MOLECULAR PATHOLOGY OF ACUTE PROMYELOCYTIC LEUKEMIA

非随机染色体易位t(15；17)(q22；q21)是一致的 急性早幼粒细胞白血病的特点。我们和其他人已经展示了 维甲酸受体α(RARA)基因参与了 断点。我们已经克隆并鉴定了融合基因的cdna。 转录本RARA/MYL、MYL/RARA和正常的MYL基因。我们的结果 表明两个融合转录本都能翻译成 融合蛋白。利用这些DNA序列信息，我们能够 用聚合酶链式反应分析8个APL基因的断裂点。我们发现 断点站点聚集在两个不同的 MYL基因。对这些断裂点的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。由于RARA是转录调节因子，推测的 融合蛋白RARA/MYL和MYL/RARA可能具有潜在的致癌作用。 MYL的氨基酸序列分析揭示了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。它是 MYL可能是一个新的转录因子。在本建议书中 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中的T(15；17)断裂点转录融合转录本Rara/MYL和 MYL/RARA是一种致癌基因，与卵巢癌的发病机制有关 APL.逆转录病毒介导的基因转移技术将用于 介绍重组逆转录病毒载体pGDRARA/MYL和 PGDmyl/RARA基因导入人骨髓细胞培养。对经济的影响 这些融合转录本在克隆形成、分化过程中的表达 并将研究其致瘤性。我们会进一步研究 重组病毒移植致急性早幼粒细胞白血病的实验研究 在动物模型中感染了骨髓。假设B：下行调节 由于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 这个假说。(1)针对RARA的反义寡核苷酸 MRNA将被用来抑制RARA在骨髓中的翻译 文化。抑制RARA翻译的作用 克隆繁殖和分化将被研究。(2)研究 RARA和MYL基因在APL细胞中表达上调的作用 细胞系NB4，通过基因转染法进行诱导分化。我们相信 这些研究将有助于理解人类免疫缺陷的分子机制。 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.