Ahn M J, Nason-Burchenal K, Moasser M M, Dmitrovsky E
Department of Medicine, Memorial Hospital, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Oncogene. 1995 Jun 15;10(12):2307-14.
The balanced t(15;17) rearrangement found in acute promyelocytic leukemia (APL) cells fuses PML on chromosome 15 to the retinoic acid receptor alpha (RAR alpha) on chromosome 17. PML/RAR alpha is expressed in APL cells with the non-rearranged alleles, PML and RAR alpha. Clinical remissions induced by all-trans-retinoic acid (RA) treatment of APL patients are linked to expression of PML/RAR apha, a transcription factor with reported dominant negative functions. The roles of PML and RAR alpha in the RA response of APL have not yet been fully explored. This study examines these roles by individually transfecting RAR alpha and PML into NB4 APL cells. NB4 is the sole APL cell line containing the t(15;17). RA treatment represses NB4 cell growth and induces a myeloid phentoype. Full length cDNAs for RAR alpha and PML were individually cloned into a CMV-driven expression vector containing the neomycin resistance gene. Surprisingly, none of the obtained stable transfectants expressed exogenous RAR alpha or PML mRNAs even when reverse transcription polymerase chain reaction (RT-PCR) detection assays were used. All clones expressed the neomycin resistance gene and were similar to parental NB4 cells in their growth and differentiation properties. An explanation explored for this lack of gene expression was that increased levels of RAR alpha or PML might suppress APL cell growth. To examine this possibility, transfection experiments were repeated using an episomal vector-based expression system containing an SV40 driven RAR alpha or PML cDNA and the hygromycin B resistance gene. A new selection strategy augmented expression of the desired cDNAs. A control episomal vector lacked a cDNA insert. Following electroporation and selection, exogenous RAR alpha expression was obtained. Compared to controls, the growth of these transfectants was markedly inhibited before and after RA-treatment and these cells more prominently induced myeloid maturation markers. In contrast, exogenous PML expression was transient since these transfectants did not appear to propagate in culture. These findings indicate: (1) a growth disadvantage for NB4 cells having increased expression of RAR alpha or PML and (2) increased RAR alpha expression augmented RA-mediated maturation of NB4 cells. This implicates a role for RAR alpha or PML in regulating the growth or differentiation of APL cells. It is hypothesized this occurs through antagonism of PML/RAR alpha actions in these leukemic cells.
在急性早幼粒细胞白血病（APL）细胞中发现的平衡型t(15;17)重排，将15号染色体上的早幼粒细胞白血病基因（PML）与17号染色体上的维甲酸受体α（RARα）融合。PML/RARα在具有未重排等位基因PML和RARα的APL细胞中表达。全反式维甲酸（RA）治疗APL患者所诱导的临床缓解与PML/RARα的表达相关，PML/RARα是一种据报道具有显性负功能的转录因子。PML和RARα在APL的RA反应中的作用尚未得到充分探索。本研究通过将RARα和PML分别转染到NB4 APL细胞中来研究这些作用。NB4是唯一含有t(15;17)的APL细胞系。RA处理可抑制NB4细胞生长并诱导髓系表型。将RARα和PML的全长cDNA分别克隆到一个含有新霉素抗性基因的CMV驱动表达载体中。令人惊讶的是，即使使用逆转录聚合酶链反应（RT-PCR）检测方法，所获得的稳定转染子中也没有一个表达外源性RARα或PML mRNA。所有克隆均表达新霉素抗性基因，并且在生长和分化特性方面与亲代NB4细胞相似。对于这种基因表达缺失所探索的一种解释是，RARα或PML水平的升高可能会抑制APL细胞生长。为了检验这种可能性，使用基于游离型载体的表达系统重复转染实验，该系统包含一个SV40驱动的RARα或PML cDNA以及潮霉素B抗性基因。一种新的选择策略增强了所需cDNA的表达。一个对照游离型载体没有cDNA插入片段。在电穿孔和选择之后，获得了外源性RARα表达。与对照相比，这些转染子在RA处理前后的生长均受到明显抑制，并且这些细胞更显著地诱导了髓系成熟标志物。相反，外源性PML表达是短暂的，因为这些转染子在培养中似乎没有增殖。这些发现表明：（1）RARα或PML表达增加的NB4细胞具有生长劣势；（2）RARα表达增加增强了RA介导的NB4细胞成熟。这暗示了RARα或PML在调节APL细胞生长或分化中的作用。据推测，这是通过拮抗这些白血病细胞中PML/RARα的作用而发生的。
