在人類罹患的各種惡性腫瘤分析中，發現p53抑癌基因在腫瘤細胞株發生異常的比例非常高。失去正常p53蛋白功能在癌症治療上常常與臨床上不良的癒後結果有關係。在本篇論文中，想探討的是在不同p53 status的非小細胞肺癌細胞株H1299細胞，以輻射線和亞砷酸鈉處理之後細胞所反應的藥物敏感性是否有不同。
將轉殖細胞以輻射線和亞砷酸鈉處理後，再培養五日，利用SRB (sulforhodamine B) 染色細胞方式觀察neo-H1299細胞、mp53-H1299細胞和wtp53-H1299細胞的細胞毒性結果。在SRB細胞毒性結果中，以50%抑制生長濃度值 (IC50) 為基準，轉殖野生型p53的H1299細胞對輻射線和亞砷酸鈉之IC50分別為2.4 Gy和169M。由轉殖pcDNA3質體的H1299細胞和轉殖野生型p53的H1299細胞相比較也發現，轉殖野生型p53的H1299細胞對輻射線和亞砷酸鈉有較高的敏感性 (wtp53-H1299細胞對於輻射線和亞砷酸鈉的敏感程度分別為neo-H1299細胞的1.5倍和1.4倍)。相對的，轉殖突變型mp53基因的H1299細胞和轉殖野生型p53的H1299細胞相比較發現，穩定轉殖dominant negative mp53質體的H1299細胞對於輻射線和亞砷酸鈉則有較低的敏感性。
將三種不同p53狀態的細胞以輻射線和亞砷酸鈉處理後，再培養三日，利用流式細胞技術分析sub-G1細胞計畫性死亡所得到的結果與利用SRB方式觀察細胞毒性的結果是相一致。細胞對輻射線或亞砷酸鈉的敏感性依序是S#40>mp53->neo-1299，這結果與利用DNA片段化分析所得到的結果相似。利用免疫螢光染色分析也發現，細胞以輻射線和亞砷酸鈉處理後，p53明顯累積在核的情形依序是S#40>mp53->neo-1299。
由西方點墨法分析中發現，轉殖野生型p53的H1299細胞會產生較多與細胞計畫性死亡相關蛋白質p53、Bak、active caspase-3和cleaved PARP的表現，並且蛋白質的表現量都有隨著處理劑量提高而增加。然而在相同的處理方式下，轉殖pcDNA3質體的H1299細胞和轉殖突變型mp53基因的H1299細胞，則表現較少(或沒有表現)與細胞計畫性死亡相關的蛋白質。
由本研究結果發現，在H1299細胞中回復p53蛋白的表現，可以增加細胞對藥物的敏感度以及誘導較大量細胞計畫性死亡的發生。未來，可將p53基因療法應用在這類p53有缺失的非小細胞肺癌治療上。

The tumor suppressor gene p53 is the most frequently mutated gene in human cancers. Loss of functional p53 leads to impaired responses of cancer cells to apoptosis induction and to poor prognosis in patients with certain types of cancer. This study examined whether X-ray and sodium arsenite (SA) sensitivities depend on various p53 gene status in H1299 cells containing stably transfected with neo-, mp53/neo- or wtp53/neo-containing vector, respectively.
The sensitivities of transfected cells to X-ray or SA were determined by sulforhodamine B (SRB) cell viability assay at day 5 after treatment. The IC50 for wtp53-H1299 (S#40) cells after 0-4 Gy X-ray or 0-30 μM SA treatment was 2.4 Gy and 16 μM, respectively. The H1299 cells transfected with wild-type p53 gene (wtp53-H1299 cells) showed a dramatic increase in susceptibility to X- ray or SA (1.5-fold to X-ray and 1.4-fold to SA) compared to neo-transfected H1299 cells. In contrast, mp53-H1299 cells showed X-ray- and SA-resistance due to the dominant negative nature of mutant p53 (R273H), compared with wtp53-H1299 cells.
The incidence of sub-G1 apoptosis by X-ray or SA was analyzed with flow cytometry at day 3 after treatment. The results of sub-G1 cells after each same dose of X-ray or SA treatment for 3 different cells were consistent with the cell survival data from SRB observation. The sensitivity order for either treatment was S#40>mp53->neo-1299. Data from DNA fragmentation assay at 24, 48 and 72 h after treatment confirmed the sensitivity order for these three cells, S#40>mp53->neo-H1299. The p53 localization of transfected cells after X-ray or SA treatment was analyzed by immuno-fluorescence stain. After treatment, the localization of p53 apparently accumulated into the nuclei in wtp53-H1299 (S#40) cells as compared with neo- and mp53-H1299 cells.
Western analyses results showed that wtp53-H1299 (S#40) cells, but not neo- and mp53-H1299 cells, p53 and pro-apoptotic protein Bak were obviously increased after X-ray or SA treatment. Fragmentation of caspase-3 and Poly (ADP-Ribose) Polymerase (PARP) was observed in wtp53-H1299 (S#40) cells, but almost no such fragmentation was observed in neo- and mp53-H1299 cells after same treatment.
These results confirmed that the restoration of p53 function by wild type p53 but not mutant p53 in null-p53 H1299 cells enhanced apparently the sensitivity to X-ray and SA and induced the occurrence of cellular apoptotic response through caspase-3 activation. In addition, our data also supported the possible clinical usage for wild type p53 gene therapy for human non small cell lung cancer (NSCLC) patients.

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