Yin Bin, Kogan Scott C, Dickins Ross A, Lowe Scott W, Largaespada David A
University of Minnesota Cancer Center, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Exp Hematol. 2006 May;34(5):631-41. doi: 10.1016/j.exphem.2006.01.015.
Chemoresistance remains a major clinical obstacle to curative chemotherapy of acute myeloid leukemia (AML), but the molecular mechanisms underlying resistance to chemotherapeutic agents used in AML are largely unknown. We have attempted to investigate genetic mechanisms causing resistance to Ara-C [1-beta-D-arabinofuranosyl-cytosine (cytarabine)], one mainstay in AML chemotherapy for decades.
Highly Ara-C-resistant murine BXH-2 strain AML cell lines were generated, and their molecular changes were compared to their sensitive parental lines. The causative changes were confirmed using a genetic approach.
We derived nine highly Ara-C-resistant murine BXH-2 strain AML sublines via in vitro selection. p21Cip1 was dramatically downregulated and p53 protein accumulation induced by Ara-C treatment was impaired in one resistant line. In this line, repeated Ara-C exposure had selected for cells that harbor a genomic deletion affecting the splicing of Trp53 mRNA. This deletion produces an aberrant Trp53 mRNA, in which exon 4 is skipped, producing a protein lacking parts of both the transactivation and DNA-binding domains. Retroviral transduction of the sensitive parental cells with a dominant-negative Trp53 cDNA caused changes in the protein levels of p21Cip1, BAX, and cleaved caspase-3, but not bcl-XL, and rendered the cells more resistant to Ara-C. Unexpectedly, we found that pifithrin-alpha (PFTalpha), a compound that has been proposed to regulate p53 protein activity, induced apoptosis in both Ara-C-sensitive and -resistant lines, and decreased Ara-C resistance in cells with either normal or mutant Trp53 genes.
These data indicate that Trp53 loss-of-function could partly explain the acquisition of AML chemoresistance, and suggest that PFTalpha could be useful in treatment of relapsed AML.
化疗耐药仍然是急性髓系白血病（AML）根治性化疗的主要临床障碍，但AML中对化疗药物耐药的分子机制很大程度上尚不清楚。我们试图研究导致对阿糖胞苷[1-β-D-阿拉伯呋喃糖基胞嘧啶（阿糖胞苷）]耐药的遗传机制，阿糖胞苷是几十年来AML化疗的主要药物之一。
构建了高度耐阿糖胞苷的小鼠BXH-2株AML细胞系，并将其分子变化与其敏感的亲代细胞系进行比较。通过遗传学方法证实了引起变化的原因。
通过体外筛选，我们获得了9个高度耐阿糖胞苷的小鼠BXH-2株AML亚系。在一个耐药细胞系中，p21Cip1显著下调，阿糖胞苷处理诱导的p53蛋白积累受损。在该细胞系中，反复阿糖胞苷暴露筛选出了携带影响Trp53 mRNA剪接的基因组缺失的细胞。这种缺失产生异常的Trp53 mRNA，其中外显子4被跳过，产生一种缺乏反式激活和DNA结合结构域部分的蛋白质。用显性负性Trp53 cDNA对敏感亲代细胞进行逆转录病毒转导，导致pCip1、BAX和裂解的caspase-3的蛋白水平发生变化，但bcl-XL未发生变化，并使细胞对阿糖胞苷更具耐药性。出乎意料的是，我们发现pifithrin-α（PFTα），一种被认为可调节p53蛋白活性的化合物，在阿糖胞苷敏感和耐药细胞系中均诱导凋亡，并降低具有正常或突变Trp53基因的细胞的阿糖胞苷耐药性。
这些数据表明Trp53功能丧失可能部分解释了AML化疗耐药的获得，并提示PFTα可能对复发AML的治疗有用。
