Quan S, Feldman E, Yang L, Wagener F A, Farley T J, Abraham N G, Ahmed T
Department of Pharmacology, Saint Vincent Hospital, New York Medical College, Valhalla 10595, USA.
J Hematother Stem Cell Res. 1999 Oct;8(5):491-502. doi: 10.1089/152581699319948.
Human interferon-alpha (IFN-alpha) has been used in the management of leukemia, but its diverse adverse effects may influence the ability of IFN-alpha to treat this disease. We constructed two retroviral vectors, LSN-IFN-alpha and LNC-IFN-alpha, in which IFN-alpha cDNA was driven by viral LTR and CMV promoters, respectively. After transduction into the PA317 and PG13 retroviral packaging cells, high titers of retrovirus were produced and were used to infect K562 and human BM CD34+ hematopoietic cells. The IFN-alpha gene expression in transduced K562 cells was confirmed by Northern blot, RT-PCR, RIA, and biologic assay. Cell proliferation and cell viability in IFN-alpha-transduced K562 cells were significantly suppressed as compared with control K562 cells. Although the IFN-alpha expression in K562 cells did not affect BCR/ABL expression, it apparently upregulated the production of adhesion molecules (VLA-4 and Mac-1). We evaluated the effect of IFN-alpha gene transfer on human CD34+ cells infected with LSN-IFN-alpha retrovirus with the aid of fibronectin (FN) fragment CH-296 and growth factors. RIA showed that IFN-alpha-transduced CD34+ cells produced 72.2+/-15 U/ml of IFN-alpha compared with 4.3+/-1.2 U/ml in control CD34+ cells. Methylcellulose clonogenic assay indicated that IFN-alpha-transduced CD34+ cells produced similar numbers of burst-forming units-erythrocytes (BFU-E)/colony-forming units-GM (CFU-GM) colonies as compared with control CD34+ cells. Selected colonies expressed IFN-alpha and neo(r) mRNA, as measured by RT-PCR. These studies indicate that retrovirus-mediated IFN-alpha gene transfer may provide a useful tool for studying the effect of IFN-alpha gene transfer on leukemic cells and long-lived CD34+ cells.
人α干扰素（IFN-α）已被用于白血病的治疗，但其多种不良反应可能会影响IFN-α治疗该疾病的能力。我们构建了两种逆转录病毒载体，LSN-IFN-α和LNC-IFN-α，其中IFN-α cDNA分别由病毒LTR和CMV启动子驱动。转导到PA317和PG13逆转录病毒包装细胞后，产生了高滴度的逆转录病毒，并用于感染K562和人骨髓CD34+造血细胞。通过Northern印迹、RT-PCR、放射免疫分析和生物学测定证实了转导的K562细胞中IFN-α基因的表达。与对照K562细胞相比，IFN-α转导的K562细胞中的细胞增殖和细胞活力受到显著抑制。虽然K562细胞中的IFN-α表达不影响BCR/ABL表达，但它明显上调了黏附分子（VLA-4和Mac-1）的产生。我们借助纤连蛋白（FN）片段CH-296和生长因子评估了IFN-α基因转移对感染LSN-IFN-α逆转录病毒的人CD34+细胞的影响。放射免疫分析显示，IFN-α转导的CD34+细胞产生72.2±15 U/ml的IFN-α，而对照CD34+细胞为4.3±1.2 U/ml。甲基纤维素克隆形成试验表明，与对照CD34+细胞相比，IFN-α转导的CD34+细胞产生的爆式红系集落形成单位（BFU-E）/粒-巨噬系集落形成单位（CFU-GM）集落数量相似。通过RT-PCR检测，所选集落表达IFN-α和新霉素抗性（neo(r)）mRNA。这些研究表明，逆转录病毒介导的IFN-α基因转移可能为研究IFN-α基因转移对白血病细胞和长寿CD34+细胞的影响提供一种有用的工具。
