Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.     

Relationship between cell density and prolidase activity in human skin fibroblasts: effects of ascorbate and fructose

To evaluate the influence of cell density on the activity of fibroblast prolidase (EC 3.4.13.9), we determined this activity in sparse and dense cultures. We also investigated, the effects of different concentrations of β-d(?) fructose and l(+) ascorbate, which both increased cell density at confluency. For a fructose concentration of 25 mM, we observed that in the absence of glucose, intracellular total proteins increased 1.5-fold and prolidase specific activity, 1.8-fold. For ascorbate, a broad optimum concentration was found (range 0.01 – 0.50 mM). Addition to cultures of 0.1 mM ascorbate increased total proteins 1.4-fold, and doubled prolidase activity. This investigation was prompted by our previous results [J. Metab. Dis. 1983, 6, 27–31], confirmed here, and suggesting that increased prolidase activity at confluency was due to a rise in cell density.     

Molecular genetics of cell death in the nematode Caenorhabditis elegans

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insightinto the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells. © 1992 John Wiley & Sons, Inc.     

Chromatin-bound and free RNA polymerase A activities in rat thymus cells following glucocorticoid treatment

Subcellular fractions from SV-40 transformed hamster lens cells, prepared by chemical extractions, were tested for the presence of T-antigen by immunoautoradiography. Most of the T-antigen was present in the nucleus and was resistant to extraction by 2 M NaCl, indicating an association with the nuclear matrix. Another part of the T-antigen was, under certain conditions, resistant to extraction of the cells with a nonionic detergent. This T-antigen could be solubilized by Ca2+ at low temperature, conditions that also cause a specific depolymerization of microtubules.     

Ultrastructural studies of endocrine-like cells in the fundic gastric mucosa of the bullfrog,Rana catesbeiana

Summary This study is concerned with electron-microscopic observations on endocrine or paracrine cells in the fundic gastric mucosa of the bullfrog. Also, an attempt was made to identify the histamine-releasing cells involved in the secretagogue response. At least three distinct endocrine-like cell types were found. The classification is based on the appearance of secretory granules and other organelles, and the relationship of endocrine-like cells with other cells in the tissue. The amphibian endocrine-like cells resemble the ECL, D and EC cells of mammals. Type-I (ECL) cells showed degranulation after repeated stimulation with tetragastrin (TG), acetylcholine (ACh) and K⁺ depolarizing solution, all of which release histamine.     

Isolation and characterization of a serially cultivated,neoplastic, epithelial cell line from theN-nitrosomethylurea induced rat mammary adenocarcinoma

Summary A new in vitro model for human breast cancer is described. Derived from anN-nitrosomethylurea (NMU) induced rat mammary adenocarcinoma, this serially cultivated cell line has been demonstrated, by a variety of criteria, to be an authentic neoplastic, rat mammary epithelial cell line. The criteria used include morphological and growth characteristics; the presence of specific cell surface antigens; steroid hormone receptors; hormone responsiveness; casein production; karyotype and isoenzyme profile analysis; anchorage independent growth and oncogenicity. Inasmuch as the NMU cell line possesses high concentrations of glucocorticoid and androgen receptors, it may provide a useful model for study of the action of these hormones in human breast cancer. In addition, the NMU line may serve as a valuable in vitro model in which to assess the effects of a variety of endogenous and exogenous agents known to influence mammary tumor growth in vivo, including drugs, nutrients, and growth factors. This work was supported by Grants CA29602 and RR05775-05 from the National Cancer Institute, Bethesda, Maryland.     

Epidermal growth factor stimulates phosphatidylinositol turnover in human foreskin fibroblasts without activation of protein kinase C

Epidermal growth factor stimulates phosphatidylinositol turnover in human foreskin fibroblasts. This is a primary cell culture with normal numbers of epidermal growth factor receptors that is stimulated to divide by epidermal growth factor. Increases are seen in the inositol phospholipids and inositol phosphates. Despite this activation of phosphatidylinositol turnover, there is no detectable activation of protein kinase C.     

Differential contribution of direct-developing and stem cell-derived melanocytes to the zebrafish larval pigment pattern

The extent of adult stem cell involvement in embryonic growth is often unclear, as reliable markers or assays for whether a cell is derived from an adult stem cell, such as the melanocyte stem cell (MSC), are typically not available. We have previously shown that two lineages of melanocytes can contribute to the larval zebrafish pigment pattern. The embryo first develops an ontogenetic pattern that is largely composed of ErbB-independent, direct-developing melanocytes. This population can be replaced during regeneration by an ErbB-dependent MSC-derived population following melanocyte ablation. In this study, we developed a melanocyte differentiation assay used together with drugs that ablate the MSC to investigate whether MSC-derived melanocytes contribute to the ontogenetic pattern. We found that essentially all melanocytes that develop before 3 dpf arise from the ErbB-independent, direct-developing population. Similarly, late-developing (after 3 dpf) melanocytes of the head are also ErbB independent. In contrast, the melanocytes that develop after 3 days postfertilization in the lateral and dorsal stripe are sensitive to ErbB inhibitor, indicating that they are derived from the MSC. We show that melanocyte regeneration mutants kit^j1e99 and skiv2l2^j24e1 that are grossly normal for the overall ontogenetic pattern also lack the MSC-derived contribution to the lateral stripe. This result suggests that the underlying regeneration defect of these mutations is a defect in MSC regulation. We suggest that the regulative functions of the MSC may serve quality control roles during larval development, in addition to its established roles in larval regeneration and growth and homeostasis in the adult.