Cytospin preparations of fine needle aspirates.
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Aged↗
Biomedical subjects
A J Yates
Publications and source records attributed to A J Yates.
An immunophenotypic survey of malignant melanomas.
An immunohistochemical study of 106 malignant melanoma specimens from 59 patients, using formalin-fixed, paraffin-embedded material, is reported. Negativity for HMB-45 was seen in 11% of specimens. The rate of positivity with CAM 5.2 was 7%. One specimen showed alpha-smooth muscle actin (alpha SMA) positivity. For 11 of the 12 cases in which anomalous immunophenotypes were seen, multiple specimens were available; nine of these showed evidence of an alteration in the immunophenotype between specimens. Comparing the primary tumours with local recurrences and metastases, there was, variously, loss of HMB-45, S-100 protein and NKI/C3 positivity, and acquisition of CAM 5.2 and alpha SMA positivity. In some cases, the change of immunophenotype appeared to occur in a single step. However, one case with six consecutive specimens showed evidence of progressive loss of HMB-45, S-100 protein and NKI/C3 with concomitant gain of CAM 5.2 staining. The implications for the use of immunophenotyping in diagnostic practice are discussed.
Biomarkers, Tumor↗
HMB-45 in non-melanocytic tumours.
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Antibodies, Neoplasm↗
A review and predictive models of ganglioside uptake by biological membranes.
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Animals↗
Gangliosides inhibit platelet-derived growth factor-stimulated receptor dimerization in human glioma U-1242MG and Swiss 3T3 cells.
We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) in a dose-responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [35S]Methionine-labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross-linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti-PDGFR antibody. Using both Swiss 3T3 and human glioma U-1242MG cells, western blots with anti-PDGFR and anti-phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350-kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF-mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor-mediated cell functions.
3T3 Cells↗
The combined effect of tumor-produced parathyroid hormone-related protein and transforming growth factor-alpha enhance hypercalcemia in vivo and bone resorption in vitro.
Humoral hypercalcemia of malignancy is a multifactorial syndrome caused by the action of tumor-produced factors on target organs of bone, kidney, and intestine to disrupt normal calcium homeostasis. Although parathyroid hormone-related protein (PTHrP) plays an integral role in the syndrome, tumors also produce other hypercalcemic factors, such as transforming growth factor-alpha (TGF-alpha), which may modulate the effects of PTHrP. In order to determine if the effects of PTHrP on calcium homeostasis can be modulated by TGF-alpha, we have used a human squamous cell carcinoma cell line (RWGT2) which produces PTHrP alone and Chinese hamster ovarian (CHO) cells expressing only transfected human TGF-alpha complementary DNA (CHO/TGF-alpha). We studied the effects of these tumors on calcium homeostasis in nude mice bearing both tumors or each tumor alone. Whole blood ionized calcium concentrations (mean +/- SEM in mmol/L) were significantly higher in mice bearing both RWGT2 and CHO/TGF-alpha tumors (3.11 +/- 0.06, P < 0.05) when compared with mice bearing either RWGT2 alone (2.02 +/- 0.06), CHO/TGF-alpha alone (1.42 +/- 0.01), or RWGT2 and nontransfected CHO tumors (1.86 +/- 0.01). This enhanced effect was also observed using continuous PTHrP-(1-34) infusion (2 micrograms/day) in mice bearing CHO/TGF-alpha tumors. In addition, tumor cell conditioned media was tested for bone resorbing activity in organ cultures of fetal rat long bones previously incorporated with 45calcium (45Ca++). Conditioned medium at 0.1% (vol/vol) from either RWGT2 or CHO/TGF-alpha had no bone resorbing activity over control (%45Ca++ release, mean +/- SEM; control 23 +/- 1, RWGT2 19 +/- 1, CHO/TGF-alpha 23 +/- 1). However, the combination of 0.1% conditioned medium from RWGT2 and CHO/TGF-alpha significantly increased bone resorption (53 +/- 2, P < 0.05). These data demonstrate that the hypercalcemic effects of tumor-produced PTHrP are enhanced by TGF-alpha and that this effect may be due to increased bone resorption.
Animals↗
Gangliosides inhibit platelet-derived growth-factor-stimulated increases in intracellular calcium in Swiss 3T3 cells.
Individual Swiss 3T3 cells stimulated by platelet-derived growth factor delivered by means of a picopump device respond with a brisk, large, and sustained increase in intracellular calcium concentration ([Ca2+]i). Preincubation of cells with either GM1 or GT1b gangliosides inhibited the proportion of responding cells and caused a dose-related diminution in the magnitude of the increase in [Ca2+]i. This effect of ganglioside is probably part of the mechanism through which gangliosides exert their biological effects, including inhibition of platelet-derived growth-factor-induced mitogenesis.
3T3 Cells↗
Reperfusion paradox: a novel mode of glial cell injury.
We have attempted to reconstruct in vitro the events that may occur in vivo during reperfusion injury after ischemia in the central nervous system. The phenomenon is induced by previous exposure to low calcium solutions ("calcium paradox") before the reperfusion episode. Intracellular calcium alterations during reperfusion of human astrocytoma U1242MG cells have been investigated with microspectrofluorimetry using the calcium-sensitive dye fura-2. Cells were perfused in calcium-free buffer solution for 30 min and then re-exposed to the control buffer solution (1.5 mM CaCl2). [Ca2+]i increased up to 3.5 times control levels during the reperfusion period. The mechanism of the increase was also investigated. Addition of TTX (2 microM) or choline chloride sodium substitution during perfusion with low calcium prevented the [Ca2+]i increase during reperfusion. Reperfusion increases in [Ca2+]i were exacerbated by low potassium in the perfusion medium, but unaltered by the calcium channel blockers cadmium (100 microM) and nickel (100 microM). In a similar manner, flunarizine (10 microM) and cadmium (100 microM) were unable to modify reperfusion [Ca2+]i alterations. Low sodium in the reperfusion medium produced significant increases in [Ca2+]i if preceded by low potassium and calcium perfusion. The viability of cells after 24 h of incubation after the insult produced by exposure to Ca(2+)-free media for 30 min was also investigated. Compared with control groups, the groups treated with Ca(2+)-free media for 30 min had a decreased number of surviving cells and morphological alterations indicative of cell pathology. The relative number of cytotoxic cells was increased by maneuvers (low potassium perfusion) that presumably blocked the Na/KATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Expression of human transforming growth factor alpha by Chinese hamster ovarian tumors in nude mice causes hypercalcemia and increased osteoclastic bone resorption.
Transforming growth factor alpha (TGF-alpha) is a polypeptide regulator of cell growth produced by many malignant tumors. It stimulates osteoclastic resorption in bone organ culture and osteoclast-like cell formation in marrow culture. To determine whether tumor production of TGF-alpha can cause hypercalcemia in vivo, we used Chinese hamster ovarian (CHO) cells transfected with the human TGF-alpha gene (TCHO), which stably express and secrete TGF-alpha. We used nontransfected CHO cells as controls (CCHO). TCHO and CCHO were inoculated intramuscularly into one hindlimb of nude mice and grew as local solid tumors. After 4 weeks of TCHO tumor growth, plasma ionized calcium (Ca2+) increased to reach 1.48 +/- 0.03 mM (mean +/- SEM), whereas mice bearing similarly sized CCHO tumors and non-tumor-bearing mice (NTB) remained normocalcemic (normal range for Ca2+, 1.15-1.30 mM). Plasma TGF-alpha was undetectable by an ELIFA assay in all NTB mice, was markedly increased in all TCHO mice (5.75 +/- 0.78 ng/ml), and was slightly increased in CCHO mice (0.50 +/- 0.22 ng/ml). Quantitative bone histomorphometry showed a prominent increase in osteoclastic bone resorption in TCHO mice. These data suggest that TGF-alpha is a mediator of hypercalcemia and increased osteoclastic bone resorption in tumors that produce it in sufficient quantity.
Animals↗
Glycolipids of a human glioma cell line bearing receptors for platelet-derived growth factor (PDGF).
Glycolipids of U-1242 MG were characterized because results of previous studies showed that exogenous gangliosides, especially GM3, inhibit PDGF-stimulated growth of this human glioma cell line. GM3 and GM2 are the major gangliosides; both separate as doublets with thin-layer chromatography. The major neutral glycolipid is glucocerebroside with nonhydroxy fatty acids, but paragloboside, ceramide dihexoside, globoside, and asialoGM2 (GA2) are also present. The coexistence in U-1242 MG of these gangliosides and the PDGF receptor, whose mitogenic signal is modulated by GM3 in these cells, suggests a possible functional relationship among them with respect to growth regulation.
Chromatography, Thin Layer↗
An overview of principles for classifying brain tumors.
The purpose of this review is to clarify for the nonneuropathologist some of the confusing issues concerning the classification of brain tumors. Following a short discussion of the commonly used methods to diagnose brain tumors clinically (frozen section, light and electron microscopy, immunohistochemistry), the general principles of classifying neural tumors are presented. Grading of tumors on the basis of histological anaplasia, and the concept that tumor cells can be related to specific cytological stages of normal cellular development (cytogenetic classification) are presented. The World Health Organization system of classifying neural tumors is an attempt to develop a standardized classification scheme with as few interpretative controversies as possible, but it has required revision as new information has been gained. The major clinical and biological features of the commonest tumor groups are then discussed. It is unlikely that improvements in the classification of brain tumors will be based solely on histological information. Definitions of tumor entities that will provide more accurate prognoses and bases for effective therapy will require considerably more information at the molecular level than is currently available.
Astrocytoma↗
Sensitivity of human glioma and brain cells to natural killer cell lysis. Effects of serum concentration, epidermal growth factor, and time in culture.
Using an in vitro monolayer natural killer (NK) cytolysis assay, the authors examined the effects of serum concentration and epidermal growth factor (EGF) on sensitivity to NK cytolysis. It was found that target cells cultured in high concentrations of serum (10% fetal bovine serum (FBS)) had higher cytotoxicity levels than those in low serum concentrations (0% to 0.5% FBS). Exposure of target cells to EGF had no effect on their sensitivity to NK cytolysis. Both glioma cell lines showed decreased NK cell sensitivity with longer times in culture. The results of cytofluorometric studies on these cell lines indicate that the differences in NK cell sensitivity may reflect the growth fraction of the target population and that a population with a higher proportion of cycling cells is more susceptible to lysis by NK cells. Whether it is possible to separate the proliferative rate of these cells from their NK cell sensitivity is unknown, but worthy of consideration.
Blood↗
Irreducible dorsal interphalangeal great toe dislocation.
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Adult↗
Inhibition of bone resorption by inorganic phosphate is mediated by both reduced osteoclast formation and decreased activity of mature osteoclasts.
High concentrations of inorganic phosphate (Pi) are known to inhibit bone resorption, although the mechanism(s) underlying this effect is unclear. To investigate whether Pi can inhibit the formation of osteoclasts we studied the effects of changes in Pi concentration between 1 and 4 mM on osteoclast-like cell formation in 1 week cultures of mouse bone marrow. Osteoclast-like cells were identified by multinuclearity, positive staining for tartrate-resistant acid phosphatase (TRAP), and contraction in response to calcitonin. Increasing concentrations of Pi inhibited formation of these cells in a dose-dependent manner. To study effects of Pi on the bone-resorbing activity of mature osteoclasts we isolated osteoclasts from calcium-deficient egg-laying hens or rat pups and incubated them on sperm whale dentine slices. High Pi concentrations markedly reduced both the number of resorption pits formed per dentine slice and the mean area of each pit in both avian and mammalian systems. These data indicate that high concentrations of Pi act on bone directly, both to inhibit generation of new osteoclasts from their precursor cells and to inhibit bone resorption by mature osteoclasts. These effects of extracellular Pi concentration may play an important modulatory role on bone turnover in vivo and have potential importance in several disease states in which Pi metabolism is perturbed.
Animals↗
Effects of GM1 and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en) on neuroblastoma (Neuro 2a) and human glioma cells (U1242 MG).
Several lines of evidence suggest that gangliosides may play a role in the regulation of growth in many cell types. Here we describe the effects on growth of two different cell lines by the addition of two different chemicals which have been reported to elevate the cellular ganglioside content through different mechanisms. Growth of neuroblastoma (Neuro 2a) cells in medium containing fetal bovine serum was inhibited in a dose-dependent fashion by both exogenous GM1 ganglioside and NeuAc2en, an inhibitor of sialidase activity. In contrast, growth of glioma cells (U-1242 MG) was not affected by exogenous GM1 or NeuAc2en in the presence of as little as 1% calf serum. However, NeuAc2en inhibited growth of U-1242 MG cells stimulated by platelet-derived growth factor in serum-free medium. These results demonstrate that the growth inhibitory effects of ganglioside on U-1242 MG but not Neuro 2a cells can be counteracted by serum, suggesting that the mechanisms through which gangliosides affect cell growth may be different for different growth factors and cell types.
Animals↗
A high performance liquid chromatography method for the analysis of glycosphingolipids using galactose oxidase/NaB3H4 labeling of intact cells and synaptosomes.
The major objective of this study was to combine an HPLC method with a galactose oxidase/NaB3H4 labeling method to allow both a chemical quantitation of individual glycolipids and analysis of their 3H labeling. Neutral glycolipids in whole cells were oxidized with galactose oxidase, and the resultant aldehydes were radiolabeled by reduction with tritiated sodium borohydride. Gangliosides, oxidized with galactose oxidase, either were reduced while in the native state in the whole cell or were first extracted and then reduced. Tritiated glycolipids were perbenzoylated and separated by HPLC. Ultraviolet detection of the derivatives was at 230 nm. Incorporated radioactivity was determined either by collecting fractions from the HPLC separation and counting on a liquid scintillation spectrometer or with a flow-through counter. The order of the derivatization and reduction is critical. Reduction of glycolipids prior to derivatization yielded sharp uv and radioactive peaks. Perbenzoylation of the oxidized glycolipids prior to reduction yielded multiple uv peaks, a noisy baseline, and broad radioactive peaks which did not always have a corresponding uv peak. The labeled neutral glycolipids were stable at -40 degrees C for at least 14 days, and gangliosides were stable at -15 degrees C for at least 14 days. When samples were stored at 20 degrees C there was a time-dependent decrease in the glycolipid/internal standard uv peak area ratio for GbOse4 and GbOse3 apparent by 28 days after perbenzoylation. The distribution of radiolabel among peaks showed no change with time or temperature. We adapted the technique to allow 3H labeling of glycolipids from monolayers of cultured glioma cells and from mouse brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Stanozolol stimulates remodelling of trabecular bone and net formation of bone at the endocortical surface.
1. We studied the mechanism of action of the anabolic steroid, stanozolol, in 23 patients with osteoporosis, using a combination of biochemical and histomorphometric techniques. 2. Treatment with stanozolol (5 mg/day) for 1 year was associated with a marked and significant decrease in the fasting urinary excretion of calcium (P less than 0.01) but not with changes in the serum concentrations of calcium and phosphate, the serum activity of alkaline phosphatase, the renal tubular reabsorption of calcium or the urinary excretion of hydroxyproline. 3. Histological examination of trabecular bone showed an increase in bone turnover and the bone formation rate increased twofold (P less than 0.02). There were no significant changes in bone volume or wall thickness after treatment. Tetracycline labelling was used to discriminate bone structural units completed before and during treatment. Measurements of the wall thickness of those bone structural units formed during treatment showed no significant change. 4. Measurements made on the endocortical surface also showed an increase in bone turnover, but in contrast to trabecular bone, the bone structural units formed at endocortical sites during treatment had a significantly greater wall thickness than those formed before treatment (P less than 0.05). 5. We conclude that stanozolol increases the turnover of trabecular bone and increases the endocortical apposition of bone.
Aged↗
Phospholipase A2 activities with a plasmalogen substrate in brain and in neural tumor cells: a sensitive and specific assay using pyrenesulfonyl-labeled plasmenylethanolamine.
We have developed a new assay method for phospholipase A2 (EC 3.1.1.4.), towards ethanolamine plasmalogen using pyrenesulfonyl-labeled plasmenylethanolamine as the substrate. This procedure is sensitive to about 3 pmol/ml per min and is absolutely specific for plasmalogen. In this method, the product of phospholipase A2, pyrenesulfonyl-labeled lysoplasmalogen, is hydrolyzed to aldehyde and labeled glycerophosphoethanolamine with hydrochloric acid exposure, and after TLC separation, the pyrenesulfonyl-glycerophosphoethanolamine is quantitated spectrofluorometrically. The excitation and emission wave lengths were 340 and 376 nm, respectively. The activity of bovine brain homogenate was 44.1 +/- 6.47 pmol/min per mg protein (n = 3). Among bovine brain subcellular fractions, the distribution and specific activity of the enzymes were highest in cytosol (38.7 +/- 1.58% and 102.6 +/- 16.2 pmol/min per mg protein, n = 3). The activities of neural tumor cells, PC12 pheochromocytoma, Neuro2A and SKNSH neuroblastoma and U1242MG glioblastoma, were 34.4 +/- 6.83 (n = 5), 7.05 +/- 0.97 (n = 4), 5.25 +/- 1.69 (n = 5), and 9.68 +/- 1.35 (n = 4), pmol/min per mg protein (M +/- S.E.M.), respectively.
Adrenal Gland Neoplasms↗