Kawagoe H, Humphries R K, Blair A, Sutherland H J, Hogge D E
British Columbia Cancer Agency, and Department of Medicine, University of British Columbia, Vancouver, Canada.
Leukemia. 1999 May;13(5):687-98. doi: 10.1038/sj.leu.2401410.
To explore the possibility that deregulated HOX gene expression might commonly occur during leukemic hematopoiesis, we compared the relative levels of expression of these and related genes in phenotypically and functionally defined subpopulations of AML blasts and normal hematopoietic cells. Initially, a semi-quantitative RT-PCR technique was used to amplify total cDNA from total leukemic blast cell populations from 20 AML patients and light density cells from four normal bone marrows. Expression of HOX genes (A9, A10, B3 and B4), MEIS1 and MLL was easily detected in the majority of AML samples with the exception of two samples from patients with AML subtype M3 (which expressed only MLL). Low levels of HOXA9 and A10 but not B3 or B4 were seen in normal marrow while MLL was easily detected. PBX1a was difficult to detect in any AML sample but was seen in three of four normal marrows. Cells from nine AML patients and five normal bone marrows were FACS-sorted into CD34+CD38-, CD34+CD38+ and CD34-subpopulations, analyzed for their functional properties in long-term culture (LTC) and colony assays, and for gene expression using RT-PCR. 93 +/- 14% of AML LTC-initiating cells, 92 +/- 14% AML colony-forming cells, and >99% of normal LTC-IC and CFC were CD34+. The relative level of expression of the four HOX genes in amplified cDNA from CD34- as compared to CD34+CD38- normal cells was reduced >10-fold. However, in AML samples this down-regulation in HOX expression in CD34- as compared to CD34+CD38- cells was not seen (P < 0.05 for comparison between AML and normal). A similar difference between normal and AML subpopulations was seen when the relative levels of expression of MEIS1, and to a lesser extent MLL, were compared in CD34+ and CD34- cells (P < 0.05). In contrast, while some evidence of down-regulation of PBX1a was found in comparing CD34- to CD34+ normal cells it was difficult to detect expression of this gene in any subpopulation from most AML samples. Thus, the down-regulation of HOX, MEIS1 and to some extent MLL which occurs with normal hematopoietic differentiation is not seen in AML cells with similar functional and phenotypic properties.
为了探究HOX基因表达失调在白血病造血过程中可能普遍存在的可能性，我们比较了这些基因以及相关基因在表型和功能明确的急性髓系白血病（AML）原始细胞亚群和正常造血细胞中的相对表达水平。最初，使用半定量逆转录聚合酶链反应（RT-PCR）技术从20例AML患者的白血病原始细胞总体中以及来自四个正常骨髓的低密度细胞中扩增总cDNA。在大多数AML样本中很容易检测到HOX基因（A9、A10、B3和B4）、MEIS1和MLL的表达，但两名AML M3亚型患者的样本除外（这两个样本仅表达MLL）。在正常骨髓中可见低水平的HOXA9和A10，但未检测到B3或B4，而MLL很容易被检测到。在任何AML样本中都很难检测到PBX1a，但在四个正常骨髓中的三个中可以检测到。对来自9例AML患者和5个正常骨髓的细胞进行荧光激活细胞分选（FACS），分为CD34+CD38-、CD34+CD38+和CD34-亚群，分析它们在长期培养（LTC）和集落测定中的功能特性，并使用RT-PCR分析基因表达。93±14%的AML LTC起始细胞、92±14%的AML集落形成细胞以及>99%的正常LTC-IC和CFC是CD34+。与CD34+CD38-正常细胞相比，CD34-扩增cDNA中四个HOX基因的相对表达水平降低了10倍以上。然而，在AML样本中，与CD34+CD38-细胞相比，CD34-细胞中HOX表达的这种下调未见（AML与正常样本比较，P<0.05）。当比较CD34+和CD34-细胞中MEIS1以及程度较轻的MLL的相对表达水平时，在正常和AML亚群之间也观察到了类似的差异（P<0.05）。相比之下，虽然在比较CD34-与CD34+正常细胞时发现了PBX1a下调的一些证据，但在大多数AML样本的任何亚群中都很难检测到该基因的表达。因此，在具有相似功能和表型特性的AML细胞中未见到正常造血分化过程中发生的HOX、MEIS1以及一定程度上MLL的下调。
