Ge Yubin, Jensen Tanya L, Stout Mark L, Flatley Robin M, Grohar Patrick J, Ravindranath Yaddanapudi, Matherly Larry H, Taub Jeffrey W
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Division of Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, Michigan, USA.
Cancer Res. 2004 Jan 15;64(2):728-35. doi: 10.1158/0008-5472.can-03-2456.
Myeloblasts from Down syndrome (DS) children with acute myeloid leukemia (AML) are significantly more sensitive in vitro to 1-beta-D-arabinofuranosylcytosine (ara-C) and generate higher 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) than non-DS AML myeloblasts. Semiquantitative reverse transcription-PCR analyses demonstrated that transcripts for cytidine deaminase (CDA) were 2.7-fold lower in DS than for non-DS myeloblasts. In contrast, transcripts of cystathionine-beta-synthase and deoxycytidine kinase were a median 12.5- and 2.6-fold higher in DS compared with non-DS myeloblasts. The ratio of deoxycytidine kinase/CDA transcripts significantly correlated with ara-C sensitivities and ara-CTP generation. In clinically relevant AML cell line models, high cystathionine-beta-synthase transcripts in DS CMK cells were accompanied by 10-fold greater ara-C sensitivity and 2.4-fold higher levels of ara-CTP compared with non-DS CMS cells. Overexpression of CDA in non-DS THP-1 cells was associated with a 100-fold decreased ara-C sensitivity and 40-fold decreased ara-CTP generation. THP-1 cells secreted CDA into the incubation media and converted extracellular ara-C completely to 1-beta-D-arabinofuranosyluracil within 30 min. Rapid amplification of 5'-cDNA ends (5'-RACE) and reverse transcription-PCR assays identified short- (sf) and long-form (lf) CDA transcripts in THP-1 cells with different 5' untranslated regions and translational start sites; however, only the latter resulted in the active CDA. Although 5' flanking sequences for both CDA transcripts exhibited promoter activity in reporter gene assays, activity for the CDAlf was low. The presence of several GATA1 binding sites in the CDAsf promoter and the uniform detection of GATA1 mutations in DS megakaryocytic leukemia suggested the potential role of GATA1 in regulating CDA transcription and the CDAsf promoter acting as an enhancer. Transfection of GATA1 into Drosophila Mel-2 cells stimulated the CDAlf promoter in a dose-dependent fashion. Additional identification of the mechanisms of differential expression of genes encoding enzymes involved in ara-C metabolism between DS and non-DS myeloblasts may lead to improvements in AML therapy.
患有急性髓系白血病（AML）的唐氏综合征（DS）儿童的成髓细胞在体外对1-β-D-阿拉伯呋喃糖基胞嘧啶（阿糖胞苷，ara-C）的敏感性显著更高，并且比非DS AML成髓细胞产生更高水平的1-β-D-阿拉伯呋喃糖基胞嘧啶5'-三磷酸（ara-CTP）。半定量逆转录-PCR分析表明，DS成髓细胞中胞苷脱氨酶（CDA）的转录本比非DS成髓细胞低2.7倍。相反，与非DS成髓细胞相比，DS成髓细胞中胱硫醚-β-合酶和脱氧胞苷激酶的转录本中位数分别高12.5倍和2.6倍。脱氧胞苷激酶/CDA转录本的比率与阿糖胞苷敏感性和ara-CTP生成显著相关。在临床相关的AML细胞系模型中，与非DS CMS细胞相比，DS CMK细胞中高胱硫醚-β-合酶转录本伴随着阿糖胞苷敏感性高10倍和ara-CTP水平高2.4倍。在非DS THP-1细胞中过表达CDA与阿糖胞苷敏感性降低100倍和ara-CTP生成降低40倍相关。THP-1细胞将CDA分泌到培养液中，并在30分钟内将细胞外阿糖胞苷完全转化为1-β-D-阿拉伯呋喃糖基尿嘧啶。5'-cDNA末端快速扩增（5'-RACE）和逆转录-PCR分析在THP-1细胞中鉴定出具有不同5'非翻译区和翻译起始位点的短型（sf）和长型（lf）CDA转录本；然而，只有后者产生有活性的CDA。尽管两种CDA转录本的5'侧翼序列在报告基因检测中均表现出启动子活性，但CDAlf的活性较低。CDAsf启动子中存在几个GATA1结合位点，以及在DS巨核细胞白血病中均匀检测到GATA1突变，提示GATA1在调节CDA转录中可能发挥作用，且CDAsf启动子作为增强子。将GATA1转染到果蝇Mel-2细胞中以剂量依赖方式刺激CDAlf启动子。进一步鉴定DS和非DS成髓细胞之间参与阿糖胞苷代谢的酶编码基因差异表达的机制可能会改善AML治疗。
