DETECTION OF ANEUPLOIDY AND ANEUPLOIDY-INDUCING AGENTS IN HUMAN-LYMPHOCYTES USING FLUORESCENCE INSITU HYBRIDIZATION WITH CHROMOSOME-SPECIFIC DNA PROBES
DETECTION OF ANEUPLOIDY AND ANEUPLOIDY-INDUCING AGENTS IN HUMAN-LYMPHOCYTES USING FLUORESCENCE INSITU HYBRIDIZATION WITH CHROMOSOME-SPECIFIC DNA PROBES
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DOI：
10.1016/0165-1161(90)90041-l
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发表时间：
1990-10-01
期刊：
影响因子：
--
通讯作者：
PINKEL, D
中科院分区：
文献类型：
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作者：
EASTMOND, DA;PINKEL, D
本文探讨了用染色体特异性DNA探针进行荧光原位杂交作为检测间期人淋巴细胞中非整倍体和非整倍体诱导剂的可行性。该分析包括对间期细胞中杂交区域的数量进行计数，以确定感兴趣的特定染色体的拷贝数。用1、7、9、17、X、Y染色体探针对22,000个未经处理的72小时淋巴细胞培养的间期核进行了检测，各常染色体含有0、1、2、3、4杂交区的核的组合频率分别为0.004、0.084、0.909、0.003和0.001。根据这些频率，对100-2000个细胞进行评分，应该可以检测到特定染色体(α)具有0.012的超二倍体频率或0.11%的亚二倍体频率的非整倍体细胞。=0.05，β。=0.80)。这种检测灵敏度的差异与带有一个明显斑点的细胞的频率较高有关。与用于分析的二维图像中杂交区与核面积的比率比较表明，这两个区域的重叠可能是在正常细胞中观察到明显单体的高频率的原因。用非整倍体诱导剂秋水仙碱、硫酸长春新碱和己烯雌酚处理后，含有3个或更多杂交区域的细胞核数量显著增加。用亚砷酸钠处理的超二倍体细胞仅略有增加，而用另一种有效的亚硝酸钠处理的电离辐射则导致含有多个杂交区的细胞核显著增加。这些结果表明，在这些条件下，电离辐射是一种非整倍体诱导剂，尽管杂交区内的染色体断裂可能是具有多个杂交区的核频率增加的一部分原因。这些结果表明，使用DNA探针的荧光原位杂交能够检测出在细胞群体中出现频率相对较低的非整倍体细胞。基于这些技术的分析应该有助于更快地鉴定非整倍体诱导的环境和治疗剂。
The feasibility of utilizing fluorescence in situ hybridization with chromosome-specific DNA probes as the basis of an assay to detect aneuploidy and aneuploidy-inducing agents in interphase human lymphocytes has been investigated. The assay involves counting the number of hybridization regions in interphase cells to determine the number of copies of a specific chromosome of interest. 22,000 interphase nuclei from untreated 72-h lymphocyte cultures were examined following hybridization with probes for chromosomes 1, 7, 9, 17, X or Y. The combined frequencies of nuclei containing 0, 1, 2, 3 and 4 hybridization regions for the various autosomal chromosomes were 0.004, 0.084, 0.909, 0.003 and 0.001, respectively. Based on these frequencies, scoring 100-2000 cells should allow detection of aneuploid cells with a 0.012 frequency of hyperdiploidy or a 0.11 frequency of hypodiploidy for a specific chromosome of interest (.alpha. = 0.05, .beta. = 0.80). This difference in test sensitivity is related to the higher frequency of cells with one apparent spot. A comparison with the ratio of hybridization region to nuclear area in the two-dimensional images used for this analysis indicates that an overlap of the two regions probably accounts for the high frequency of apparent monosomy observed in normal cells. Treatment with the aneuploidy-inducing chemicals, colchicine, vincristine sulfate and diethylstilbestrol resulted in significant dose-related increases in the number of nuclei containing 3 or more hybridization regions. Treatment with the clastogen sodium arsenite produced only a minor increase in apparently hyperdiploid cells whereas treatment with ionizing radiation, another potent clastogen, resulted in a significant increase in nuclei containing multiple hybridization regions. These results suggest that ionizing radiation is an aneuploidy-inducing agent under these conditions although chromosomal breakage within the hybridization region may account for a portion of the increased frequency of nuclei with multiple hybridization regions. These results indicate that the use of fluorescence in situ hybridization with DNA probes is capable of detecting aneuploid cells occurring at relatively low frequencies within a population of cells. Assays based on these techniques should facilitate a more rapid identification of aneuploidy-inducing environmental and therapeutic agents.