Identification and partial biological characterization of autocrine growth inhibitory activity in Nb2 lymphoma cell conditioned medium.

Abstract

The purpose of these studies was to determine whether lactogen-dependent Nb2-11c cells and lactogen-independent Nb2-SP cells differ with respect to morphology and autocrine growth control. To this end, the ultrastructural and surface morphology of both Nb2 cell lines was analyzed and the autocrine growth modulatory activity of Nb2 cell conditioned medium (Nb2-CM) was determined. The autocrine growth inhibitory activity of Nb2-CM was biologically characterized and attempts were made to biochemically characterize and purify the Nb2 cell autocrine growth inhibitor as well as to determine its mechanism of action. Quantitative analysis of transmission electron micrographs reveals that the ultrastructural morphology of lactogen-dependent Nb2-11c cells differs from that of lactogen-independent Nb2-SP cells. Nb2-11c cells exhibit a greater incidence and volume density of nuclear pockets, whereas the incidence and volume density of lipid droplets is greater in the Nb2-SP cell line. Surface feature of Nb2-11c and Nb2-SP cells, as examined with scanning electron microscopy, and indistinguishable. Nb2-11c and Nb2-SP cells share a common mode of growth control in the form of constitutive secretion of an autocrine inhibitory factor. Medium conditioned by either Nb2-11c or Nb2-SP cells inhibits the growth of both cell lines. Nb2-CM-mediated growth inhibition is dose-dependent and reversible. Nb2-CM does not induce quiescence or cell death, but rather, causes a delay in the progression of cells through all phases of the cell cycle. Nb2 cell proliferation stimulated by a variety of mitogens is inhibited by Nb2-CM. Nb2-CM also has the ability to inhibit the growth of normal rat splenocytes as well as MCF-7 human breast cancer cells. Biochemical analysis of Nb2-CM was equivocal; however, indirect evidence suggests that the autocrine growth inhibitory factor produced by Nb2 cells may be a prostaglandin or another arachadonic acid metabolite since the growth inhibitory activity of Nb2-CM is reduced when CM is prepared in the presence of indomethacin. Interestingly, levels of prostaglandin F₁(α) are elevated in CM-treated culture supernatants. Examination of other signal transduction systems in Nb2 cells suggests that neither cAMP activation, polyamine biosynthesis, nor protein kinase C activation mediate or influence the inhibitory effect of Nb2-CM.