Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies.     

慢病毒介导的稳定表达LBH的前列腺癌细胞株的建立

目的:构建稳定表达LBH基因的人前列腺癌细胞株PC-3M-LBH,探讨LBH基因对PC-3M细胞增殖能力的影响。方法:构建表达LBH基因的重组慢病毒载体并制备出相应的慢病毒,感染低表达LBH基因的人前列腺癌PC-3M细胞后,经嘌呤霉素筛选获得细胞克隆;实时荧光定量PCR和蛋白印迹法(Western-Blot)分别检测细胞株中LBH的mRNA、蛋白表达水平;采用CCK-8法检测表达LBH基因后细胞增殖能力的改变。结果:成功构建了重组慢病毒表达质粒p Lenti-LBH并包装出了慢病毒,感染前列腺癌细胞后经嘌呤霉素筛选得到PC-3M-LBH细胞株;PC-3M-LBH细胞株中LBH基因的mRNA和蛋白表达显著上调;相对母细胞和NC对照组,PC-3M-LBH细胞在接种后第4天即出现明显的生长抑制,到第6天其生长抑制率达到19.7%。结论:构建的细胞株能稳定表达LBH基因,该基因的表达能显著抑制前列腺癌PC-3M细胞的体外增殖。     

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.     

Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies.     

Elevation of YAP promotes the epithelial-mesenchymal transition and tumor aggressiveness in colorectal cancer

Tumor metastasis is the leading cause of death in cancer patients. Identifying metastatic biomarkers in tumor cells would help cancer diagnoses and the development of therapeutic targets. Yes-associated protein (YAP) plays an important role in organ development and has gained much attention in tumorigenesis. However, the role of YAP and the underlying mechanism in tumor metastasis of colorectal cancer (CRC) is still unclear. In this study, we generated metastatic 116-LM cells from the HCT116 CRC cell line. We found that the capacity for tumor aggressiveness was elevated in 116-LM cells and identified that YAP and its mRNA level were upregulated in 116-LM cells. Moreover, expression of YAP was found to correlate with epithelial-mesenchymal transition (EMT) marker expressions, whereas suppression of YAP decreased EMT marker expressions and impeded tumor migration and invasion. Additionally, upregulation of YAP was identified in colon cancer patients, and it was correlated with EMT gene expressions. Furthermore, we identified LBH589, a histone deacetylase inhibitor, that was capable of inhibiting tumor growth and aggressiveness in both HCT116 and 116-LM cells. LBH589 potentially inhibited YAP and its mRNA expression, accompanied by diminished expressions of YAP downstream genes and EMT markers. Together, YAP plays a crucial role in aggressiveness and metastasis of CRC, and YAP may be an attractive therapeutic target.