Deciphering the molecular mode of action of histone deacetylase inhibitors in human germ cell cancer cell lines in vitro and in vivo

我们之前的分析表明，ISTODAX在极低浓度下可导致gem细胞癌（GCC）细胞系中组蛋白H3和H4的超乙酰化、细胞凋亡和G2/ m阻滞。在人成纤维细胞中，只有G2/ m阻滞，但未检测到凋亡。此外，我们确定了驱动细胞对ISTODAX反应的推定关键因素。我们假设SWI/ snf调节因子ARID1A的下调以及应激传感器GADD45B、ATF3、ID2、ZFP36和DUSP1的上调诱导了GCC细胞系的凋亡。此外，我们发现5个基因在GCC细胞系和成纤维细胞之间共同表达（DHRS2、RHOB、CRISPLD2、BAIPA2和p21）。我们假设其中一个因素诱导p21表达，导致G2/ m阻滞。在这项研究中，我们希望通过cDNA过表达或CRISPR/ cas介导的敲低这些因子在GCC细胞和成纤维细胞中阐明istodax级联的层次结构和每个关键参与者的功能作用，然后分析其分子效应。我们将分别利用qRT-PCR-和western blot-以及染色质免疫沉淀-和共免疫沉淀-分析来筛选基因表达的变化和检测蛋白质- dna - /蛋白质-蛋白质相互作用。诱导凋亡或G2/ m阻滞将通过基于facs的方法来测量。接下来，将表达luciferase、过表达cdna和CRISPR/ cas敲除的细胞系移植到裸鼠精管中。在两个月的ISTODAX应用期间，将通过实时成像技术测量肿瘤生长情况。此外，如前所述，将分离肿瘤并分析ISTODAX体内治疗的分子效应。最后，收集到的数据将与体外分析的结果相关联。这些实验将破译ISTODAX在体外和体内的作用模式，并深入了解GCC细胞对ISTODAX的反应机制，这可能会影响治疗的成功。精管注射将证明ISTODAX能够通过血液-睾丸屏障，突出ISTODAX作为一种有价值的GCC体内治疗选择。

Our previous analyses have shown that ISTODAX causes hyperacetylation of histones H3 and H4, apoptosis and G2/M-arrest at very low concentrations in gem cell cancer (GCC) cell lines. In human fibroblasts, only a G2/M-arrest, but no apoptosis was detectable. Additionally, we identifed putative key factors driving the cellular response to ISTODAX. We postulate that downregulation of the SWI/SNF-regulator ARID1A as well as upregulation of stress-sensors GADD45B, ATF3, ID2, ZFP36 and DUSP1 induced apoptosis in GCC cell lines. Furthermore, we found 5 genes to be commonly expressed between GCC cell lines and fibroblasts (DHRS2, RHOB, CRISPLD2, BAIPA2 and p21). We assume that one of these factors induces p21 expression, causing G2/M-arrest.In this study, we would like to shed light on the hierarchical structure of the ISTODAX-cascade and the functional role of each key player by cDNA overexpression or CRISPR/Cas-mediated knock down of these factors in GCC cells and fibroblasts, followed by analyses of the molecular effetcs. We will utilize qRT-PCR- and western blot- as well as chromatin-immunoprecipitation- and co-immunoprecipitation-analyses to screen for changes in gene expression and detect protein-DNA- / protein-protein-interactions, respectively. Induction of apoptosis or G2/M-arrest will be measured by FACS-based methods.Next, Luciferase-expressing, cDNA-overexpressing and CRISPR/Cas-knock out cell lines will be transplanted into the seminiferous tubule of nude mice. During a two month ISTODAX application, tumor growth will be measured by live imaging technology. Additionally, tumors will be isolated and analyzed for the molecular effects of an ISTODAX in vivo treatment as described before. Finally, gathered data will be correlated to the results of the in vitro analyses.These experiments will decipher the mode of action of ISTODAX in vitro and in vivo and provide insight into the response mechanisms of GCC cells towards ISTODAX that might influence the success of a therapy. The seminiferous tubule injections will demonstrate that ISTODAX is able to pass the blood-testis-barrier, highlighting ISTODAX as a valuable GCC in vivo therapy option.