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Biomedical subjects

G Eilon

Publications and source records attributed to G Eilon.

At least 19 recordsLinked to original sources
PubMed1995

The role of strain/vendor differences on the outcome of focal ischemia induced by intraluminal middle cerebral artery occlusion in the rat.

This study investigated the role of strain and vendor differences on the outcome of focal cerebral ischemia in the rat induced by occlusion of the middle cerebral artery by an intraluminal thread. The cortical infarct volumes (Mean +/- S.E.M.) were: 14.2 +/- 6.2 mm3 in Simonsen Laboratories Sprague-Dawley rats; 84.0 +/- 22.9 mm3 in Simonsen Laboratories Wistar rats; 223.3 +/- 23.6 mm3 in Taconic Laboratories Sprague-Dawley rats; and 239.5 +/- 30.7 mm3 in Charles River Laboratories Sprague-Dawley rats. Middle cerebral artery occlusion combined with bilateral common carotid artery occlusion for 60 min increased cortical infarct volumes to: 113.0 +/- 18.8 mm3; 152.4 +/- 21.1 mm3; 227.8 +/- 19.3 mm3; and 248.4 +/- 24.0 mm3, respectively. To control the effect of blood pressure as a variable contributing to the outcome of ischemia, additional experiments where performed in which the blood pressure in Simonsen Laboratories Sprague-Dawley rats was lowered to the level of the blood pressure in Taconic Laboratories Sprague-Dawley rats. Although this manipulation increased the cortical infarct volumes in Simonsen Laboratories Sprague-Dawley rats, the volumes were still less than those in Taconic Laboratories Sprague-Dawley rats. The results of the present study indicate that the outcome of ischemia in the intraluminal thread model may dramatically differ depending on the strain and vendor of animal used.

Animals↗
PubMed1990

Correlation of protein kinase C translocation, P-glycoprotein phosphorylation and reduced drug accumulation in multidrug resistant human KB cells.

Treatment of drug-resistant human KB carcinoma cells (KB-V1) with 0.2 microM phorbol 12-myristate 13-acetate (PMA) resulted in increases of 4-fold in both membrane-associated protein kinase C activity and phosphorylation of P-glycoprotein. The response was essentially complete after 30 min and was relatively stable, since both of these parameters remained elevated above basal levels in cells exposed to PMA for 24 hours. In contrast, long-term PMA treatment of drug-sensitive KB-3 cells caused complete depletion of protein kinase C. The rate of accumulation of [3H]vinblastine in KB-V1 cells was 0.8 +/- 0.1 pmol/mg/30 min in the absence, and 1.9 +/- 0.2 pmol/mg/30 min in the presence, of 20 microM verapamil. Preincubation of cells with PMA resulted in a time-dependent decrease, up to 60% after 24 hours, in both of these values. These results suggest that protein kinase C mediated phosphorylation stimulates the drug transport activity of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Protein kinase C phosphorylates P-glycoprotein in multidrug resistant human KB carcinoma cells.

Studies were undertaken to identify the protein kinase(s) responsible for P-glycoprotein phosphorylation in multidrug-resistant (KB-V1) human carcinoma cells and to elucidate the functional role of phosphorylation. P-glycoprotein migrated on sodium dodecyl sulfate gels with apparent Mr 150,000 and is termed P150. When KB-V1 membrane vesicles were incubated with [gamma-32P] ATP, P150 was phosphorylated by an endogenous kinase that exhibited properties of membrane-inserted protein kinase C (PKC). Both membrane-bound P150 and purified P150 served as effective substrates for highly purified rat brain PKC which incorporated approximately 0.6 mol of phosphate/mol of P150. Enzyme assays showed that KB-V1 cells exhibit 4-fold higher PKC activity compared with the drug-sensitive KB-3 cell line. The basal phosphorylation of P150 observed in 32P-labeled cells was increased 2-fold by phorbol ester (PMA) treatment and reduced 30% by treatment with the isoquinolinsulfonamide H-7. Phosphopeptide maps of partially digested P150, phosphorylated either in vitro with PKC or in intact 32P-labeled control or PMA-stimulated cells, were indistinguishable from one another. Drug accumulation assays revealed that PMA treatment of KB-V1 cells significantly reduced [3H]vinblastine accumulation induced by verapamil or by tetrandrine. The results suggest that PKC is primarily responsible for P150 phosphorylation in KB-V1 cells and that phosphorylation may play a modulatory role in the drug transport process.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity.

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.

Amino Acid Sequence↗
PubMed1988

Heart protein kinase C activity increases during progression of disease in the cardiomyopathic hamster.

Protein kinase C activity in the particulate fraction of the heart increases two-fold during mid-stage of disease in the cardiomyopathic hamster. No change in the corresponding enzyme activity occurs with aging in healthy control hamsters. In the solubilized particulate fraction of hearts from both myopathic and control animals, Ca++/phospholipid-dependent endogenous phosphorylation of proteins of Mr 26, 31, 45, 53, 69, 98, 105 and 126 kDa are observed. All of these proteins are more highly phosphorylated in the protein kinase C-enriched preparation from the myopathic heart compared to the control. No significant differences between myopathic and control hamsters are observed in the activities of protein kinase C or phosphoinositide-specific phospholipase C from heart cytosol.

Age Factors↗
PubMed1988

The cysteine protease inhibitor, E-64, stimulates the polarization and locomotor responses of endothelial cells to wounding.

To clarify the role of proteases and protease inhibitors in the initiation and execution of endothelial cell movement, we observed the effect of several protease inhibitors on the polarization and locomotor responses of an endothelial cell monolayer subjected to experimental wounding. We found that the thiol protease inhibitor, E-64 (L-transepoxysuccinyl-leucylamido-[4-guanidino]butane) stimulated both cellular processes. The stimulatory effect of E-64 on the polarization response of cells to wounding required a preincubation period of at least 1 hr, calcium-calmodulin interaction, protein kinase C activation, and was blocked by cyclic AMP analogs. The chemokinetic action of E-64 appears to be unique among the protease inhibitors tested and may represent a novel role for this cysteine protease inhibitor or its endogenous counterpart in the modulation of cell locomotion.

Animals↗
PubMed1985

Estrogens and antiestrogens stimulate release of bone resorbing activity by cultured human breast cancer cells.

Patients with advanced breast cancer may develop acute, severe hypercalcemia when treated with estrogens or antiestrogens. In this study, we examined the effects of estrogens and related compounds on the release of bone resorbing activity by cultured human breast cancer cells in vitro. We found that the estrogen receptor positive breast cancer cell line MCF-7 releases bone resorbing activity in response to low concentrations of 17 beta-estradiol. Bone resorbing activity was also released in response to the antiestrogen nafoxidine. Other steroidal compounds had no effect on the release of bone resorbing activity. Estrogen-stimulated release of bone resorbing activity occurred with live bone cultures, but not with devitalized bones, indicating that the effect was bone cell mediated. The breast cancer cell line MDA-231, which does not have estrogen receptors, did not release bone resorbing activity in response to 17 beta-estradiol or nafoxidine. Release of the bone resorbing activity by MCF-7 cells incubated with 17 beta-estradiol was inhibited by indomethacin (10 microM) and flufenamic acid (50 microM), two structurally unrelated compounds that inhibit prostaglandin synthesis. Concentrations of 17 beta-estradiol and nafoxidine that caused increased release of bone resorbing activity by the breast cancer cells caused a four- to fivefold increase in release of prostaglandins of the E series by MCF-7 cells. These data may explain why some patients with advanced breast cancer develop acute hypercalcemia when treated with estrogens or antiestrogens, and why bone metastases are more common in patients with estrogen receptor positive tumors.

Biological Products↗
PubMed1985

Constitutive biosynthesis of bone Gla protein in a human osteosarcoma cell line.

A human osteosarcoma cell line derived from cells obtained from a patient with Paget's disease is shown to synthesize and secrete bone Gla protein (BGP); (osteocalcin), a noncollagenous bone matrix protein. Using a human BGP-specific RIA, we show that the human osteosarcoma cells synthesize significant amounts of BGP without any prior induction of BGP synthesis by 1,25-dihydroxyvitamin D. After specific immunoprecipitation of poly-A+ RNA in vitro translation products with antibodies to BGP, we found that BGP is synthesized as a precursor with an apparent mol wt of 13.5K, as demonstrated on 15% sodium dodecyl sulfate-polyacrylamide gels. Finally, pulse labeling of the osteosarcoma cells with [3H]proline reveals that the cells synthesize mature BGP of 12,000 mol wt as well as a higher mol wt precursor (13,500) of the protein.

Autoradiography↗
PubMed1985

Identification of a messenger ribonucleic acid fraction in human prostatic cancer cells coding for a novel osteoblast-stimulating factor.

Prostatic cancer is frequently associated with new bone formation although the tumor-derived factors responsible for changes in bone cell function have not been identified. We have examined the synthesis of osteoblast-stimulating factors in a cultured human prostatic cancer cell line (PC-3) and show that conditioned medium from PC-3 cells stimulate mitogenesis and alkaline phosphatase in cells with the osteoblast phenotype (cultured rat osteosarcoma cells) and collagen synthesis in fetal rat calvaria. In order to characterize tumor-derived gene products which stimulate cells of the osteoblast phenotype messenger RNA (mRNA) was isolated from PC-3 cells and microinjected into Xenopus laevis oocytes. mRNA-directed translation products which were secreted into the oocyte medium were collected and assayed for a number of osteoblast stimulating properties. Translation products from PC-3 mRNA-injected oocytes stimulated division of cultured osteosarcoma cells by 8-fold and increased DNA synthesis as measured by incorporation of [3H]thymidine into these cells. In addition, tumor-derived translation products stimulated the production of alkaline phosphatase activity, a marker enzyme for bone formation, in cultured osteosarcoma cells. Oocytes injected either with water or with mRNA from a tumor not associated with bone formation were devoid of these activities. Total mRNA from the human prostatic cancer cells was then denatured and fractionated by size by agarose gel electrophoresis. When individual fractions of mRNA were eluted from the gel, translated in Xenopus oocytes, and the secreted translation products were tested for alkaline phosphatase-stimulating activity on osteoblast-like cells, the majority of the activity could be recovered in a mRNA fraction which was approximately 1800 bases in length. These results indicate that the PC-3 prostatic cancer cell line synthesizes a mRNA of approximately 1800 bases which codes for a heretofore unrecognized osteoblast-stimulating factor.

Alkaline Phosphatase↗
PubMed1984

New bone resorption stimulation factor elaborated by a human osteosarcoma cell line.

The FM-2 cell line is a cloned, immortalized cell line derived from a human osteosarcoma. Conditioned medium from FM-2 cultures contains a factor which stimulates calcium mobilization from fetal rat bone organ cultures. Treated bones contain increased numbers of osteoclasts and decreased bone matrix. This factor has a molecular weight of approximately 29,000 as determined by gel filtration. Its biological activity is dependent on a protein moiety and is completely inhibited by calcitonin. Its synthesis by the FM-2 line is dependent on cell density and replenishment of fresh medium. This factor is not parathyroid hormone, a vitamin D metabolite, prostaglandin E, epidermal growth factor, or osteoclast-activating factor, all of which have bone-resorbing activities. Also, FM-2-conditioned medium inhibits collagen synthesis in fetal rat calvaria cells and decreases alkaline phosphatase levels in an osteoblastic cell line, and these two properties coelute with the calcium-mobilizing factor from a hydroxylapatite column. These biological products, synthesized by a cell line derived from a tumor, may represent physiological factors normally synthesized by a subpopulation of bone cells.

Animals↗
PubMed1983

Association of increased cyclic adenosine 3':5'-monophosphate content in cultured human breast cancer cells and release of hydrolytic enzymes and bone-resorbing activity.

The established human breast cancer cell line MCF-7 resorbs bone directly in vitro independently of viable endogenous bone cells, and this resorption of bone is closely correlated with the release of hydrolytic enzymes by the cultured tumor cells. In this study we have examined the effects of a number of drugs which increase the intracellular cyclic adenosine 3':5'-monophosphate (cyclic AMP) content on the release of hydrolytic enzymes by the tumor cells and their capacity to resorb bone. When MCF-7 cultures were treated with cholera toxin (0.05 to 5 micrograms/ml), a potent adenylate cyclase inducer, mineral-releasing, lysosomal enzyme, and collagenolytic activities increased more than 2-fold in the cell culture medium. Prostaglandin E1 (0.1 microM), 8-bromocyclic AMP (10 mM), and isobutylmethylxanthine (30 microM) caused similar effects. These data suggest that adenylate cyclase activation and increases in cyclic AMP content in the tumor cells caused the release of lysosomal enzymes and collagenolytic activity and caused the resorption of bone. We have previously found that colchicine, a drug which inhibits microtubule assembly, also increased hydrolytic enzyme release and release of bone-resorbing activity. Direct measurements of cyclic AMP were made in MCF-7 cells 3 hr after treatment with colchicine (10 microM) as well as MCF-7 cells treated with prostaglandin E1, cholera toxin, and isobutylmethylxanthine. MCF-7 cells showed a 2-fold increase in cyclic AMP content after treatment with all of these agents, although there was no similar increase in mouse 3T3 cells which do not produce bone-resorbing activity under these conditions. These data suggest that increases in cyclic AMP concentrations in human breast cancer cells lead to release of hydrolytic enzymes and resorption of bone.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Characteristics of a calcitonin-responsive cell line derived from a human osteosarcoma.

Although the primary cell type in human osteosarcoma is usually a neoplastic osteoblast, numerous other mesenchymal cell types may coexist in the same tumor. Previously described cloned, long-term osteosarcoma cell lines have had an osteoblastic phenotype. In this report, we describe a nonosteoblastic, long-term cell line derived from an osteosarcoma in a patient with Paget's disease. The cell line (FM-2) is nontransformed in having a low saturation density and anchorage-dependent growth, and it is nontumorigenic in nude mice. Important features of its fine structure include numerous elongated mitochondria, abundant Golgi and lysosomes, and a poorly developed rough endoplasmic reticulum. The line has high levels of lysosomal enzymes (acid phosphatase and N-acetylglucosaminidase) and low levels of alkaline phosphatase. It lacks numerous macrophage markers (lysozyme, C3, Fc receptors, and M1 antigen). The FM-2 line had a dose-dependent cyclic AMP response (7-fold increase) following treatment with calcitonin but not with parathormone. In 125I-calcitonin-binding experiments, we calculated approximately 5.3 +/- 0.2 X 10(3) receptor sites/cell with a kd of 1.8 +/- 0.1 X 10(-9) M. Conditioned medium from the FM-2 line was a potent stimulator of calcium release as assayed in a 45Ca-labeled fetal rat bone organ culture. This activity was not prostaglandin, vitamin D, parathormone, or epidermal growth factor, which are known stimulators of bone resorption. The FM-2 line does not appear to be derived from an osteoblast, macrophage, or fibroblast and may represent a calcitonin-responsive bone stem cell.

Animals↗
PubMed1982

Effects of two bacterial products, muramyl dipeptide and endotoxin, on bone resorption in organ culture.

We have compared two components of bacterial cell walls, muramyl dipeptide (MDP) and lipopolysaccharide (LPS), for their effects on bone resorption as measured by the release of previously incorporated 45Ca. MDP is the smallest active component of peptidoglycan, whereas LPS is the active component of endotoxin. Fetal rat long bones were cultured for 5 days in a chemically defined medium supplemented with bovine serum albumin (BSA) or serum. LPS increased 45Ca release at concentrations of 0.03-1.0 microgram/ml. LPS further purified by electrolytic dialysis (ED-LPS) was active at 0.01 microgram/ml. ED-LPS was ineffective at such low concentrations in the presence of serum. The response to MDP was more variable than that to LPS, but bone resorption was stimulated at concentrations of 10(-7)-10(-5) M. MDP was less effective or inactive in medium supplemented with serum. Stereoisomers of MDP that do not have adjuvant activity caused minimal stimulation of bone resorption, whereas 6-0-steroyl MDP stimulated resorption at 10(-8) M. The stimulation of bone resorption by LPS and MDP was not inhibited by indomethacin. Both LPS and MDP increased lysosomal enzyme release in proportion to their effects on 45Ca release. LPS also markedly increased collagenase activity in the medium, but MDP did not. These results indicate that chemically different products of bacterial cell walls can stimulate bone resorption in vitro. These products may be distinguished by differences in dose response curve, serum inhibition, and collagenase release.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1982

Chloroquine, hydroxystilbamidine, and dapsone inhibit resorption of fetal rat bone in organ culture.

Three potential inhibitors of lysosomal enzyme release, chloroquine, hydroxystilbamidine, and dapsone were tested for their effects on the release of previously incorporated 45Ca and beta (beta)-glucuronidase from fetal rat long bones cultured in a chemically defined medium. At concentrations of 10(-5) to 10(-8)M, all three agents were able to inhibit the stimulation of bone resorption by parathyroid hormone (PTH) or prostaglandin E2 (PGE2). Inhibition was seen at concentrations which did not alter the uptake of (3H)-2-deoxy-glucose or the incorporation of (3H)-thymidine in bone. While the inhibitors blocked the stimulation of beta-glucuronidase release by PTH and PGE2, they could also cause a direct increase in total beta-glucuronidase content and release. Hence the usual strong correlation between the release of beta-glucuronidase and 45Ca was no longer seen in the presence of inhibitors. These data indicate that chloroquine, hydroxystilbamidine, and dapsone are potent inhibitors of bone resorption which may act by blocking the release of lysosomal enzymes in cells stimulated by PTH or PGE2, but may have a different effect on other cell populations.

Animals↗
PubMed1981

The ultrastructure of osteoclasts in microphthalmic mice.

Osteoclasts in microphthalmic (mi/mi) mice were significantly smaller than in their heterozygous (mi/+) or normal (+/+) littermates as measured by electronmicroscopic morphometry. Clear zones were also significantly smaller and normal ruffled borders were absent. The cytoplasm was not fully differentiated. No differences were seen between osteoclasts from mi/+ and +/+ mice. The number of osteoclasts was not different in mi/mi, mi/+ or +/+ mice. Lysosomal enzymes and collagenase content of bones in the three genotypes did not show marked differences.

Animals↗
PubMed1981

Effects of inhibition of microtubule assembly on bone mineral release and enzyme release by human breast cancer cells.

When supernates from the established human breast cancer cell line MCF-7 were applied to fetal rat long bones that had been labeled with 45Ca and devitalized to remove endogenous bone cells, mineral was released from the bones. The release of bone mineral by MCF-7 supernates was associated with increased basal release of hydrolytic enzyme activity by the tumor cells. The basal release of lysosomal enzymes and collagenolytic activity by MCF-7 cells with approximately twice that of mouse 3T3 cells, which did not cause mineral release by the fetal rat bones. Release of hydrolytic enzymes and bone mineral-releasing activity was increased by colchicine and vinblastine, drugs that inhibit microtubule assembly, but not affected by lumicolchicine. Time-course experiments performed on MCF-7 cells with or without colchicine showed that release of cathepsin D and collagenolytic activity was associated more closely with release of bone mineral and degradation of bone matrix than was the release of N-acetylglucosaminidase. The release of previously incorporated [3H]proline from the bones exposed to MCF-7 cell cultures was more closely associated with release of collagenolytic activity by MCF-7 cells than with release of cathepsin D or N-acetylglucosaminidase. These data suggest that breast cancer-mediated bone resorption in vitro is positively correlated with release of hydrolytic enzymes by the tumor cells, and release of these enzymes is enhanced by disassembly of microtubules.

Acetylglucosaminidase↗