人鼻咽癌顺铂耐药细胞系(CNE2/DDP)及其亲代细胞(CNE2)的比较基因组杂交研究

Comparative Genomic Hybridization Analysis of Nasopharyngeal Carcinoma Drug-Resistant Cell CNE2/DDP and Its Parent Cell CNE2

背景与目的:比较基因组杂交(comparativegenomichybridization,CGH)是一种在荧光原位杂交(FISH)技术上发展起来的,用于检测两个基因组间相对DNA拷贝数的改变(缺失或扩增),并将这些变化在染色体上进行定位的分子细胞遗传学方法。为全面了解鼻咽癌耐药细胞与药物敏感细胞在基因组DNA水平上可能存在的差异,以及这种差异在肿瘤耐药性产生中的意义,我们用CGH技术对鼻咽癌耐药细胞系(CNE2/DDP)和其亲代药物敏感细胞(CNE2)的基因组DNA进行检测和分析。方法:提取两种癌细胞及正常胎盘组织的基因组DNA,以随机引物法进行荧光标记(CNE2/DDP和CNE2DNA以Fluorescein-12-dUTP标记,探针显绿色荧光;正常胎盘组织以Tetramethylrhodamine-5-dUTP标记,探针显红色荧光),将标记的DNA探针同时与正常淋巴细胞分裂中期染色体进行杂交,杂交信号在荧光显微镜下经CCD(chargecoupleddevice)摄像装置摄取,并通过荧光数字图像分析系统(quipsCGHprogram)进行数据处理,计算两种荧光的比率并绘制分析图。结果:CNE2细胞存在广泛的染色体改变,主要表现在1q,3q,5p,6p,7p,8q,9q,11p,12q,19q的扩增和4q,12p,13p,14p,15p,18,20q,21p,22的缺失。从CNE2诱导的耐药细胞系CNE2/DDP恒定表现为8q,19q的扩增和8p的缺失,其它的染色体均未发现明显异常的?

BACKGROUND & OBJECTIVE: Developed from fluorescence in situ hy bridization (FISH), comparative genomic hybridization (CGH) is a new molecular a nd cellular technique,used to examine the genomic imbalances,especially the loss and amplification of chromosomes,and to locate these alterations in certain chr omosome regions. For a comprehensive understanding of the possible differences i n genomic DNA between nasopharyngeal carcinoma drug- resistant and drug- sensi tive cells, we analyzed the genomic DNA of a nasopharyngeal carcinoma drug- res istant cell line CNE2/DDP and its parent cell line CNE2 with CGH. METHODS: Genom ic DNA was extracted from CNE2/DDP and CNE2 cells, as well as from normal placen ta tissue. Fluorescent random primed labeling method was used to label the DNA p robes (CNE2 and CNE2/DDP cells with green fluorescein- 12- dUTP and normal pla centa tissue with red tetramethylrhodamine- 5- dUTP).The labeled DNA probes we re then co- hybridized with normal lymphocyte metaphase chromosomes. Signals we re taken by charge coupled device (CCD) and processed with Quips CGH Program aft er fluorescent hybridization. The green- to- red fluorescent ratio was calcula ted automatically and showed with graphs. RESULTS: There were extensive chromoso me changes in CNE2, mainly the gain of 1q, 3q, 5p, 6p, 7p, 8q, 9q, 11p, 12q and 19q, and the loss of 4q, 12p, 13p, 14p, 15p, 18, 20q, 21p and 22. However, the C NE2/DDP cells, which come from the CNE2, showed a relatively normal karyotype ex cept loss of 8p and gain of 8q and 19q. Consistent result was achieved after the CNE2/DDP cells were cultured in the medium free of any drugs for over one month . It appears that the CNE2/DDP cell has relative normal and much more stable chr omosome constitution than its parent CNE2 cell. CONCLUSION: The CNE2/DDP is a si ngle drug- resistant clone selected from CNE2,which is a blend of sub- clones that have different sorts of chromosome number and structure. It strongly sugges ts that the appearance of tumor drug- resistance is mainly a select process in which the would- be drug- resistant cell clone be selected from unfavorable su rvival condition.