All-trans retinoic acid (ATRA)-based differentiation therapy has been unsuccessful in treating t(15;17) negative acute myeloid leukemia (AML) patients, motivating interest in combination therapies using ATRA plus other agents. Using the t (15, 17) negative HL-60 human myeloblastic leukemia model, we find that the cyclin-dependent kinase (CDK) inhibitor, roscovitine, augments signaling by an ATRA-induced macromolecular signalsome that propels differentiation and enhances ATRA-induced differentiation. Roscovitine co-treatment enhanced ATRA-induced expression of pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src Family Kinases (SFKs) Lyn and Fgr and SFK Y416 phosphorylation, adaptor proteins c-Cbl and SLP-76, Vav, and acetylated 14-3-3 in the signalsome. Roscovitine enhanced ATRA-induced c-Raf interaction with Lyn, Vav, and c-Cbl. Consistent with signalsome hyper-activation, roscovitine co-treatment enhanced ATRA-induced G1/0 arrest and expression of differentiation markers, CD11b, ROS and p47 Phox. Because roscovitine regulated Lyn expression, activation and partnering, a stably transfected Lyn knockdown was generated from wt-parental cells to investigate its function in ATRA-induced differentiation. Lyn-knockdown enhanced ATRA-induced up-regulation of key signalsome molecules, c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, Vav1, SLP-76, and Fgr, but with essentially total loss of pY416-SFK. Compared to ATRA-treated wt-parental cells, differentiation markers p47 phox, CD11b, G1/G0 arrest and ROS production were enhanced in ATRA-treated Lyn-knockdown stable transfectants, and addition of roscovitine further enhanced these ATRA-inducible markers. The Lyn-knockdown cells expressed slightly higher c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, and SLP-76 than wt-parental cells, and this was associated with enhanced ATRA-induced upregulation of Fgr and cell differentiation, consistent with heightened signaling, suggesting that enhanced Fgr may have compensated for loss of Lyn to enhance differentiation in the Lyn-knockdown cells.

中文翻译：

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Roscovitine 增强全反式视黄酸 (ATRA) 诱导的白血病细胞分化：对假定 Cdk2 抑制剂的信号分子的新作用。

基于全反式视黄酸 (ATRA) 的分化疗法在治疗 t(15;17) 阴性急性髓性白血病 (AML) 患者方面未成功，这激发了人们对使用 ATRA 和其他药物的联合疗法的兴趣。使用 t (15, 17) 阴性 HL-60 人成髓细胞白血病模型，我们发现细胞周期蛋白依赖性激酶 (CDK) 抑制剂 roscovitine 通过 ATRA 诱导的大分子信号体增强信号传导，促进分化并增强 ATRA 诱导的分化. Roscovitine 联合处理增强了 ATRA 诱导的 pS259-pS289/296/301-pS621-c-Raf、pS217/221-Mek、Src 家族激酶 (SFKs) Lyn 和 Fgr 以及 SFK Y416 磷酸化、衔接蛋白 c-Cbl 和SLP-76、Vav 和信号体中的乙酰化 14-3-3。Roscovitine 增强了 ATRA 诱导的 c-Raf 与 Lyn、Vav 和 c-Cbl 的相互作用。与信号体过度激活一致，roscovitine 联合治疗增强了 ATRA 诱导的 G1/0 期阻滞和分化标志物 CD11b、ROS 和 p47 Phox 的表达。因为roscovitine 调节Lyn 表达、激活和配对，从wt-亲本细胞产生稳定转染的Lyn 敲低以研究其在ATRA 诱导的分化中的功能。Lyn 敲低增强了 ATRA 诱导的关键信号体分子 c-Raf、pS259-c-Raf、pS289/296/301-c-Raf、Vav1、SLP-76 和 Fgr 的上调，但基本上完全丧失了pY416-SFK。与 ATRA 处理的野生型亲本细胞相比，ATRA 处理的 Lyn-knockdown 稳定转染子的分化标志物 p47 phox、CD11b、G1/G0 停滞和 ROS 产生增强，并且添加 roscovitine 进一步增强了这些 ATRA 诱导标记。