MOLECULAR PATHOLOGY OF ACUTE PROMYELOCYTIC LEUKEMIA

描述：(改编自调查人员的摘要)非随机 染色体易位在骨肉瘤的发病机制中起重要作用 急性白血病。然而，其发病的分子机制是 相对不为人所知。急性胰腺炎的分子病理学研究 早幼粒细胞白血病(APL)强烈支持 融合蛋白PML-RARpha，编码自t(15；17)，在 APL的发展。根据调查人员的发现，PML 是一种生长抑制因子，从其他人那里报告的结果是APL的模型 对其发病机制进行了探讨。在该模型中，PML-RARpha扮演着核心角色 作为PML和RXR的显性负向抑制因子。自动减支 这两种蛋白质中的一种可以促进生长和分化 阻滞于早幼粒细胞阶段，导致APL发病。这 模型代表了第一个强调显性的 负抑制物在急性白血病发生发展中的作用。这个 研究人员发现PML是一种生长抑制因子可能有助于 更好地了解APL的发病机制。关于生物多样性的研究 PML的功能将为进一步了解PML提供关键信息 APL.这项建议的两个主要目标是：(1)澄清 APL发病的分子机制。实验的目的是为了 使用显性负抑制物支持所提出的模型 PML、RARpha(或RXR)和由诱导物驱动的突变体PML-RARpha 推动者。将建立这些突变体的稳定转基因株以 探讨它们对人白血病细胞生长和分化的影响 细胞和原代成纤维细胞。RXR和RXR基因过表达对细胞生长的影响 APL来源NB4细胞中PML对克隆形成、分化和分化的影响 将对增长情况进行调查。PML、RARpha基因的显性负性突变体 这项研究的实验室中提供了细胞系。(2)至 研究PML的生物学功能。结果显示了最高的数字 在G1期出现胞核弥漫型；在S期出现核弥漫型PML 与豆荚的减少相吻合；豆荚的显著增加 在伽马射线照射后不久，酪氨酸和丝氨酸 PML的残基被磷酸化。这些发现表明， PML在细胞周期进程中的修饰可能对 它的生物功能。与会者提议调查这一角色 磷酸化对PML生物学功能的影响。站点定向 将进行诱变以鉴定和改变磷酸氨基 酸转变为非磷酸化形式。它们在NIH/3T3中形成豆荚的能力 细胞以及neu抑制转化的作用将被研究。会的 关于PML是否被细胞周期磷酸化的研究 相关的激酶。已经创建了PML缺失突变体，我们发现 PML形成POD的能力是其转化的关键 抑制器功能。调查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 PML在NB4细胞中的转染体将被用于研究PML 诱导分化、凋亡或细胞周期停滞。它对中国的影响 致瘤性和克隆性也将被调查。这个 研究人员发现，PML可提高辐射后细胞的存活率 ，并将调查PML是否影响细胞周期分布 并抑制这些细胞的凋亡。最后，身份识别和 ~(32)P标记PML探针鉴定PML相关蛋白 并由酵母双杂交系统进行。

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.