He Yong, Fan Shi-Zhi, Jiang Yao-Guang, Chen Jian-Ming, Li Zhi-Ping, Zhou Ping, Zhou Yuan-Guo
Thoracic Surgery Center of People's Liberation Army, The Third Military Medical University, Chongqing, PR China.
Ai Zheng. 2004 Jun;23(6):645-9.
BACKGROUND & OBJECTIVE: Recent studies have shown that wild-type p53 gene can enhance the chemosensitivity of the majority of non-small cell lung cancers. p73 gene and its homologue p53 gene have significant sequence and functional similarities. This study was designed to investigate the effect of p73 gene on chemosensitivity of human lung adenocarcinoma cells H1299.
The pcDNA3-HA-p73alpha plasmid were transferred into in vitro cultured human lung adenocarcinoma cell line H1299 with Dosper. The p73alpha positive cells which were resistant to G418 were selected out. The expression of exogenous P73alpha protein were examined by Western blot analysis. MTT assay were used to analyze the response of transfected cells to cisplatin (cDDP),adriamycin (ADM). The drug-induced apoptosis of transfected cells was measured by flow cytometry and TUNEL technique. The biological behavior changes of the cells were observed by colony formation assay.
The transfected lung adenocarcinoma cell line H1299 overexpressed P73alpha protein stably. MTT assay showed that the IC(50) values for cDDP and ADM were reduced to approximate 86.2% and 99.2% in the transfected cells compared with the untransfected cells, respectively. The low concentration of chemotherapeutic drugs (1.25 micromol/L cDDP or 0.05 micromol/L ADM) without inhibition essentially suppressed the growth of the transfected cells markedly. The cDDP-induced apoptosis rate increased from 10.1% to 38.4%(P< 0.01). The ADM-induced apoptosis rate increased from 12.1% to 49.3%(P< 0.01). Colony-formation assays showed that the colony number in transfected cells was decreased significantly by chemotherapeutic drugs compared with parental cells(P< 0.01). The sensitive enhancement ratios for cDDP or ADM were 1.8 and 2.6, respectively.
p73 gene was capable of enhancing the sensitivity of H1299 cells to chemotherapeutic drugs such as cDDP and ADM, and the increased sensitivity was associated with the induction of apoptosis undepend p53 gene. It suggests the possibility of using p73 gene combined with anticancer agents to improve the effectiveness of chemotherapy.
近期研究表明，野生型p53基因可增强大多数非小细胞肺癌的化疗敏感性。p73基因与其同源物p53基因在序列和功能上具有显著相似性。本研究旨在探讨p73基因对人肺腺癌细胞H1299化疗敏感性的影响。
采用脂质体转染法将pcDNA3-HA-p73α质粒导入体外培养的人肺腺癌细胞系H1299。筛选出对G418耐药的p73α阳性细胞。采用蛋白质免疫印迹法检测外源性P73α蛋白的表达。采用MTT法分析转染细胞对顺铂（cDDP）、阿霉素（ADM）的反应。采用流式细胞术和TUNEL技术检测转染细胞的药物诱导凋亡情况。通过集落形成试验观察细胞生物学行为的变化。
转染后的肺腺癌细胞系H1299稳定过表达P73α蛋白。MTT法显示，与未转染细胞相比，转染细胞中cDDP和ADM的IC50值分别降至约86.2%和99.2%。低浓度化疗药物（1.25 μmol/L cDDP或0.05 μmol/L ADM）对未转染细胞生长无明显抑制作用，但对转染细胞生长有明显抑制作用。cDDP诱导的凋亡率从10.1%增至38.4%（P<0.01）。ADM诱导的凋亡率从12.1%增至49.3%（P<0.01）。集落形成试验显示，与亲代细胞相比，化疗药物可显著降低转染细胞的集落数（P<0.01）。cDDP或ADM的敏感增强率分别为1.8和2.6。
p73基因能够增强H1299细胞对cDDP和ADM等化疗药物的敏感性，且敏感性增加与诱导凋亡有关，不依赖p53基因。这提示联合应用p73基因与抗癌药物提高化疗疗效的可能性。
