Wang Xiao-Lei, Qian Xin-Lai, Zhao Qing-Zheng, Xu Zhen-Gang, Tang Ping-Zhang
Department of Head and Neck Surgery, Cancer Hospital, Chinese Academy of Medical Sciences & Union Medical University, Beijing, PR China.
Ai Zheng. 2003 Nov;22(11):1140-6.
BACKGROUND & OBJECTIVE: Adenovirus type 5 early region 1A (E1A) gene has been found to be a tumor suppression gene recently. The protein of E1A gene regulates the expression of many cellular genes positively or negatively, and possesses the activities of inducing differentiation of tumor cell, reversing of malignant phenotype, anti-carcinogenesis and anti-metastasis. Study of E1A protein on the treatment of lymph node metastasis of human head and neck squamous cell carcinoma (HNSCC) was not reported. This study was designed to investigate the growth inhibition and radiochemosensitivity of E1A gene on human lymph node metastasis cell line 686LN-1 derived from the patient with human tongue squamous cell carcinoma in vitro and its mechanism.
The pcDNA3-E1A recombinant plasmid, designed for high-level expression of E1A gene in a variety of eukaryotic cell lines,was transfected into 686LN-1 cells mediated by lipofectamine. To observe the growth inhibition of E1A gene on the cells, the growth curve and doubling time were investigated. Cells before and after transfection were treated with cisplatin, paclitaxel, bleomycin, and 5-fluorouracil (5-FU) for 24 hours or irradiation, respectively, then the changes of sensitivity were tested by MTT assay. The redistributions of cell cycle were analyzed by flow cytometry. Immunocytochemical staining was used to detect the expression of p53 and HER-2/neu.
Compared with the vector-transfected cells (686LN-1-vect cells), the E1A-transfected cells (686LN-1-E1A cells) grew slowly, and the doubling time elongated (1.41-fold). 686LN-1-E1A cells showed distinct sensitivity to the anticancer drugs and irradiation. According to the IC(50) value, the sensitivity of 686LN-1-E1A cells increased approximately 8-fold to cisplatin, 20-fold to bleomycin, 10-fold to paclitaxel, 1-fold to irradiation compared with 686LN-1-vect cells. However, the sensitivity to 5-FU did not change. The cell cycle was dramatically arrested at G(2)/M phase in the 686LN-1-E1A cells. E1A gene remarkably suppressed the expression of HER-2/neu gene in 686LN-1-E1A cells.
E1A gene can significantly inhibit the growth rate of lymph node metastasis cell line 686LN-1 of HNSCC. Moreover, it also slightly enhance the cell sensitivity to antitumor drugs and irradiation. These functions of E1A gene may be associated with its ability to suppress the HER-2/neu expression and to arrest the cell at G(2)/M phase.
5型腺病毒早期区域1A（E1A）基因近来被发现是一种肿瘤抑制基因。E1A基因的蛋白质可正向或负向调节许多细胞基因的表达，并具有诱导肿瘤细胞分化、逆转恶性表型、抗癌和抗转移的活性。关于E1A蛋白对人头颈鳞状细胞癌（HNSCC）淋巴结转移治疗作用的研究未见报道。本研究旨在探讨E1A基因对人舌鳞状细胞癌患者来源的人淋巴结转移细胞系686LN-1的体外生长抑制及放化疗敏感性及其机制。
设计用于在多种真核细胞系中高水平表达E1A基因的pcDNA3-E1A重组质粒，通过脂质体介导转染入686LN-1细胞。为观察E1A基因对细胞的生长抑制作用，研究其生长曲线和倍增时间。转染前后的细胞分别用顺铂、紫杉醇、博来霉素和5-氟尿嘧啶（5-FU）处理24小时或进行照射，然后通过MTT法检测敏感性变化。采用流式细胞术分析细胞周期的重新分布。免疫细胞化学染色用于检测p53和HER-2/neu的表达。
与载体转染细胞（686LN-1-vect细胞）相比，E1A转染细胞（686LN-1-E1A细胞）生长缓慢，倍增时间延长（1.41倍）。686LN-1-E1A细胞对抗癌药物和照射表现出明显的敏感性。根据半数抑制浓度（IC50）值，与686LN-1-vect细胞相比，686LN-1-E1A细胞对顺铂的敏感性增加约8倍，对博来霉素增加20倍，对紫杉醇增加10倍，对照射增加1倍。然而，对5-FU的敏感性未改变。686LN-1-E1A细胞的细胞周期在G2/M期显著停滞。E1A基因显著抑制686LN-1-E1A细胞中HER-2/neu基因的表达。
E1A基因可显著抑制HNSCC淋巴结转移细胞系686LN-1的生长速度。此外，它还可轻微增强细胞对抗肿瘤药物和照射的敏感性。E1A基因的这些功能可能与其抑制HER-2/neu表达及使细胞停滞在G2/M期的能力有关。
