John F. DiStefano

The anaemia of chronic disorders: studies of iron reutilization in the anaemia of experimental malignancy and chronic inflammation.

The purpose of this study was to evaluate the contribution of defective reutilization of iron in the pathogenesis of the anaemia of chronic disease (ACD). Normal rats with or without splenectomy and rats with Walker 256 carcinosarcoma with or without splenectomy were studied. The reutilization of 59Fe labelled heat‐damaged red cells (59Fe DRBC), the sequestration of 59Fe DRBC in major organs, red cell mass, 51Cr red cell survival, serum iron, plasma erythropoietin and routine haematological parameters were measured.

Mechanism of BCG-Activated Macrophage-Induced Tumor Cell Cytotoxicity: Evidence for Both Oxygen-Dependent and Independent Mechanisms

Activated peritoneal macrophages have been shown to be cytotoxic to cancer cells. Bacillus Calmette-Guerin (BCG)-activated rat peritoneal macrophages have a basal cytolytic potential for 3H-thymidine-labelled Walker 256 cancer cells in vitro that can be markedly enhanced by digitonin. This stimulation of cytotoxicity can be partially inhibited by catalase and the combination of superoxide dismutase plus catalase. This suggests that digitonin stimulates activated macrophages to produce superoxide, hydrogen peroxide and possibly other free radicals which can augment macrophage-induced tumor cell cytotoxicity. After a 2-hour incubation with digitonin, macrophages are no longer stimulated by digitonin. However, after a 2-hour drug preincubation period, inhibitors of serine protease activity (DFP, TLCK, SBTI) and inhibitors of protein synthesis (cycloheximide) are potent inhibitors of basal macrophage-induced tumor cytotoxicity. We suggest that BCG-activated macrophages have two mechanisms for destroying cancer cells: one mediated by proteolytic activity, and a second mechanism dependent on the generation of oxygen-derived free radicals.

Characterization of Tumor Cell Membrane Serine Proteases by Polyacrylamide Gel Electrophoresis

Studies in our laboratory have indicated that tumor cell membrane-bound proteases are responsible for the ability of tumor cells to lyse normal cells in vitro. In order to evaluate the tumor cell membrane enzymes, a purified tumor cell membrane preparation was prepared and the nonionic detergent Triton X-100 was used to extract active enzymes from the cell membranes. The solubilized membrane enzymes were then studied by Triton X-100 polyacrylamide gel electrophoresis under non-denaturing conditions. Using this technique the tumor cell membranes were shown to contain esterproteases that reacted with the substrates alpha-naphthyl acetate and naphthol-AS-aminocaproate. These esterproteases were inhibited by diisopropyl fluorphosphate and tosyl lysine chloromethyl ketone but not by tosylamide phenylethyl chloromethyl ketone, soybean trypsin inhibitor p-chloromercuribenzene sulfonic acid; N-ethylmaleimide choline iodide, alpha-1-anti-trypsin. NaF, epsilon-aminocaproic acid, ethylenediamine tetraacetic acid, or eserine. SBTI affinity chromatography of the tumor cell membrane extract revealed that some of the serine esterproteases bound to the SBTI column. The proteolytic activity of the tumor cell membrane extract and a fraction eluted from the SBTI affinity column was demonstrated using casein. We conclude that the tumor cell membranes contain previously undescribed serine proteases that are identifiable by their esterase activity and inhibitor profiles in polyacrylamide gels.

Analysis of the Cell Membrane Proteolytic Enzymes of the B16, Fl, F10, and BL6 Melanoma and Their Role in Target Cell Destruction

The tumor-induced red blood cell (RBC) cytolysis assay has been used to demonstrate that three B16 melanoma sublines, the F1, F10, and BL6, cause the cytolysis of normal red blood cells in vitro. RBC cytolysis was inhibited for all three sublines by metalloprotease inhibitors. Cell membrane preparations have been prepared for all three sublines and tumor cell membrane-induced RBC cytolysis was also shown to be inhibited by metalloprotease inhibitors. The F10 and BL6 sublines were shown to have cell membrane-bound proteases. The BL6 subline has a cell membrane enriched in an enzyme with a trypsin-like arginine specificity. The trypsin-like protease may have a metal dependence. The BL6 subline has a collagenolytic cell membrane enzymes and a chymotrypsin-like cell membrane enzyme. B16 cell membrane enzymes may be responsible for RBC cytolysis in vitro in a process requiring divalent cations.

LIPOPROTEIN INHIBITOR OF BQNE MARROW CELLS IN TUMOR‐BEARING RATS

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker‐256 carcinoma (W‐256). Plasma from tumor‐bearing rats was treated by gel filtration chromatography (Sephadex G‐150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. 59Fe‐heme synthesis, [3H]thymidine DNA synthesis, and 14C‐leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (>400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipo‐protein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.