NUTRIT DETERMIN OF PROTEIN METABOLISM IN TUMOR & HOST

最初的拨款题为《蛋白质代谢的营养决定因素》 《肿瘤与宿主》的重点是验证体内的同位素模型 同时测量组织蛋白质合成和降解率。 这项工作意义重大，因为它提供了量化和机械化的 研究影响蛋白质代谢的营养变量的方法 肿瘤及其宿主。在前两年的资助中，我们有 证明了这种方法在生长组织以及 在接近稳定状态的组织中。为了验证这种方法，我们已经开始 应用流式细胞术联合溴脱氧尿嘧啶核苷(BrdUrd) 标记以获得对肿瘤细胞周期动力学的估计。使用这个 方法联合14C-亮氨酸静脉滴注治疗肿瘤坏死因子 荷瘤大鼠模型显示细胞裂解与 并提高肿瘤蛋白降解率。我们的工作是第一次 肿瘤坏死因子抗肿瘤作用机制的体内实验研究。认识 蛋白质降解过程的重要性，我们建议 通过开发细胞分选技术的应用来继续这项工作 以确定特定周期的细胞内蛋白质降解。我们会 并利用多参数流式细胞仪检测相关指标 细胞DNA、RNA和蛋白质。这些参数的比率，确定 在每个细胞中，将进一步表征蛋白质的调节 肿瘤组织中的代谢。这些研究将澄清生理上的 蛋白质降解与肿瘤生长和CALL的关系及其意义 循环动力学。 多参数流式细胞术在肿瘤活体分选中的应用 细胞，结合体内BrdUrd脉冲标记和14C-亮氨酸 输注，将决定肿瘤坏死因子对蛋白质作用的周期特异性 生物降解及其生理意义的关系 对肿瘤坏死因子的抗肿瘤作用。 在资助的头两年中完成的其他工作表明 鱼油喂饲对肿瘤生长抑制的显著作用 与蛋白质合成减少有关。我们还表明， 膳食脂肪对荷瘤大鼠肿瘤坏死因子的调节作用 鱼油；在这些饮食条件下，肿瘤坏死因子的主要作用是 减少了肿瘤蛋白质的合成，而不是促进了降解。 上述方法也适用于确定机制 鱼油对肿瘤生长的抑制作用。我们提议的续建计划 我将检验提出的三个替代假说，以解释 喂鱼油。而肿瘤蛋白质合成的减少可能是 鱼油浸泡过程中肿瘤细胞增殖能力下降 喂食，拟议的实验将提供一个更明确的答案，因为 关于膳食脂肪对细胞周期动力学(增殖)的影响，宿主 天然细胞毒性(巨噬细胞活性)和肿瘤细胞敏感性 这一宿主防御机制。

The original grant titled "Nutritional Determinant of Protein Metabolism in Tumor and Host" has focused on validating an in vivo isotope model for simultaneous measurement of tissue protein synthesis and degradation rates. This work is significant in that it provides quantitative and mechanistic means to study nutritional variables affecting protein metabolism in the tumor and its host. In the first two years of funding, we have demonstrated the applicability of this method in growing tissues as well as in tissues at near-steady state. To validate this method, we have begun to apply flow cytometry in conjunction with bromodeoxyuridine (BrdUrd) labelling to derive estimates of tumor cell-cycle kinetics. Use of this method, together with 14C-leucine infusion, in the TNF-treated tumor-bearing rat model has demonstrated a correlation between cell lysis and enhanced tumor protein degradation rate. Our work represents the first in vivo demonstration of the antitumor mode of action of TNF. Recognizing the importance of the process of protein degradation, we propose to continue this work by developing the application of cell sorting techniques to determine cycle-specific intracellular protein degradation. We will also utilize multiparameter flow cytometric methods to measure correlated cellular DNA, RNA, and protein. Ratios of the these parameters, determined in each cell, will further characterize the regulation of protein metabolism in tumor tissue. These studies will clarify the physiological significance of protein degradation in relation to tumor growth and call cycle kinetics. Application of multiparameter flow cytometry and viable sorting of tumor cells, in conjunction with in vivo BrdUrd pulse labelling and 14C-leucine infusion, will determine the cycle-specificity of TNF action on protein degradation and the physiological significance of this process in relation to the antitumor effect of TNF. Other work completed during the first two years of funding has demonstrated a significant effect of fish-oil feeding on tumor growth retardation associated with a decrease in protein synthesis. We have also shown that dietary fat can modulate the effect of TNF in the tumor-bearing rat fed fish-oil; the predominant effect of TNF under these dietary conditions is reduced tumor protein synthesis rather than enhanced degradation. The above methodologies are also applicable for determining the mechanism of tumor growth retardation by fish-oil. Our proposed continuation plan will test three alternative hypotheses put forth to explain the effect of fish oil feeding. While the decrease in tumor protein synthesis can be related to diminished tumor cell proliferation potential during fish-oil feeding, the proposed experiments will provide a more definitive answer as to the effects of dietary fat on cell-cycle kinetics (proliferation), host natural cytotoxicity (macrophage activity), and tumor cell susceptibility to this host defense mechanism.