ZBTB16, a glucocorticoid response gene in acute lymphoblastic leukemia, interferes with glucocorticoid-induced apoptosis. Homo sapiens

Glucocorticoids (GCs) cause apoptosis in lymphoid lineage cells and are therefore widely used in the therapy of lymphoid malignancies. The molecular mechanisms of the anti-leukemic GC effects are, however, poorly understood. We have previously defined a list of GC-regulated candidate genes by Affymetrix-based whole genome comparative expression profiling in children with acute lymphoblastic leukemia (ALL) during systemic GC monotherapy and in experimental systems of GC-induced apoptosis. ZBTB16, a Zink finger and BOZ-domain containing transcriptional repressor, was one of the most promising candidates derived from this screen. To investigate its possible role in GC-induced apoptosis and cell cycle arrest, we performed conditional over-expression experiments in CCRF-CEM childhood ALL cells. Transgenic ZBTB16 alone had no detectable effect on survival, however, it reduced sensitivity to GC-induced apoptosis. This protective effect was not seen when apoptosis was induced by antibodies against Fas/CD95 or 3 different chemotherapeutics. To address the molecular mechanism underlying this protective effect, we performed whole genome expression profiling in cells with conditional ZBTB16 expression. Surprisingly, ZBTB16 induction did not significantly alter the expression profile, however, it interfered with the regulation of several GC response genes. One of them, BCL2L11/Bim, has previously been shown to be responsible for cell death induction in CCRF-CEM cells. Thus, ZBTB16´s protective effect can be attributed to interference with transcriptional regulation of apoptotic genes, at least in the investigated model system. Overall design: To determine ZBTB16 response genes, C7H2-2C8-ZBTB16#19 and #58 cells (expressing ZBTB16 in a doxycycline-dependent manner) were cultured in duplicates in the absence (treatment “none”) or presence of 400ng/ml doxycycline (treatment “Dox”) for 2h, 6h or 24h. Total RNA was prepared and 1.5 µg RNA subjected to expression profiling on Exon 1.0 microarrays (total of 24 arrays). To assess the effect of ZBTB16 on the GC response, the above cell lines were cultured for 24h in the absence (treatment “Dex”) or presence (treatment “DexDox”) of 200ng/ml doxycycline and subsequently exposed to 10-8M dexamethasone for 6h and 24h and expression-profiled as above resulting in a total of 15 arrays (one of the four 6h replicates (0997_001_58pp6S2_E01_MB_110708.CEL) had to be removed from the following analysis because it didn´t pass quality control).

糖皮质激素（Glucocorticoids, GCs）可诱导淋巴系细胞凋亡，因此被广泛应用于淋巴系统恶性肿瘤的治疗。然而，糖皮质激素发挥抗白血病作用的分子机制仍不甚明确。此前，我们通过基于Affymetrix平台的全基因组比较表达谱分析，在接受全身糖皮质激素单药治疗的急性淋巴细胞白血病（acute lymphoblastic leukemia, ALL）患儿以及糖皮质激素诱导凋亡的实验模型中，筛选得到了一系列受糖皮质激素调控的候选基因。ZBTB16作为一种含有锌指结构与BOZ结构域的转录抑制因子，是该筛选中最具潜力的候选基因之一。为探究ZBTB16在糖皮质激素诱导凋亡及细胞周期阻滞中的潜在作用，我们在CCRF-CEM儿童急性淋巴细胞白血病细胞中开展了条件性过表达实验。单独过表达ZBTB16对细胞存活无明显可检测到的影响，但可降低细胞对糖皮质激素诱导凋亡的敏感性。而当使用抗Fas/CD95抗体或3种不同化疗药物诱导凋亡时，并未观察到这一保护效应。为阐明该保护效应的分子机制，我们对条件性表达ZBTB16的细胞开展了全基因组表达谱分析。令人意外的是，单独诱导ZBTB16表达并未显著改变细胞的表达谱，但却干扰了多个糖皮质激素应答基因的调控。其中，BCL2L11/Bim此前已被证实可介导CCRF-CEM细胞的死亡诱导。因此，至少在所研究的模型系统中，ZBTB16的保护效应可归因于其对凋亡相关基因转录调控的干扰。

总体实验设计：为鉴定ZBTB16应答基因，我们将C7H2-2C8-ZBTB16#19与#58细胞（以强力霉素依赖的方式表达ZBTB16）分为两组进行重复培养：一组不添加任何处理（标记为"none"），另一组添加400ng/ml强力霉素（标记为"Dox"），分别培养2h、6h或24h。提取总RNA后，取1.5μg RNA在Exon 1.0微阵列上进行表达谱分析，共计24张芯片。为评估ZBTB16对糖皮质激素应答的影响，我们将上述细胞系分别培养24h：一组不添加强力霉素但后续加入地塞米松处理（标记为"Dex"），另一组预先添加200ng/ml强力霉素后再加入地塞米松（标记为"DexDox"）；随后分别于6h和24h收取样本，按前述方法进行表达谱分析，共计获得15张芯片（其中1份6h时间点的重复样本0997_001_58pp6S2_E01_MB_110708.CEL因未通过质量控制，被移除后续分析）。