A hypothesis was formed that it would be possible to isolate an adequate amount of protein from a patient, having normal renal function, to identify biological markers of a particular disease state using a variety of proteomics techniques. To support this hypothesis, three samples of urine were collected from a volunteer: first when healthy, later when experiencing acute inflammation due to a pilonidal abcess, and again later still after successful recovery from the condition. The urine from these samples was processed by solid-phase extraction to concentrate and desalt the endogenous proteins and peptides. The proteins and peptides from these urine samples were analyzed in three different experiments: (1) traditional two-dimensional gel electrophoresis followed by proteolysis and mass spectrometric identification of various protein spots, (2) whole mixture proteolysis followed by one-dimensional packed capillary liquid chromatography and tandem mass spectrometry, (3) whole mixture proteolysis followed by two-dimensional capillary liquid chromatography and tandem mass spectrometry. In all three cases, a set of proteins was identified representing putative biomarkers. Each of these proteins was then found to have been previously linked in the scientific literature to inflammation. One acute phase reactant in particular, orosomucoid, was readily observed in all three experiments to dramatically increase in abundance, thereby supporting the hypothesis.

Purpose: For many tumor cells, de novo lipogenesis is a requirement for growth and survival.A considerable body of work suggests that inhibition of this pathway may be a powerful approach to antineoplastic therapy. It has recently been shown that inhibition of various steps in the lipogenic pathway individually can induce apoptosis or loss of viability in tumor cells. However, it is not clear whether quantitative differences exist in the ability of lipogenic enzymes to control tumor cell survival. We present a systematic approach that allows for a direct comparison of the control of lipogenic pathway enzymes over tumor cell growth and apoptosis using different cancer cells. Experimental Design: RNA interference-mediated, graded down-regulation of fatty acid synthase (FAS) pathway enzymes was employed in combination with measurements of lipogenesis, apoptosis, and cell growth. Results: In applying RNA interference titrations to two lipogenic enzymes, acetyl-CoA carboxylase 1 (ACC1) and FAS, we show that ACC1and FAS both significantly control cell growth and apoptosis in HCT-116 cells. These results also extend to PC-3 and A2780 cancer cells. Conclusions: Control of tumor cell survival by different steps in de novo lipogenesis can be quantified. Because ACC1 and FAS both significantly control tumor cell growth and apoptosis, we propose that pharmacologic inhibitors of either enzyme might be useful agents in targeting cancer cells that critically rely on fatty acid synthesis. The experimental approach described here may be extended to other targets or disease-relevant pathways to identify steps suitable for therapeutic intervention.

Exposure of LLC‐PK1 epithelial cell cultures to phorbol ester tumor promoters causes immediate translocation of protein kinase C‐α (PKC‐α) from cytosolic to membrane‐associated compartments. With a very similar time course, a dramatic and sustained increase in tight junctional (paracellular) permeability occurs. This increased permeability extends not only to salts and sugars but macromolecules as well. Fortyfold increases of transepithelial fluxes of biologically active EGF and insulin occur. Recovery of tight junction barrier function coincides with proteasomal downregulation of PKC‐α. The failure to downregulate activated membrane‐associated PKC‐α has correlated with the appearance of multilayered cell growth and persistent leakiness of tight junctions. Accelerated downregulation of PKC‐α results in only a partial and transient increase in tight junction permeability. Transfection of a dominant/ negative PKC‐α results in a slower increase in tight junction permeability in response to phorbol esters. In a separate study using rat colon, dimethylhydrazine (DMH)‐induced colon carcinogenesis has been preceded by linear increases in both the number of aberrant crypts and transepithelial permeability, as a function of weeks of DMH treatment. Adenocarcinomas of both rat and human colon have been found to have uniformly leaky tight junctions. Whereas most human colon hyperplastic and adenomatous polyps contain nonleaky tight junctions, adenomatous polyps with dysplastic changes did possess leaky tight junctions. Our overall hypothesis is that tight junctional leakiness is a late event in epithelial carcinogenesis but will allow for growth factors in luminal fluid compartments to enter the intercellular and interstitial fluid spaces for the first time, binding to receptors that are located on only the basal‐lateral cell surface, and causing changes in epithelial cell kinetics. Tight junctional leakiness is therefore a promotional event that would be unique to epithelial cancers.

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