El-Khoury Victoria, Gomez Dennis, Liautaud-Roger Françoise, Trussardi-Régnier Aurélie, Dufer Jean
Unité MéDIAN-CNRS UMR 6142, IFR 53, Faculté de Pharmacie, Reims, France.
Cytometry A. 2004 Dec;62(2):109-17. doi: 10.1002/cyto.a.20088.
Texture analysis of chromatin patterns by image cytometry can be used in the development and refinement of diagnosis and prognosis of cancers and in the follow-up of therapies. However, little is known about the biological mechanisms underlying these patterns. Epigenetic mechanisms as histone posttranslational modifications and particularly histone acetylation could play a major role in the determination of these chromatin patterns and then influence nuclear texture measurements.
This study examined the consequences of treatment by the histone deacetylase inhibitor trichostatin A (TSA) on the nuclear texture in human cell lines sensitive and resistant to chemotherapy. Small cell lung carcinoma H69 cells and their variant H69-VP, which is resistant to etoposide, were incubated with 100 ng/ml of TSA for 0 to 24 h. Nuclear texture was evaluated by image cytometry and compared with the histone H4 acetylation level measured by western blotting and expression of c-jun gene evaluated by reverse transcription and real-time polymerase chain reaction.
TSA treatment induced an increase in histone H4 acetylation level in both cell lines. However, at the level of chromatin texture, sensitive H69 cells displayed a progressive chromatin decondensation up to 24 h, whereas resistant H69-VP showed rapid (8 h) but transient changes. Similarly, expression of c-jun increased regularly in TSA-treated H69 cells. In H69-VP cells, an increase was also observed up to 12 h followed by a decrease after 24 h of treatment.
Analysis of nuclear texture appeared to be a sensitive technique to detect chromatin pattern alterations induced by the histone deacetylase inhibitor TSA in the H69 cell line and enabled the observation of chromatin pattern discrepancies between chemotherapeutic drug-sensitive and drug-resistant cells during this treatment. When c-jun gene expression was analyzed as gene sensitive to epigenetic control, these textural differences seemed to be correlated to gene expression.
通过图像细胞术对染色质模式进行纹理分析可用于癌症诊断、预后的发展与完善以及治疗随访。然而，对于这些模式背后的生物学机制知之甚少。表观遗传机制，如组蛋白翻译后修饰，尤其是组蛋白乙酰化，可能在这些染色质模式的决定中起主要作用，进而影响细胞核纹理测量。
本研究检测了组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）处理对化疗敏感和耐药人细胞系细胞核纹理的影响。将小细胞肺癌H69细胞及其对依托泊苷耐药的变体H69-VP与100 ng/ml的TSA孵育0至24小时。通过图像细胞术评估细胞核纹理，并与通过蛋白质印迹法测量的组蛋白H4乙酰化水平以及通过逆转录和实时聚合酶链反应评估的c-jun基因表达进行比较。
TSA处理导致两种细胞系中组蛋白H4乙酰化水平升高。然而，在染色质纹理水平上，敏感的H69细胞在24小时内呈现出渐进性的染色质解聚，而耐药的H69-VP细胞则表现出快速（8小时）但短暂的变化。同样，在TSA处理的H69细胞中，c-jun的表达呈规律性增加。在H69-VP细胞中，在处理12小时时也观察到增加，随后在处理24小时后下降。
细胞核纹理分析似乎是一种检测组蛋白去乙酰化酶抑制剂TSA在H69细胞系中诱导的染色质模式改变的灵敏技术，并且能够观察到在这种处理过程中化疗药物敏感和耐药细胞之间的染色质模式差异。当将c-jun基因表达作为对表观遗传控制敏感的基因进行分析时，这些纹理差异似乎与基因表达相关。
