He Yong, Fan Shi-Zhi, Jiang Yao-Guang
Thoracic Surgery Center of People's Liberation Army, Daping Hospital/Institute of Surgery Research, The Third Military Medical University, Chongqing, 400042, P. R. China.
Ai Zheng. 2006 Aug;25(8):925-32.
BACKGROUND & OBJECTIVE: It is important to overcome gene therapy resistance caused by wt-p53 in non-small cell lung cancer (NSCLC) . The p53 family member p73 is a p53 homolog. This study was to observe the apoptosis and chemosensitivity effect of p53-resistant human lung adenocarcinoma cell line A549 following wild-type p73 gene transfection alone or combined with chemotherapeutic agents.
Plasmids pcDNA3-HA-p53 or pcDNA3-HA-p73alpha were transfected into A549 cells with Dosper. Positive cell clones were selected using G418. The exogenous p53 or p73alpha gene expressions were examined by Western blot. MTT assay was used to analyze the response of transfected cells to cisplatin (DDP) or adriamycin (ADM). The drug-induced apoptosis of transfected cells was measured by flow cytometry, TUNEL technique and DNA fragmentation. The biological behavior change of transfected cells was investigated by colony formation assay.
Transfected A549 cells stably overexpressed p53 or p73alpha. Low concentration of chemotherapeutic agents (6.25 micromol/L DDP or 0.25 micromol/L ADM) which had no obvious effects on non-transfected cells, suppressed p73-transfected cell growth significantly; the 50% inhibitory concentration (IC(50)) of DDP for A549 cells decreased from 22.65 micromol/L to 3.75 micromol/L, and the IC(50) of ADM decreased from 4.20 micromol/L to 0.06 micromol/L after p73alpha transfection. p73, but not p53, sensitized A549 cells to DDP and ADM: DDP-induced apoptosis rate was increased from 10.6% to 36.8% (P<0.01), ADM-induced apoptosis rate was increased from 13.0% to 41.1% (P<0.01) after p73 transfection. DDP and ADM significantly suppressed colony formation of p73-transfected cells compared with parental cells (P<0.01). The sensitive enhancement ratios for DDP and ADM were 2.0 and 2.4, respectively.
Exogenous p73 gene enhances the sensitivity of A549 cells to chemotherapeutic agents by inducing apoptosis through p53-independent pathway. p73 gene could be used to treat p53-resistant tumors.
克服非小细胞肺癌（NSCLC）中由野生型p53引起的基因治疗耐药性很重要。p53家族成员p73是p53的同源物。本研究旨在观察单独转染野生型p73基因或联合化疗药物后，对p53耐药的人肺腺癌细胞系A549的凋亡及化疗敏感性的影响。
用脂质体转染试剂将质粒pcDNA3-HA-p53或pcDNA3-HA-p73α转染至A549细胞。用G418筛选阳性细胞克隆。通过蛋白质免疫印迹法检测外源性p53或p73α基因的表达。采用MTT法分析转染细胞对顺铂（DDP）或阿霉素（ADM）的反应。通过流式细胞术、TUNEL技术和DNA片段化检测药物诱导的转染细胞凋亡。通过集落形成试验研究转染细胞的生物学行为变化。
转染的A549细胞稳定过表达p53或p73α。低浓度化疗药物（6.25 μmol/L DDP或0.25 μmol/L ADM）对未转染细胞无明显影响，但能显著抑制p73转染细胞的生长；p73α转染后，DDP对A549细胞的半数抑制浓度（IC50）从22.65 μmol/L降至3.75 μmol/L，ADM的IC50从4.20 μmol/L降至0.06 μmol/L。p73而非p53使A549细胞对DDP和ADM敏感：p73转染后，DDP诱导的凋亡率从10.6%升至36.8%（P<0.01），ADM诱导的凋亡率从13.0%升至41.1%（P<0.01）。与亲代细胞相比，DDP和ADM显著抑制p73转染细胞的集落形成（P<0.01）。DDP和ADM的敏感增强率分别为2.0和2.4。
外源性p73基因通过非p53依赖途径诱导凋亡，增强A549细胞对化疗药物的敏感性。p73基因可用于治疗p53耐药肿瘤。
