Liu Xiao-Qiong, Pan Zhi-Gang, Li Man-Zhi, Jiang Ju-Hong, Fu Jie, Long Qing-Xin, Wang Xun-Zhang, Zeng Yi-Xin
Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong, 510060, PR China.
Ai Zheng. 2003 Feb;22(2):128-35.
BACKGROUND & OBJECTIVE: Nonrandom allelic loss at chromosome 3p21.3 is a common and early event in nasopharyngeal carcinoma (NPC), which implicates the presence of tumor suppressor genes (TSGs) that may be involved in the pathogenesis of NPCs. BLU gene, containing a MYND domain and located at 3p21.3, has been considered as a NPC associated candidate tumor suppressor gene (TSG) due to the occurrence of loss of its expression and aberrant promoter hypermethylation in most NPCs. This study was designed to construct expression vectors containing either wild type BLU gene and its mutants and to analyze the effect of BLU gene on proliferation of NPC cells by transfection assays.
The full-length cDNA of BLU gene was amplified by RT-PCR. The expression vectors containing various BLU mutants were constructed by site-directed mutagenesis by overlapping PCR. These mutants include a MYND domain deletion mutant, a Ser402Phe and del405Cys, del406Ser mutant, and a Gly160Arg mutant. The wild type BLU gene and the MYND domain deletion mutant were transfected into NPC cell lines CNE1 and CNE2. The effect on apoptosis was determined by TUNEL assay. Cellular proliferation of the stably-transfected cells was examined with cell growth curve and by colony formation assays. Tumorigenicity in nude mice of CNE2 stably-transfected with BLU was investigated.
No significant difference in apoptosis index (AI) was observed between cells transfected with wild type or MYND domain deleted BLU gene and cells transfected with plasmid controls. Exogenous expression of wild type BLU gene had no effect on growth rate and colony formation ability of CNE1 and CNE2. BLU gene showed no suppressor ability in CNE2 tumorigenicity.
Although BLU gene was frequently altered in NPCs, its suppressor role in NPC cells proliferation was not evident. Thus, the possibility of BLU gene as a TSG involved in NPC development remained to be elucidated by further studies.
3p21.3染色体上的非随机等位基因缺失是鼻咽癌（NPC）常见的早期事件，这意味着可能存在参与鼻咽癌发病机制的肿瘤抑制基因（TSG）。BLU基因含有一个MYND结构域，位于3p21.3，由于在大多数鼻咽癌中其表达缺失和启动子异常高甲基化的发生，已被认为是一个与鼻咽癌相关的候选肿瘤抑制基因（TSG）。本研究旨在构建含有野生型BLU基因及其突变体的表达载体，并通过转染实验分析BLU基因对鼻咽癌细胞增殖的影响。
通过RT-PCR扩增BLU基因的全长cDNA。通过重叠PCR定点诱变构建含有各种BLU突变体的表达载体。这些突变体包括一个MYND结构域缺失突变体、一个Ser402Phe和del405Cys、del406Ser突变体以及一个Gly160Arg突变体。将野生型BLU基因和MYND结构域缺失突变体转染到鼻咽癌细胞系CNE1和CNE2中。通过TUNEL法测定对凋亡的影响。用细胞生长曲线和集落形成实验检测稳定转染细胞的细胞增殖情况。研究了稳定转染BLU的CNE2在裸鼠中的致瘤性。
转染野生型或MYND结构域缺失的BLU基因的细胞与转染质粒对照的细胞之间，凋亡指数（AI）无显著差异。野生型BLU基因的外源表达对CNE1和CNE2的生长速率和集落形成能力无影响。BLU基因在CNE2致瘤性方面未显示出抑制能力。
尽管BLU基因在鼻咽癌中经常发生改变，但其在鼻咽癌细胞增殖中的抑制作用并不明显。因此，BLU基因作为参与鼻咽癌发生发展的TSG的可能性仍有待进一步研究阐明。
