Intracellular alkaline phosphatase activity in cultured human cancer cells

Summary The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum, Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.This work was supported by National Institutes of Health Grant No. CA 21967     

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.     

Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase.

Human SK-N-MC neurotumor cells express beta 1- but not beta 2-adrenergic receptors. Following exposure of the cells to isoproterenol, there was no reduction in the maximum response of adenylyl cyclase to the agonist but a 3-fold shift to less sensitivity in the concentration response. This desensitization was very rapid and dose dependent; half-maximal effects occurred at 10 nM isoproterenol. A similar shift was observed when membranes from control cells were incubated with ATP and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). No shift, however, was observed in intact cells exposed to either dibutyryl cyclic AMP or dopamine, which stimulates adenylyl cyclase in these cells through D1 dopamine receptors. To pursue the role of protein kinases in the desensitization process, cells were made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), and exposed to isoproterenol. The PKA inhibitor but not heparin blocked the agonist-mediated desensitization. In contrast, desensitized human tumor cells (HeLa and A431), which express beta 2-adrenergic receptors, exhibited both a shift in concentration response and a reduction in maximum response; the former was blocked by the PKA inhibitor and the latter by heparin. Our results indicated that whereas both human beta 1- and beta 2-adrenergic receptors are susceptible to PKA, only the beta 2 receptors are susceptible to beta ARK. These differences in desensitization may be due to differences in receptor structure as the human beta 1 receptor has fewer potential phosphorylation sites for beta ARK in the carboxyl terminus than the human beta 2 receptor.     

Cytolytic activities of activated macrophages versus paraformaldehyde-fixed macrophages; soluble versus membrane-associated TNF.

Membrane-associated tumor necrosis factor (TNF) and soluble TNF were compared as to their lytic activities, and as to the kinetics of their expression by macrophages activated with LPS and/or IFN-gamma in the presence or absence of cycloheximide. EL 4 tumor cells, resistant and sensitive to lysis by recombinant TNF or membrane-associated TNF (paraformaldehyde (PF)-fixed activated macrophages) were used as targets. In the presence of cycloheximide the TNF-resistant S-EL4 cells were lysed by both TNFs. PF-fixed macrophages was cytolytic after 1 hr activation but not after 3 or more hours of activation. Their activity was totally inhibited by anti-TNF antibodies and was a composite of transmembrane (integral) TNF and soluble TNF conjugated to macrophage membrane TNF receptors. Treatment of the macrophages with glycine pH 3.0 buffer dissociated the conjugated TNF without affecting the integral membrane TNF. When macrophages were activated with LPS +/- IFN-gamma in the presence of cycloheximide or activated just with IFN-gamma their activity after fixation with paraformaldehyde was no longer detected. Nonfixed macrophages under these conditions still remained cytotoxic. Tumor cell susceptibility to membrane-associated TNF activity, in contrast to recombinant (soluble) TNF, was greatly reduced in the presence of nicotinamide, an inhibitor of ADP-ribosyltransferase, suggesting that the mechanisms of lysis by these TNFs may be different. The lytic activity of both TNFs was found to be receptor-dependent in that tumor cells, whose TNF binding sites were "down-regulated" by TPA, were rendered resistant to lysis by both membrane-associated and soluble TNFs.     

Specific gangliosides increase rapidly in rat liver following partial hepatectomy

Rat liver gangliosides (sialic acid containing glycosphingolipids) were analyzed by HPTLC and HPLC following either partial hepatectomy or sham operation. Analysis of whole liver gangliosides by HPTLC demonstrated that within 6 h after partial (68%) hepatectomy, there was a significant increase in GM1 compared to both sham and control animals. By 48 h, GM1 was further increased and the polysialylgangliosides GD1a, GD1b and GT1b had also risen significantly, whereas changes in GM3 were negligible. Gangliosides associated with the plasma membrane were increased up to 3.5-fold in regenerating liver compared to sham-hepatectomized controls as assessed by HPLC. Although elevations in membrane gangliosides were associated with hepatocyte proliferation, they did not closely follow the growth curve. The time course of changes in ganglioside biosynthesis suggests differential upregulation of GM3 synthase and GD3 synthase in regenerating livers.     

Neoglycolipid analogues of ganglioside GM1 as functional receptors of cholera toxin.

We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective. The distance between GM1OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination. Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

TPA induction of EL4 resistance to macrophage-released TNF: role of ADP-ribosylation in tumoricidal activities of TNF and other factors

Activated macrophages synthesize and release numerous tumoricidal soluble factors that can be divided into receptor- or nonreceptor-dependent agents. Tumor necrosis factor (TNF) would be an example of the former. In our experimental model the killing of EL4 thymoma cells by syngeneic activated macrophages involves, but not exclusively, TNF. Our results show that approximately 50% of the anti-EL4 activity expressed by macrophages can be specifically inhibited with rabbit anti-mouse TNF antibody. EL4 variants resistant to the lytic activity of TNF were still susceptible to macrophage-mediated lysis. A tumor-promoting phorbol ester, TPA, rendered TNF-sensitive and -insensitive EL4 cells resistant to M phi-mediated lysis. However, TPA down-regulated TNF-specific binding sites on both TNF-sensitive and -resistant cell surface membranes, suggesting that resistance to TNF involves postligand:receptor events. Tumor cell G-protein involvement (ADP-ribosylation), as a result of TNF-TNF receptor interactions, was investigated. The results showed that pertussis toxin was cytotoxic against TNF-sensitive and -resistant EL4 cells but not against TPA-treated target cells. Inhibitors of ADP-ribosyltransferase inhibited pertussis toxin cytotoxicity and macrophage-mediated lysis but did not interfere with recombinant TNF lytic activity.     

Competition by Estrogens for Catecholamine Receptor Binding In Vitro

Abstract: We have examined the ability of various steroids to compete for high-affinity binding of ³H-labeled ligands to catecholamine receptors in membranes prepared from rat cerebral cortex, striatum, and anterior pituitary. Ligands employed were: [³H]WB4101, [³H]prazosin, [³H]yohimbine, and [³H]clonidine (alpha-noradrenergic); [³H]dihydroalprenolol (beta-noradrenergic); [³H]spiperone and [³H]ADTN (dopaminergic). Only the 17β estrogens were effective and only binding of [³H]spiperone and [³H]ADTN in striatum and [³H]WB4101 and [³H]prazosin in cerebral cortex was reduced. Thus putative dopaminergic and alpha₁-noradrenergic sites alone appear to recognize estrogens. A slight competitive effect on [³H]spiperone binding to anterior pituitary membranes was also observed. Among the 17β estrogens tested, the most effective in all cases was the catechol estrogen 2-hydroxyestradiol (2-OHE₂). The ability of 2-OHE₂ (IC₅₀= 20–30 μM) to inhibit ligand binding to alpha₁ receptors was comparable to that of norepinephrine (IC₅₀= 10–20 μM), whereas for dopamine receptors in striatum and pituitary 2-OHE₂ was an order of magnitude less effective than dopamine (IC₃₀= 12 μM) in reducing binding of ³H ligands. Estradiol-17β and 2-hydroxyestrone were also able to inhibit binding, but the order of steroid potency was different for alpha₁ and dopaminergic receptors. Progesterone, testosterone, and corticosterone were without effect in all cases. These results show that there is specificity of steroid interactions with catecholamine receptors in the brain, both in terms of steroid structure and receptor type. The possible relevance of these interactions to neuroendocrine function is discussed.     

Induction of immunity and specific unresponsiveness in vitro by cell-bound antigen: I. Symmetry in enhancement of both responses

Pulse treatment of lymphoid cells from rabbits with solubilized antigens from T2 phage results in the firm binding of small but highly active amounts of antigen. Binding of phage antigens to viable, nonviable, or disrupted cells enhances their ability to evoke antibody formation or specific unresponsiveness in the primary in vitro response of rabbit spleen cells. Transfer of sonicate containing the equivalent of 10₂ to 10₃ antigen-pulsed cells carrying 10^?8 to 10^?7 μg phage protein nitrogen into spleen cell cultures regularly evokes antibody formation, while introduction to such cultures of 10^?3 μg phage protein nitrogen in cell-bound form evokes unresponsiveness. These findings indicate a 10- to 100-fold amplification of tolerogenic and immunogenic activities of cell-bound over soluble T2 antigen.