W. Ross Allen

Alterations of Lipid Metabolism in Response to Nerve Growth Factor

Abstract: In response to nerve growth factor (NGF), clonal pheochromocytoma cells flatten and extend neurites capable of making functional synapses. Although no significant changes in overall phospholipid composition occur in the presence of NGF, there is increased incorporation of 32PO4 into phosphatidylinositol and phosphatidic acid within 10 min after the addition of NGF. NGF stimulates the incorporation of 32PO4 into other lipids, such as phosphatidylcholine, to a lesser extent. The kinetics of the NGF‐induced phosphatidylinositol responses are different when the cells are in suspension from when they are attached to culture dishes. These changes in phospholipid metabolism are discussed with respect to their role in NGF‐induced nerve differentiation.

Stimulation of the release of two glycoproteins from mouse 3T3 cells by growth factors and by agents that increase intralysosomal pH.

Abstract Peptide growth factors selectively increase the amount of mitogen-regulated protein (MRP) and major excreted protein (MEP) released by mouse 3T3 cells. Balb c 3T3 cells release mainly MEP and Swiss 3T3 cells release mainly MRP. Fibroblast growth factor, epidermal growth factor, nerve growth factor, serum, and concanavalin A increase the extracellular appearance of both MEP and MRP, but to different extents. Several agents that have been shown to, or would be expected to increase, intralysosomal pH also selectively increase the release of MEP and MRP from both Balb c and Swiss 3T3 cells. The effective agents are monensin, nigericin, ammonium chloride, methylamine, chloroquine, and high extracellular pH.

Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells

Using an improved method to quantify the level of phosphorylation of the S6 ribosomal protein, we have analyzed the effect of growth stimuli on S6 phosphorylation in quiescent murine Swiss/3T3 cells to see if it can be dissociated from the later increase in DNA synthesis. Saturating concentrations of epidermal growth factor (EGF), insulin and serum each stimulate phosphorylation of the S6 ribosomal protein to the same maximal level; this is not so for DNA synthesis. Subsaturating concentrations of EGF and insulin act synergistically to stimulate both S6 phosphorylation and DNA synthesis, but qualitatively the two synergistic interactions are expressed differently. Insulin increases the maximal response of DNA synthesis to EGF, whereas it decreases the concentration of EGF required for half-maximal stimulation of S6 phosphorylation. We conclude that S6 phosphorylation is not a principal regulator of DNA synthesis, and that insulin and EGF regulate both S6 phosphorylation and DNA synthesis through different, but interacting, pathways of action.

Rapid and efficient method for analyzing phosphorylation of the S6 ribosomal protein in 32Pi-labeled, tissue culture cells

We describe a method for studying the phosphorylation of the S6 ribosomal protein in intact cells. The procedure has the advantage of using few cells, little 32Pi, and by using an air-driven centrifuge, many samples can be processed in a short time. Metabolically labeling the ribosomes with [3H]uridine before the experiment provides a measure of ribosome yield. The amount of 32Pi incorporated into proteins other than S6, which cosediment with the ribosomes, increases by the same amount as the specific activity of [32P]ATP increases, when the cells are stimulated by prostaglandin F2α, insulin, epidermal, or fibroblast growth factor, or serum; whereas the 32Pi incorporated into S6 increases by a factor greater than the increase in the specific activity of [32P]ATP. We show that the phosphate on S6 turns over at least as rapidly as does the phosphate on ATP. This last observation allows us to use a procedure, which we have outlined for determining the absolute amount of phosphate added to S6 due to a stimulus.

A monoclonal antibody recognizes a 39 kDa protein expressed in atretic granulosa cells

A major problem in ovarian physiology is the lack of conveniently quantifiable markers of atresia. Towards this end, we identified a monoclonal antibody (anti-OA-2) that selectively recognizes granulosa cells in atretic follicles. When cryostat sections of rat ovaries were incubated with anti-OA-2, granulosa cells in atretic follicles showed intense immunofluorescent labeling. In contrast, no anti-OA-2 immunoreactivity was observed in the granulosa of the healthy follicles. The amount of anti-OA-2 binding was significantly enhanced when atresia was stimulated by treatment with human chorionic gonadotropin, testosterone, or estrogen withdrawal. The results of immunoprecipitation and Western blot analyses indicated that the OA-2 antigen is a 39 kDa protein which is actively synthesized by the granulosa during atresia. The 39 kDa protein is localized at or near the inner surface of the plasma membrane. We conclude that the anti-OA-2 monoclonal will prove useful as a convenient analytical tool to study the regulation of granulosa atresia.

Monoclonal Antibodies in the Study of Mammary Development and Neoplasia

We discuss briefly the general characteristics of the monoclonal antibodies (MoAbs) useful for identifying cell types in the mammary gland, and the problem of cross-reactivity. We discuss the cell types recognized in the rat mammary gland by MoAbs and polyclonal sera and their possible connections in two separate lineages, lumenal and basal-myoepithelial. Emphasis is given to the identification of stem cells present in the end buds. On the basis of marker distribution the very early ducts are made up of stem cells. Stem cells are not present only in end buds. Transplantation experiments show that they are also present in ducts, where they can be identified by marker distribution. There is some evidence that in addition to these pluripotent stem cells, there is a second type, committed to producing ducts and alveoli. We have studied the distribution of the markers in rat mammary cancers induced by NMU. They show that cancers derive from stem cells, but it cannot be decided which kind. Even in cancers with relatively typical mammary organization, the expression of the markers is irregular. Transplanted cancers are much more abnormal. They express Thy-1, which is also expressed by cells of early ducts. This suggests that cancer progesssion follows backwards the developmental pathway. We also describe two MoAbs with considerable specificity for human breast cancer of possible clinical usefulness.

SELECTIVE INDUCTION BY PEPTIDE GROWTH FACTORS OF THE SYNTHESIS OF SECRETED PROTEINS IN 3T3 CELLS

Publisher Summary The changes in cellular metabolism needed for the initiation of DNA synthesis in quiescent cells require the synthesis of new proteins and probably involve the activation of genes. It has been found that peptide mitogens, such as epidermal growth factor and fibroblast growth factor, selectively increase the extracellular appearance of two glycoproteins secreted by 3T3 cells. These proteins are mitogen-regulated protein (MRP) and major excreted protein (MEP). The mitogens regulate at transcription or translation, not at glycosylation or secretion. It is speculated that these mitogen-induced proteins could participate in the regulation of cell growth in one of several ways. MEP or MRP could be internal growth regulating proteins. These proteins, being secreted, could be communicatory molecules for coordinating the growth of a tissue.