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.
Showing the most recent 10 out of 15 publications
