Derradji Hanane, Bekaert Sofie, Van Oostveldt Patrick, Baatout Sarah
Laboratory of Radiobiology, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium.
Anticancer Res. 2005 Mar-Apr;25(2A):1039-50.
The end of eukaryotic chromosomes terminates with nucleoprotein structures called telomeres. They insure several functions including capping the end of the chromosomes, ensuring their stability and protecting them from end-to-end fusion and preventing the activation of the DNA damage checkpoints.
A flow-FISH methodology, i.e. quantitative fluorescence in situ hybridization (Q-FISH) in combination with flow cytometry, has been developed in our laboratory in order to estimate telomere length in three human cancer cell lines: K-562 (chronic myelogenous leukaemia), IM-9 (multiple myeloma) and 1301 (T cell lymphoblastic leukaemia). Telomeres were visualised after hybridisation with FITC-labelled PNA (Peptide Nucleic Acid) probes. We evaluated the most critical steps of the flow-FISH protocol to ensure reproducibility. Different methodological set ups were compared. Three fixation procedures (ethanol 80%, methanol 80% and formaldehyde 4%) were tested besides different fixation times (15 min and 60 min) as well as hybridization times (2 h and overnight). For each of these protocols the following parameters were compared: forward scatter (related to the cell size), side scatter (related to the cell granularity), DNA (FL3 and FL4 fluorescence) and PNA content (FL1 fluorescence) using an EPICS XL flow cytometer.
Regarding the fixation procedures, methanol proved to be the best, followed by ethanol and formaldehyde, with respect to the efficiency to measure the different parameters cited above. Indeed, fixation using methanol gave the optimal PNA signal compared to using ethanol and formaldehyde in two of the studied cell lines (K-562 and 1301); the difference observed was highly significant in the 1301 cell line. The duration of fixation did not show significant interference in the reproducibility of the results for the three cell lines studied. An overnight hybridization appeared to be more effective when compared to the 2-h hybridization in the case of the K-562 cell line.
The most important steps of the flow-FISH technique, namely the fixative procedure, as well as the hybridization and the fixation times, were investigated. Considering the latter, suitable protocols were set up for routine and fast telomere length estimation in the cancer cell lines.
真核染色体的末端以称为端粒的核蛋白结构终止。它们确保多种功能，包括封闭染色体末端、确保其稳定性、保护它们免于端对端融合以及防止DNA损伤检查点的激活。
我们实验室开发了一种流式荧光原位杂交方法，即定量荧光原位杂交（Q-FISH）与流式细胞术相结合，以估计三种人类癌细胞系中的端粒长度：K-562（慢性粒细胞白血病）、IM-9（多发性骨髓瘤）和1301（T细胞淋巴细胞白血病）。用异硫氰酸荧光素（FITC）标记的肽核酸（PNA）探针杂交后观察端粒。我们评估了流式荧光原位杂交方案中最关键的步骤以确保可重复性。比较了不同的方法设置。除了不同的固定时间（15分钟和60分钟）以及杂交时间（2小时和过夜）外，还测试了三种固定程序（80%乙醇、80%甲醇和4%甲醛）。对于这些方案中的每一个，使用EPICS XL流式细胞仪比较以下参数：前向散射（与细胞大小有关）、侧向散射（与细胞颗粒度有关）、DNA（FL3和FL4荧光）和PNA含量（FLI荧光）。
关于固定程序，就测量上述不同参数的效率而言，甲醇被证明是最佳的，其次是乙醇和甲醛。事实上，在两个研究的细胞系（K-562和1301）中，与使用乙醇和甲醛相比，用甲醇固定产生了最佳的PNA信号；在1301细胞系中观察到的差异非常显著。固定时间对所研究的三种细胞系的结果可重复性没有显示出显著干扰。在K-562细胞系的情况下，与2小时杂交相比，过夜杂交似乎更有效。
研究了流式荧光原位杂交技术的最重要步骤，即固定程序以及杂交和固定时间。考虑到后者，为癌细胞系中常规和快速的端粒长度估计建立了合适的方案。
