Simona Ronzoni, Mario Faretta, Marco Ballarini, Piergiuseppe Pelicci, Saverio Minucci
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Histone acetylation and transcription levels were monitored during differentiation induced by retinoic acid alone or in combination with TSA. Blood samples from patients were analyzed with the described protocol to monitor the effects of HDAC inhibitors in vivo and validate the developed protocol for clinical usage.</p><p><strong>Results: </strong>Flow cytometric detection of acetylation status can successfully detect modifications induced by HDAC inhibitor treatment in vivo as demonstrated by analysis of various blood samples from patients treated with valproic acid. Changes in acetylation levels during the cell cycle demonstrated a reproducible increase in histone acetylation during the replication phase that was subsequently decreased at the G2M entrance, thus paralleling the behavior of DNA replication and transcriptional activity.</p><p><strong>Conclusions: </strong>Multiparameter analysis of histone acetylation and expression of molecular markers, DNA ploidy, and/or cell cycle kinetics can provide a quick and statistically reliable tool for the diagnosis and evaluation of treatment efficacy in clinical trials using HDAC inhibitors.</p>","PeriodicalId":520601,"journal":{"name":"Cytometry. 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引用次数: 61
摘要
背景:可逆性组蛋白乙酰化影响染色质结构组织,从而调节基因表达和其他核事件。在正常细胞中,组蛋白乙酰化水平受到严格调节,其调节机制的改变已被证明与肿瘤发生有关。方法:我们开发了一种新的流式细胞技术检测组蛋白乙酰化,基于特异性单克隆抗体识别乙酰化组蛋白尾部。对组蛋白乙酰化水平和DNA进行双变量分析,以研究在细胞周期和组蛋白去乙酰化酶(HDAC)抑制剂trichostatin A (TSA)诱导组蛋白超乙酰化后染色质组织的调节。在维甲酸单独或联合TSA诱导的分化过程中监测组蛋白乙酰化和转录水平。采用所述方案分析患者的血液样本,以监测体内HDAC抑制剂的效果,并验证所开发的方案的临床应用。结果:流式细胞术检测乙酰化状态可以成功检测体内HDAC抑制剂治疗引起的修饰,这一点通过对丙戊酸治疗患者的各种血液样本的分析得到证实。细胞周期中乙酰化水平的变化表明,在复制阶段组蛋白乙酰化可重复增加,随后在G2M入口降低,从而平行于DNA复制和转录活性的行为。结论:组蛋白乙酰化、分子标记表达、DNA倍体和/或细胞周期动力学的多参数分析可以为临床试验中使用HDAC抑制剂的治疗效果的诊断和评估提供快速和统计可靠的工具。
New method to detect histone acetylation levels by flow cytometry.
Background: Reversible histone acetylation affects chromatin structural organization, thus regulating gene expression and other nuclear events. Levels of histone acetylation are tightly modulated in normal cells, and alterations of their regulating mechanisms have been shown to be involved in tumorigenesis.
Methods: We developed a new flow cytometric technique for detection of histone acetylation, based on a specific monoclonal antibody that recognizes acetylated histone tails. Bivariate analysis for histone acetylation levels and DNA were performed to study modulation of chromatin organization during the cell cycle and after induction of histone hyperacetylation by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Histone acetylation and transcription levels were monitored during differentiation induced by retinoic acid alone or in combination with TSA. Blood samples from patients were analyzed with the described protocol to monitor the effects of HDAC inhibitors in vivo and validate the developed protocol for clinical usage.
Results: Flow cytometric detection of acetylation status can successfully detect modifications induced by HDAC inhibitor treatment in vivo as demonstrated by analysis of various blood samples from patients treated with valproic acid. Changes in acetylation levels during the cell cycle demonstrated a reproducible increase in histone acetylation during the replication phase that was subsequently decreased at the G2M entrance, thus paralleling the behavior of DNA replication and transcriptional activity.
Conclusions: Multiparameter analysis of histone acetylation and expression of molecular markers, DNA ploidy, and/or cell cycle kinetics can provide a quick and statistically reliable tool for the diagnosis and evaluation of treatment efficacy in clinical trials using HDAC inhibitors.