Ndoye Alioune, Bouali Sanae, Dolivet Gilles, Leroux Agnes, Erbacher Patrick, Behr Jean-Paul, Berg Kristian, Guillemin François, Merlin Jean-Louis
Laboratoire de Recherche en Oncologie, EA 3452, Centre Alexis Vautrin, Vandoeuvre-les-Nancy, France.
Int J Oncol. 2004 Dec;25(6):1575-81.
p53 is frequently mutated in head-and-neck squamous cell carcinoma. Wild-type p53 gene transfer induces apoptosis in vitro and tumor regression in vivo and clinical investigations of p53 gene therapy have been reported, mostly using viral vectors. Non-viral vectors are increasingly being used as an alternative to viral vectors and photochemical internalisation (PCI) of non-viral vectors has been reported to yield high gene transfer efficiency. The p53-mutated status of FaDu human pharynx carcinoma cell line was first assessed by DNA sequencing and the cells were transfected using tetraglucosylated polyethylenimine (PEI-Glu4) in conjunction with photochemical internalisation (PCI). The green fluorescent protein (GFP) was used as a reporter for determination of the transgene expression kinetics with or without PCI. p53 gene transfer was performed in these optimised conditions, and subsequent induction of apoptosis was investigated by flow cytometric determination of the phosphatidylserine externalization. Long-term cell death was assessed using colony forming assays. DNA sequencing in FaDu cells showed a G/T point mutation at codon 248 in exon 7 of p53 gene, resulting in an arginine-to-leucine substitution. As a consequence, P53 was shown to be expressed in >90% of untreated cells using immunocytochemistry. Using PEI-Glu4 as vector, PCI was found to significantly enhance GFP gene transfer whatever the formulation solution. Transfection efficiency was significantly increased with PCI. GFP expression kinetics (24-144 h) demonstrates that PCI induces sustained transgene expression with >10% of cells remaining transfected after 144 h. In such conditions, p53 gene transfer using PEI-Glu4 and PCI, resulted in spontaneous induction of apoptosis. As a consequence, long-term cell death was significantly enhanced after wt-p53 gene transfer when PCI was used, reaching up to 50% cell death. Wild-type p53 gene transfer using PEI-Glu4/DNA complexes and PCI, yields sustained transgene expression and induces cell death in p53-mutated FaDu cells.
p53在头颈部鳞状细胞癌中经常发生突变。野生型p53基因转移在体外可诱导细胞凋亡，在体内可使肿瘤消退，并且已有关于p53基因治疗的临床研究报道，大多使用病毒载体。非病毒载体正越来越多地被用作病毒载体的替代品，并且据报道非病毒载体的光化学内化（PCI）可产生高基因转移效率。首先通过DNA测序评估FaDu人咽癌细胞系的p53突变状态，并使用四葡糖基化聚乙烯亚胺（PEI-Glu4）结合光化学内化（PCI）转染细胞。绿色荧光蛋白（GFP）用作报告基因，用于测定有无PCI时转基因的表达动力学。在这些优化条件下进行p53基因转移，随后通过流式细胞术测定磷脂酰丝氨酸外化来研究细胞凋亡的诱导情况。使用集落形成试验评估长期细胞死亡情况。FaDu细胞中的DNA测序显示p53基因第7外显子248密码子处存在G/T点突变，导致精氨酸到亮氨酸的替换。因此，使用免疫细胞化学方法显示，超过90%的未处理细胞中P53表达。使用PEI-Glu4作为载体，发现无论制剂溶液如何，PCI均可显著增强GFP基因转移。PCI可显著提高转染效率。GFP表达动力学（24 - 144小时）表明，PCI可诱导持续的转基因表达，144小时后仍有超过10%的细胞保持转染状态。在这种条件下，使用PEI-Glu4和PCI进行p53基因转移，导致细胞凋亡的自发诱导。因此，当使用PCI时，野生型p53基因转移后长期细胞死亡显著增强，细胞死亡率高达50%。使用PEI-Glu4/DNA复合物和PCI进行野生型p53基因转移，可在p53突变的FaDu细胞中产生持续的转基因表达并诱导细胞死亡。
