DRUG RESISTANCE OF NORMAL HEMATOPOIETIC STEM CELLS

正常造血干细胞的耐药性

• 批准号: 2100259
• 负责人: JAN S MOREB
• 金额: $ 9.94万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2100259
• 关键词: aldehyde dehydrogenases; antisense nucleic acid; bone marrow; chemoprevention; cyclophosphamide; cytoplasm; drug resistance; enzyme activity; enzyme inhibitors; gene expression; hematopoietic stem cells; interleukin 1; messenger RNA; neoplastic cell; northern blottings; polymerase chain reaction; tissue /cell culture; transfection; tumor necrosis factor alpha; western blottings

The broad long-term objective of this research is to understand the mechanism of drug resistance in normal hematopoietic stem cells in order to develop new therapies aimed at increasing their resistance and thus the therapeutic index of chemotherapeutic agents used to treat cancer patients. The overall goal of the present proposal is to investigate the role of cytosolic aldehyde dehydrogenase (ALDH1) in the protection of normal hematopoietic progenitors from 4-hydroperoxycyclophosphamide (4- HC), an active derivative of cyclophosphamide. ALDH1 is the enzyme responsible for the inactivation of 4-HC. Diethylaminobenzaldehyde, an inhibitor of ALDH1, can prevent the cytoprotection induced by IL-1 and TNFalpha. The first specific aim will investigate the effect of IL-1 and TNFalpha on ALDH1 mRNA and protein in normal as well as malignant cells. Northern analysis, mapping the ALDH1 mRNA extracted from purified cells with radiolabeled RNA probes, and transcriptional run on assay will be performed. In addition, Western blotting and ALDH1 activity assay will evaluate the ALDH1 protein. Indeed, in preliminary experiments, I have shown that the ALDH1 mRNA and protein are increased in bone marrow cells after incubation with IL-1 and TNFalpha for 20hrs. The second specific aim is to study the isolated effects of ALDH1 in certain cell lines. Full-length ALDH1 cDNA will be synthesized by reverse transcriptase PCR, incorporated in an appropriate retroviral vector and used to infect candidate cell lines to study its effect on their resistance to 4-HC. The third specific aim is to determine the effect of overexpression of ALDH1 in normal hematopoietic progenitors in terms of their in vitro resistance to 4-HC. In the second as well as the third specific aim, the cloning of ALDH1 cDNA into the retroviral vector will be in the orientation to act as a sense or antisense to ALDH1 which will result in the increase or decrease of the ALDH1 protein, respectively, in the transduced cells. These studies will allow me to verify the exact role of ALDH1 in the protection against 4-HC toxicity in normal and malignant cells. It will also give insight into the mechanisms of drug resistance in normal hematopoietic progenitors and whether its amplification may give a therapeutic advantage for the normal progenitors over some malignant cells. The result of these experiments may lead to future studies that will evaluate the ability of human stem cells engineered to overexpressed ALDH1 to increase the therapeutic index of cyclophosphamide in vivo.

这项研究的长期目标是了解 正常造血干细胞的耐药机制 开发新的疗法，旨在增加他们的抵抗力， 用于治疗癌症的化学治疗剂的治疗指数 患者 本提案的总体目标是调查 细胞质醛脱氢酶（ALDH 1）在保护 正常造血祖细胞从4-氢过氧环磷酰胺（4- HC），环磷酰胺的活性衍生物。 ALDH1是一种酶， 负责4-HC的失活。 二乙氨基苯甲醛 ALDH 1抑制剂，可阻止IL-1诱导的细胞保护作用， TNF α。 第一个具体目标是研究IL-1和IL-12对人乳腺癌细胞的影响。 TNF α对正常和恶性细胞中ALDH1 mRNA和蛋白的影响。 北方分析，定位从纯化细胞中提取的ALDH 1 mRNA 使用放射性标记的RNA探针，并将在检测中进行转录运行 执行。 此外，蛋白质印迹和ALDH 1活性测定将 检测ALDH1蛋白。 事实上，在初步实验中，我 显示ALDH 1 mRNA和蛋白在骨髓细胞中增加， 用IL-1和TNF α孵育20小时后。 第二特定 目的是研究ALDH 1在某些细胞系中的分离作用。 全长ALDH 1 cDNA将通过逆转录酶PCR合成， 将其掺入合适的逆转录病毒载体中并用于感染 候选细胞系，以研究其对4-HC抗性的影响。 第三个具体目的是确定过表达的影响， ALDH 1在正常造血祖细胞中的体外表达 对4-HC的耐受性。 在第二和第三个具体目标中， 将ALDH 1 cDNA克隆入逆转录病毒载体将在 作为ALDH 1的有义或反义，这将导致 ALDH1蛋白的增加或减少，分别在 转导的细胞。 这些研究将使我能够证实 ALDH 1在正常和恶性肿瘤中对4-HC毒性的保护作用 细胞 它还将使人们深入了解耐药性的机制 在正常造血祖细胞中，其扩增是否可以 给予正常祖细胞治疗优势， 恶性细胞 这些实验的结果可能会导致未来 这些研究将评估人类干细胞的能力， 过表达ALDH1以增加环磷酰胺的治疗指数 in vivo.