MOLECULAR PATHOLOGY OF ACUTE PROMYELOCYTIC LEUKEMIA

非随机染色体易位t（15;17）（q22; q2 l）与染色体易位的发生有一致性。 急性早幼粒细胞白血病（APL） 我们和其他人已经证明 维甲酸受体α（RARA）基因参与了 断点。 我们克隆了融合蛋白的cDNA并进行了鉴定 转录本RARA/myl、myl/RARA和正常myl cDNA。 我们的结果 表明这两种融合转录本都能够翻译成 融合蛋白 利用这些DNA序列信息，我们能够 PCR扩增分析8个APL的断裂点。 我们发现 断裂点聚集在两个不同的内含子内， myl基因。 这些断裂点的DNA序列分析表明， 所有APL的融合转录本都能够利用正确的 阅读框架。 由于RARA是一种转录调节因子， 融合蛋白RARA/myl和myl/RARA可能是潜在致癌的。 氨基酸序列分析揭示了MYL的一个富含半胱氨酸的结构域 类似于在一个新的DNA结合蛋白家族中发现的那些。 是 myl可能是一种新的转录因子。 本提案中 我们计划研究t（15;17）易位断裂点在 APL的发病机制。 提出了两个假设。假设A： APL转录融合转录物RARA/myl中的t（15;17）断点和 myl/RARA是致癌的，并负责发病机制， APL。 逆转录病毒介导的基因转移技术将用于 引入重组逆转录病毒构建体pGDRARA/myl， 将pGDmyl/RARA导入人骨髓培养物中。 的效果 这些融合转录本的表达对克隆形成、分化 和致瘤性进行研究。 我们会进一步研究 重组病毒移植治疗急性早幼粒细胞白血病 在动物模型中感染骨髓。 假设B：下调 t（15;17）易位导致的RARA，负责 APL的发病机制。 将设计两个不同的实验来研究 这个假设。(1)抗RARA的反义寡核苷酸 mRNA将用于抑制骨髓中RARA的翻译 文化 对RARA翻译抑制的影响 将研究克隆形成和分化。(2)研究 APL细胞中RARA和myl基因表达增加的作用 细胞株NB 4，通过基因转染，诱导分化。 我们认为 这些研究应该有助于理解的分子机制， APL的发病机制。

Non-random chromosomal translocation t(15;17)(q22;q2l) is a consistent feature of acute promyelocytic leukemia (APL). We and others have shown that the retinoic acid receptor alpha (RARA) gene is involved in the breakpoint. We have cloned and characterized the cDNA of the fusion transcript RARA/myl, myl/RARA and the normal myl cDNA. Our results indicated that both fusion transcripts are able to translate into a fusion protein. Using these DNA sequence information, we are able to analyze the breakpoint sites of eight APL by PCR amplification. We found that the breakpoint sites clustered within two different introns of the myl gene. DNA sequence analysis of these breakpoint sites demonstrated that the fusion transcripts of all APL are able to utilize the correct reading frame. Since RARA is a transcription regulator, the putative fusion proteins RARA/myl and myl/RARA may be potentially oncogenic. Amino acid sequence analysis of myl revealed a cysteine-rich domain similar to those found in a new family of DNA-binding proteins. It is possible that myl may be a novel transcription factor. In this proposal we plan to study the role of t(15;17) translocation breakpoint in the pathogenesis of APL. Two hypotheses are proposed. Hypothesis A: The t(15;17) breakpoint in APL transcribes fusion transcripts RARA/myl and myl/RARA which are oncogenic and is responsible for the pathogenesis of APL. Retroviral mediated gene transfer technique will be used to introduce the recombinant retrovirus constructs pGDRARA/myl and pGDmyl/RARA into the human bone marrow culture. The effect on the expression of these fusion transcripts on clonogenicity, differentiation and tumorigenicity will be studied. We will further study the leukemogenesis of APL by transplantation of the recombinant virus infected bone marrow in an animal model. Hypothesis B: Down regulation of the RARA as a result of t(15;17) translocation, is responsible for the pathogenesis of APL. Two different experiments will be designed to study this hypothesis. (1) Antisense oligodeoxynucleotides against the RARA mRNA will be used to inhibit the translation of RARA in bone marrow culture. The effect on the suppression of RARA translation on clonogenicity and differentiation will be studied. (2) To study the effect of increased expression of RARA and myl genes in the APL cell line, NB4, by gene transfection, on differentiation. We believe that these studies should help understand the molecular mechanism of the pathogenesis of APL.