Hanania E G, Fu S, Zu Z, Hegewisch-Becker S, Korbling M, Hester J, Durett A, Andreeff M, Mechetner E, Holzmayer T
Department of Hematology, University of Texas MD Anderson Cancer Center, Houston 77030, USA.
Gene Ther. 1995 Jun;2(4):285-94.
A retrovirus containing the multiple drug resistance (MDR-1) cDNA, was used to transduce cultures of CD34 selected human marrow cells, on stromal monolayers in the presence of hematopoietic growth factors IL-3 and IL-6, following collection from patients recently recovered from chemotherapy-induced myelosuppression. In one experiment, these CD34 selected cells were grown in Dexter cultures for 35 days or more following MDR-1 transduction, and then plated in methylcellulose. Polymerase chain reaction (PCR) analysis of colonies picked after 10-14 days of methylcellulose culture, using a set of primers that are specific for the endogenous or the retrovirally transduced MDR-1, showed that the long-term culture initiating cells (LTCICs) were transduced by the MDR-1 virus. Analysis of the colonies from the CD34 selected MDR-1 transduced cells, with a reverse transcription (RT) PCR assay that could distinguish viral MDR-1 mRNA from endogenous MDR-1 mRNA, showed that the viral MDR-1 mRNA levels were much higher than that of the MDR-1 mRNA from the endogenous MDR-1 gene in the transduced CD34 selected cells. Fluorescence activated cell sorting (FACS) analysis of the CD34 selected transduced marrow cells within 48 h after the transduction, using the C219 and UIC2 monoclonal antibodies for p-glycoprotein, showed that the transduction frequency under these conditions varied from 7 to 20%. Rhodamine efflux studies showed that this additional p-glycoprotein was functional and that the frequency of cells with high p-glycoprotein levels was higher in the transduced cells than in the non-transduced cells. The resistance to taxol of the CD34 selected transduced cells, as judged by the plating efficiency of clonogenic progenitor cells derived from these cells by growth in methylcellulose supplemented with taxol was much higher in the transduced cells than in untransduced cells. In order to test the reproducibility of the transduction frequency of the retroviral supernatants from PA317 MDR-1 viral producer cells on CD34 selected cells, the virus produced from 12 different lots of supernatants from the PA317 MDR-1 producer cell line was used to transduce CD34 selected marrow cells from four different patients, and to transduce the peripheral blood cells of two additional patients collected following chemotherapy-induced myelosuppression. The supernatant lots used for these transduction experiments were tested by Microbiological Associates (Rockville, MD, USA), by the Mus dunni co-cultivation and amplification tests in the S+L-assay and found to be negative for replication-competent retrovirus, and later approved for human use by the Food and Drug Administration.(ABSTRACT TRUNCATED AT 400 WORDS)
从近期化疗诱导骨髓抑制恢复的患者采集细胞后，在造血生长因子IL - 3和IL - 6存在的情况下，使用含有多药耐药（MDR - 1）cDNA的逆转录病毒转导在基质单层上的CD34分选的人骨髓细胞培养物。在一项实验中，这些CD34分选的细胞在MDR - 1转导后在德克斯特培养物中培养35天或更长时间，然后接种于甲基纤维素中。使用一组对内源性或逆转录病毒转导的MDR - 1特异的引物，对甲基纤维素培养10 - 14天后挑选的集落进行聚合酶链反应（PCR）分析，结果显示长期培养起始细胞（LTCICs）被MDR - 1病毒转导。用能区分病毒MDR - 1 mRNA和内源性MDR - 1 mRNA的逆转录（RT）PCR检测法分析CD34分选的MDR - 1转导细胞的集落，结果显示在转导的CD34分选细胞中，病毒MDR - 1 mRNA水平远高于内源性MDR - 1基因的MDR - 1 mRNA水平。转导后48小时内，使用针对P - 糖蛋白的C219和UIC2单克隆抗体，对CD34分选的转导骨髓细胞进行荧光激活细胞分选（FACS）分析，结果显示在这些条件下转导频率为7%至20%。罗丹明外排研究表明，这种额外的P - 糖蛋白具有功能，且转导细胞中P - 糖蛋白水平高的细胞频率高于未转导细胞。通过在补充有紫杉醇的甲基纤维素中生长，从这些细胞衍生的克隆形成祖细胞的接种效率来判断，CD34分选的转导细胞对紫杉醇的抗性在转导细胞中比未转导细胞高得多。为了测试PA317 MDR - 1病毒生产细胞的逆转录病毒上清液对CD34分选细胞的转导频率的可重复性。使用从PA317 MDR - 1生产细胞系的12个不同批次上清液产生的病毒转导来自4名不同患者的CD34分选骨髓细胞，以及转导另外2名化疗诱导骨髓抑制后采集的患者的外周血细胞。用于这些转导实验的上清液批次由美国马里兰州罗克维尔的微生物学协会通过S + L检测中的Mus dunni共培养和扩增试验进行检测，发现对具有复制能力的逆转录病毒呈阴性，随后被美国食品药品监督管理局批准用于人体。（摘要截断于400字）
