MOLECULAR PATHOLOGY OF ACUTE PROMYELOCYTIC LEUKEMIA

急性早幼粒细胞白血病的分子病理学

• 批准号: 2330801
• 负责人: KUN-SANG CHANG
• 金额: $ 17.5万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330801
• 关键词: 3T3 cells; acute myelogenous leukemia; apoptosis; cell differentiation; cell growth regulation; chimeric proteins; chromosome translocation; disease /disorder model; genetic promoter element; growth factor receptors; growth inhibitors; human tissue; molecular oncology; molecular pathology; mutant; neoplastic cell; phosphorylation; site directed mutagenesis; tissue /cell culture; transfection

DESCRIPTION: (adapted from the investigator's abstract) Nonrandom chromosomal translocation plays a major role in the pathogenesis of acute leukemia. However, the molecular mechanism of pathogenesis is relatively unknown. Studies on the molecular pathology of acute promyelocytic leukemia (APL) strongly support the importance of the fusion protein PML-RARalpha, encoded from the t(15;17), in the development of APL. Based on the finding by the investigators that PML is a growth suppressor and results reported from others, a model of APL pathogenesis was proposed. In this model, PML-RARalpha plays a central role as a dominant negative inhibitor against PML and RXR. Sequestration of these two proteins results in growth stimulation and differentiation block at the promyelocyte stage which leads to APL pathogenesis. This model represents the first to emphasize the importance of a dominant negative inhibitor in the development of acute leukemia. The investigator's finding that PML is a growth suppressor may contribute to a better understanding of APL pathogenesis. Studies on the biologic function of PML will provide critical information to further understand APL. The two major goals of this proposal are: (1) to elucidate the molecular mechanism of APL pathogenesis. Experiments are designed to support the proposed model using dominant negative inhibitors against PML, RARalpha (or RXR), and a mutant PML-RARalpha driven by an inducible promoter. Stable transfectants of these mutants will be established to investigate their effect on growth and differentiation of human leukemia cells and primary fibroblasts. The effect of over-expression of RXR and PML in the APL-derived NB4 cells on clonogenicity, differentiation and growth will be investigated. Dominant negative mutants of PML, RARalpha and cell lines are available in the laboratory for this study. (2) To study the biologic function of PML. Results demonstrated a highest number of PODs at the G1 phase; a nuclear diffused PML pattern at the S phase coincided with a decreased in PODs; a significant increased in PODs shortly after gamma-irradiation and that both tyrosine and serine residues of PML are phosphorylated. These findings suggest that modification of PML during cell cycle progression may be important for its biologic function. The participants propose to investigate the role of phosphorylation on the biologic function of PML. Site directed mutagenesis will be performed to identify and to alter the phosphoamino acid to a nonphosphorylated form. Their ability to form PODs in NIH/3T3 cells and to suppress transformation by neu will be investigated. It will be investigated as to whether PML is phosphorylated by a cell cycle related kinase. PML deletion mutants have been created, we found that the ability of PML to form POD is essential for its transformation suppressor function. The investigators will continue to study their effect on suppressing transcription activity of EGFR promoter. Stable transfectants of PML in NB4 cells will be used to investigate whether PML induces differentiation, apoptosis, or cell cycle arrest. Its effect on tumorigenicity and clonogenicity will also be investigated. The investigators have found that PML enhances cell survival after radiation exposure, and will investigate whether PML affect cell cycle distribution and inhibits apoptosis in these cells. Finally, identification and characterization of the PML associated proteins by 32P-labelled PML probe and by the yeast two-hybrid system will carried out.

描述：（改编自研究者摘要）非随机 染色体易位在遗传病的发病机制中起主要作用。 急性白血病 然而，发病的分子机制是 相对未知。 急性脑梗死的分子病理学研究 早幼粒细胞白血病（APL）强烈支持的重要性 融合蛋白PML-RAR α，由t（15;17）编码，在 APL的发展。根据研究者的发现，PML 是一种生长抑制因子，其他人报道的结果是APL的模型 提出了发病机理。在这个模型中，PML-RAR α起着核心作用。 作为PML和RXR的显性负性抑制剂。 封存 这两种蛋白质的结合导致生长刺激和分化 早幼粒细胞期阻滞导致APL发病。这 模型代表了第一个强调占主导地位的重要性， 在急性白血病的发展中起负性抑制作用。的 研究人员发现PML是一种生长抑制因子，这可能有助于 更好地了解APL的发病机制。生物学研究 PML的功能将提供关键信息，以进一步了解 APL。这一建议的两个主要目标是：（1）阐明 APL发病的分子机制。实验旨在 支持所提出的模型，使用显性负抑制剂， PML、RAR α（或RXR）和由诱导型 启动子将建立这些突变体的稳定转染子， 观察它们对人白血病细胞生长和分化影响 细胞和原代成纤维细胞。RXR过表达的影响， APL衍生的NB 4细胞中的PML对克隆形成、分化和 将对增长进行研究。PML、RAR α显性阴性突变体 并且细胞系在实验室中可用于本研究。(2)到 研究PML的生物学功能。结果显示， 在G1期出现核扩散的PML模式， 与POD减少一致; POD显著增加 在γ射线照射后不久，酪氨酸和丝氨酸 PML的残基被磷酸化。这些发现表明 在细胞周期进展过程中PML的修饰可能对 其生物学功能。与会者建议调查的作用 对PML生物学功能的影响。定点 将进行诱变以鉴定和改变磷酸氨基 酸转化为非磷酸化形式。它们在NIH/3 T3中形成POD的能力 细胞并抑制由neu引起的转化。它将 研究PML是否被细胞周期磷酸化 相关激酶PML缺失突变体已经产生，我们发现， PML形成POD能力是其转化所必需的 抑制功能研究人员将继续研究他们的 抑制EGFR启动子转录活性的作用。稳定 在NB 4细胞中的PML的转染子将用于研究PML是否 诱导分化、凋亡或细胞周期停滞。效应 还将研究致瘤性和克隆形成性。的 研究人员发现，PML可增强辐射后的细胞存活率， 暴露，并将研究PML是否影响细胞周期分布 并抑制这些细胞的凋亡。最后，识别和 用~（32）P标记的PML探针鉴定PML相关蛋白 并通过酵母双杂交系统进行。