REGULATION OF ALV EXPRESSION AND PATHOGENESIS

已经开发了一个模型系统来研究逆转录病毒肿瘤发生， 调节肿瘤诱导易感性的遗传因素。 禽 白血病病毒（ALV）整合后可诱发鸡法氏囊淋巴瘤 的病毒长末端重复序列（LTR）序列旁边的c-myc原， 癌基因 不稳定或寿命短的蛋白质似乎调节c-myc 高表达，因为LTR增强的转录是特异性的， 蛋白质合成抑制后下降。 观察到这种不稳定性 仅在前B细胞类型中，这表明它在B细胞中可能是重要的 ALV肿瘤诱导的特异性。 此外，抗淋巴瘤鸡 菌株表现出稳定的LTR增强的转录（不受抑制的影响 蛋白质合成），这表明LTR增强c- myc基因转录可能在淋巴瘤易感性中起重要作用。 5个LTR 来自囊淋巴瘤细胞的结合蛋白已经用 凝胶位移测定。 前B细胞中有三种蛋白质是特别不稳定的 （蛋白质合成抑制后结合减少），因此可能是 对于LTR增强至关重要。 体内足迹技术将是 用于确定体内是否也发生不稳定蛋白质结合。 的 每个蛋白结合序列对LTR增强的贡献将是 通过转染野生型或突变型LTR报告基因进行分析 构建成囊淋巴瘤细胞。 编码主要不稳定蛋白质的基因正在被克隆， 测序，以获得DNA和抗体探针，用于分析作用， 这些蛋白质在淋巴瘤发生中的作用。 不稳定的蛋白质表达将是 在不同的细胞类型中进行比较，以确定为什么 在前B细胞中不稳定。 Western和北方印迹分析将确定 如果这种不稳定性是由蛋白质半衰期的变化或后 翻译修饰如磷酸化。 结合蛋白 在B细胞发育期间，通过蛋白质印迹法分析表达 和免疫荧光染色，以确定蛋白质是否不稳定， ALV靶向前B细胞，在成熟B细胞中稳定，不再敏感 淋巴瘤 蛋白表达将在淋巴瘤易感的 和耐药菌株，以确定如何稳定表达可以 参与抵抗淋巴瘤。 这些研究将使人们了解 肿瘤诱导过程中c-myc高表达的调节， LTR结合蛋白在肿瘤易感性中的作用。 的理解 ALV淋巴瘤易感性的机制可应用于研究 癌基因激活和淋巴瘤的发生。

A model system has been developed to study retroviral oncogenesis, and the genetic factors regulating susceptibility to tumor induction. Avian leukosis virus (ALV) induces bursal lymphoma in chickens, after integration of viral long terminal repeat (LTR) sequences next to the c-myc proto- oncogene. Labile or short-lived proteins appear to regulate c-myc hyperexpression, because LTR-enhanced transcription is specifically decreased after inhibition of protein synthesis. This lability is observed only in pre-B cell types, suggesting that it may be important in the B cell specificity of ALV tumor induction. Moreover, lymphoma-resistant chicken strains exhibit stable LTR-enhanced transcription (unaffected by inhibition of protein synthesis), suggesting that labile regulation of LTR-enhanced c- myc transcription may be important in lymphoma susceptibility. Five LTR binding proteins from bursal lymphoma cells have been characterized using the gel shift assay. Three proteins are specifically labile in pre-B cells (decreased binding after protein synthesis inhibition) and thus may be essential for LTR enhancement. An in vivo footprinting technique will be used to determine if labile protein binding also occurs in vivo. The contribution of each protein binding sequence to LTR enhancement will be analyzed by transfection of wild type or mutant LTR-reporter gene constructs into bursal lymphoma cells. The genes encoding the major labile proteins are being cloned and sequenced, to obtain DNA and antibody probes for analysis of the role of these proteins in lymphomagenesis. Labile protein expression will be compared in different cell types, to determine why it is specifically labile in pre-B cells. Western and Northern blot analyses will determine if this lability is regulated by changes in protein half life or by post- translational modifications such as phosphorylation. Binding protein expression will be analyzed during B cell development, by Western blotting and immunofluorescent staining, to determine if the proteins are labile in ALV target pre-B cells, and stable in mature B cells no longer susceptible to lymphoma. Protein expression will be compared in lymphoma-susceptible and -resistant strains, to determine how stable expression could be involved in resistance to lymphoma. These studies will give insight to the regulation of c-myc hyperexpression during tumor induction, and the role of the labile LTR binding proteins in tumor susceptibility. An understanding of the mechanism of ALV lymphoma susceptibility can be applied to studies of oncogene activation and lymphomagenesis in humans.