Kamashev Dmitrii, Vitoux Dominique, De Thé Hugues
CNRS UPR9051, Hôpital St. Louis, Laboratoire associé N degrees 11, 1, Av. C. Vellefaux, 75475 Paris, Cedex 10, France. email:
J Exp Med. 2004 Apr 19;199(8):1163-74. doi: 10.1084/jem.20032226.
PML-RARA was proposed to initiate acute promyelocytic leukemia (APL) through PML-RARA homodimer-triggered repression. Here, we examined the nature of the PML-RARA protein complex and of its DNA targets in APL cells. Using a selection/amplification approach, we demonstrate that PML-RARA targets consist of two AGGTCA elements in an astonishing variety of orientations and spacings, pointing to highly relaxed structural constrains for DNA binding and identifying a major gain of function of this oncogene. PML-RARA-specific response elements were identified, which all conveyed a major transcriptional response to RA only in APL cells. In these cells, we demonstrate that PML-RARA oligomers are complexed to RXR. Directly probing PML-RARA function in APL cells, we found that the differentiation enhancer cyclic AMP (cAMP) boosted transcriptional activation by RA. cAMP also reversed the normal silencing (subordination) of the transactivating function of RXR when bound to RARA or PML-RARA, demonstrating that the alternate rexinoid/cAMP-triggered APL differentiation pathway also activates PML-RARA targets. Finally, cAMP restored both RA-triggered differentiation and PML-RARA transcriptional activation in mutant RA-resistant APL cells. Collectively, our findings directly demonstrate that APL cell differentiation parallels transcriptional activation through PML-RARA-RXR oligomers and that those are functionally targeted by cAMP, identifying this agent as another oncogene-targeted therapy.
PML-RARA被认为通过PML-RARA同二聚体触发的抑制作用引发急性早幼粒细胞白血病（APL）。在此，我们研究了APL细胞中PML-RARA蛋白复合物及其DNA靶点的性质。使用一种筛选/扩增方法，我们证明PML-RARA靶点由两个AGGTCA元件组成，它们具有惊人多样的方向和间距，这表明DNA结合的结构限制高度宽松，并确定了该致癌基因的一个主要功能获得。鉴定出了PML-RARA特异性反应元件，它们仅在APL细胞中对维甲酸（RA）传递主要转录反应。在这些细胞中，我们证明PML-RARA寡聚体与维甲酸X受体（RXR）复合。直接在APL细胞中探究PML-RARA的功能，我们发现分化增强剂环磷酸腺苷（cAMP）增强了RA的转录激活作用。当RXR与RARA或PML-RARA结合时，cAMP还逆转了RXR反式激活功能的正常沉默（从属关系），表明替代类视黄酸/cAMP触发的APL分化途径也激活PML-RARA靶点。最后，cAMP在突变的RA抗性APL细胞中恢复了RA触发的分化和PML-RARA转录激活。总体而言，我们的发现直接证明APL细胞分化与通过PML-RARA-RXR寡聚体的转录激活平行，并且这些寡聚体在功能上被cAMP靶向，确定该药物为另一种靶向致癌基因的疗法。
