MDM2mRNA發生選擇性短小變異。本研究分析多株正常肺細胞與肺癌細胞中毒物代謝酵素-麩胱甘轉移脢酵素活性，發現在正常肺細胞株中以MRC-5表現量最高，肺癌細胞株中以A549表現量最高而H1355表現量最低。比較H1355及MRC-5細胞在BPDE處理下，MDM2mRNA發生選擇性短小變異的情形，結果發現僅H1355細胞發生MDM2mRNA選擇性短小變異，而MRC-5細胞則無此現象。因此，以RT-PCR增幅MRC-5細胞中GST--M2及M4的基因表現，並將GST-M2或M4基因穩定表現於H1355肺癌細胞株。Nested-RTPCR及競爭性ELISA分析發現，穩定表現GST-M2之H1355細胞株可降低BPDE所造成的DNA-adducts，同時也可減緩經由處理BPDE後所誘發的MDM2mRNA選擇性短小變異。綜合以上結果，本研究證實穩定表現GST-M2於H1355肺癌細胞株可有效的降低經由BPDE所造成的DNAadducts，並減緩MDM2mRNA發生選擇性短小變異；同時也說明了GST-M2酵素活性對於降低BPDE造成之DNA傷害可能扮演重要的角色。
The collective results from laboratories of Professors Huei Lee, and Jiunn-Liang Ko have shown that the frequency of MDM2 splice variants in lung cancer patients (31 of 117; 26%) was significantly higher than that of nontumor patients (6 of 70; 8.57%). Interestingly, there was a higher frequency of MDM2 splice variants for smokers and males. while the major splice variant was MDM2-657, a splice variant that was significant associated with tumor malignance. First of all, the study investigated whether the toxins of cigarette smoking would induce alternative splicing of MDM2 mRNA. After H1355 cells being treated in vitro with PAHs, B[a]P or the B[a]P metabolite benzo[a]pyrene diolepoxide (BPDE)-mediaated MDM2 splicing were detected by nested RT-PCR. It was demonstrated that splicing occurred at cryptic splice donor and acceptor sites in regions with high sequence homology. In addition, BPDE-induced MDM2 splicing was prevented with the actinomycin D treatment prior to BPDE exposure. Finally, to investigate whether the formation of DNA adducts could increase the frequency of MDM2 splicing and explore cellular factors able to prevent DNA adduct formation and further alleviate MDM2 splicing, the enzyme activities of total GST of normal and tumor lung cell lines were analyzed. Results showed that the GST activity was the highest in MRC-5, among normal lung cells, and in A549 among lung cancer cells. Interestingly, the catalytic activity of H1355 was undetectable. Therefore, on the same dosage of BPDE-2μM, the alternative splicing of MDM2 was only detected by nested RT-PCR in H1355 cells but not in MRC-5 cells. The results of nested RT-PCR and competitive ELISA demonstrated that overexpression of GST-M2 could prevent DNA adduct formation and further alleviate MDM2 splicing.
