Treatment of Philadelphia leukemia in severe combined immunodeficient mice by combination of cyclophosphamide and bcr/abl antisense oligodeoxynucleotides.
Treatment of Philadelphia leukemia in severe combined immunodeficient mice by combination of cyclophosphamide and bcr/abl antisense oligodeoxynucleotides.
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通过环磷酰胺和 bcr/abl 反义寡脱氧核苷酸的组合治疗严重联合免疫缺陷小鼠的费城白血病。
DOI：
10.1093/jnci/89.2.124
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Calabretta,B
中科院分区：
文献类型：
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作者：
Skorski,T;Nieborowska-Skorska,M;Wlodarski,P;Perrotti,D;Hoser,G;Kawiak,J;Majewski,M;Christensen,L;Iozzo,RV;Calabretta,B
背景：费城1细胞是人类慢性粒细胞白血病（CML）细胞，含有BCR/ABL癌基因（BCR和ABL基因的融合体）。通过与特异性靶向该癌基因的反义硫代磷酸寡脱氧核苷酸（[S]ODNs）联合治疗，可以在体外实现这些细胞的选择性根除（bcr/abl [S] ODN）和次优（用作单药）剂量的马磷酰胺（环磷酰胺的体外活性形式）。目的：我们评估了bcr/ abl反义[S] ODN单独或在单次注射环磷酰胺治疗后，为了抑制Philadelphia 1细胞在严重联合免疫缺陷（SCID）小鼠中诱导的白血病过程（即，原发性CML母细胞危象[CML-BC]细胞）。此外，我们还研究了bcr/abl反义[S]ODN-mafosfamide联合应用对Philadelphia 1细胞体外杀伤作用的可能机制。（25毫克/公斤体重; 25%的剂量）和/或bcr/abl反义[S] ODN通过存活分析进行评估，通过检查骨髓中是否存在白血病细胞（使用集落形成试验或使用偶联逆转录和聚合酶链反应筛选bcr/abl信使RNA），以及通过检查各种组织中是否存在浸润性白血病细胞。通过使用商业测定法监测在用bcr/abl反义[S]ODNs处理加上或减去先前用马磷酰胺处理之后原代CMLBC细胞中的体外细胞凋亡诱导（细胞死亡程序）。通过使用共聚焦显微镜和流式细胞术（对于荧光[S] ODN）或通过使用采用放射性标记探针的印迹技术（对于提取的未标记[S] ODN），测定体外经或未经马磷酰胺预处理的CML-BC细胞对[S] ODN的相对细胞摄取。通过使用蛋白质印迹法测定处理和未处理细胞中的特异性蛋白质水平。结果：用环磷酰胺和特异性bcr/abl反义[S]ODNs联合治疗显著延缓了白血病小鼠的疾病进程（P<0.001，相对于单独用特异性反义[S]ODNs、单独用环磷酰胺或环磷酰胺加非特异性[即，对照]反义[S]ODNs）; 50%用环磷酰胺和特异性反义[S]ODNs处理的小鼠似乎治愈了白血病。联合治疗与细胞凋亡诱导增加相关。此外，细胞摄取bcr/abl反义[S] ODN似乎增加了2倍至6倍，通过预先处理与马磷酰胺。这种增加的摄取[S]ODNs与增强抑制p210 bcr/abl蛋白levels.Conclusions和影响：联合治疗与反义[S]ODNs针对特定的癌基因和毒性较小剂量的抗癌药物可能是一个合理的策略，追求治疗人类白血病。
Background:Philadelphia1cells are human chronic myelogenous leukemia (CML) cells that contain the BCR/ABL oncogene (a fusion of the BCR and ABL genes). Selective eradication of these cells in vitro can be achieved by combined treatment with antisense phosphorothioate oligodeoxynucleotides ([S]ODNs) specifically targeted to this oncogene (bcr/abl [S]ODNs) and a suboptimal (for use as a single agent) dose of mafosfamide (the in vitro active form of cyclophosphamide).Purpose:We evaluated the ability of bcr/ abl antisense [S]ODNs, alone or subsequent to treatment with a single injection of cyclophosphamide, to suppress the leukemic process induced in severe combined immunodeficient (SCID) mice by Philadelphia1cells (i.e., primary CMLblast crisis [CML-BC] cells). In addition, we studied potential mechanisms that might explain the efficacy of the bcr/abl antisense [S]ODN-mafosfamide combination against Philadelphia1cells in vitro.Methods:The effects of treating leukemic mice with cyclophosphamide (25 mg/kg body weight; 25% of the dose required to eradicate evidence of leukemia in SCID mice) and/or bcr/abl antisense [S]ODNs were assessed by analysis of survival, by examination of bone marrow for the presence of leukemia cells (using a colony formation assay or using coupled reverse transcription and the polymerase chain reaction to screen for bcr/abl messenger RNA), and by examination of a variety of tissues for the presence of infiltrating leukemia cells. The induction of apoptosis (a cell death program) in vitro in primary CMLBC cells following treatment with bcr/abl antisense [S]ODNs plus or minus prior treatment with mafosfamide was monitored by use of a commercial assay. Relative cellular uptake of [S]ODNs by CML-BC cells treated in vitro with or without prior treatment with mafosfamide was determined by use of confocal microscopy and flow cytometry (for fluorescent [S]ODNs) or by use of blotting techniques that employed radioactively labeled probes (for extracted, unlabeled [S]ODNs). Levels of specific proteins in treated and untreated cells were determined by use of western blotting methods. ReportedPvalues are two-sided.Results:The disease process in leukemic mice was retarded substantially by combination treatment with cyclophosphamide and specific bcr/abl antisense [S]ODNs (P<.001, relative to treatment with specific antisense [S]ODNs alone, cyclophosphamide alone, or cyclophosphamide plus nonspecific [i.e., control] antisense [S]ODNs); 50% of the mice treated with cyclophosphamide and specific antisense [S]ODNs appeared to be cured of leukemia. The combination treatment was associated with increased induction of apoptosis. In addition, cellular uptake of bcr/abl antisense [S]ODNs appeared to be increased twofold to sixfold by prior treatment with mafosfamide. This increased uptake of [S]ODNs was associated with enhanced suppression of p210bcr/abl protein levels.Conclusions and Implications:Combination therapy with antisense [S]ODNs targeted to specific oncogenes and less toxic doses of anticancer drugs may represent a rational strategy to pursue for the treatment of human leukemias.