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B F Sloane

Publications and source records attributed to B F Sloane.

At least 73 records · Page 4Linked to original sources
PubMed1990

Cathepsin B to cysteine proteinase inhibitor balance in metastatic cell subpopulations isolated from murine tumors.

Our laboratories have previously demonstrated that the malignancy of human and animal tumors is associated with increases in cathepsin B activity, due in part to increases in cathepsin B-specific RNA transcripts and in part to decreased regulation by the endogenous low molecular weight cysteine proteinase inhibitors (CPIs). In this study we have extended these observations to tumor cell subpopulations of B16 amelanotic melanoma (B16a) and Lewis lung carcinoma (3LL) isolated by centrifugal elutriation. B16a subpopulations exhibited a 10-fold differential in lung colonization potential, whereas 3LL subpopulations exhibited no differential. In the B16a subpopulations, cathepsin B activities, total cellular and plasma membrane-associated, corresponded positively (4- and 10-fold increase, respectively) with their lung colonization potentials. CPI activities, total cellular and plasma membrane-associated, corresponded inversely (2- and 5-fold decrease, respectively) with the lung colonization potential of the B16a subpopulations. In the 3LL subpopulations, neither cathepsin B nor CPI activities changed. In the plasma membrane fractions of all 3LL subpopulations the ratio of cathepsin B activity to CPI activity was less than 1, whereas in the plasma membrane fractions of all B16a subpopulations the ratio was 1 or greater. In the plasma membrane fractions of the B16a subpopulations of higher lung colonization potential the ratios were 2.5 and 7, indicating that the levels of endogenous CPIs in these fractions may not be sufficient to regulate cathepsin B activity. Cathepsin B mRNA levels were not increased in the B16a subpopulations expressing increased cathepsin B activity. Thus increased cathepsin B activity in these subpopulations was apparently due not to increased synthesis but to decreased regulation by the endogenous CPIs. These results suggest that membrane-associated cathepsin B and CPIs may both play a role in the expression of the experimental metastatic phenotype.

Animals↗
PubMed1990

Cathepsin B and its endogenous inhibitors: the role in tumor malignancy.

Several lysosomal proteinases including the cysteine proteinase cathepsin B have been implicated in malignant progression of tumors. Many investigators have demonstrated correlations between increased activity of cathepsin B and increased metastatic capability of animal tumors or malignancy of human tumors. Such increases in cathepsin B activity in malignant tumors may reflect alterations in synthesis, in activation and processing, and/or in intracellular trafficking and delivery as well as in the endogenous inhibitors of cathepsin B. Increases in mRNA transcripts for cathepsin B have been observed in both murine and human tumors and multiple transcripts for cathepsin B have been identified, but an association of multiple transcripts with malignancy has not been confirmed. Cathepsin B precursors found in human malignant ascites fluid do not possess mannose-rich carbohydrates suggesting that a defect in the post translational processing of carbohydrate moieties on tumor cathepsin B may be responsible for the release of cathepsin B observed in many tumor systems. However, the intracellular trafficking of cathepsin B responsible for its association with plasma membrane/endosomal systems and for its release will require further study as both latent, precursor forms of cathepsin B and native forms of cathepsin B are involved. We speculate that malignant tumor cells adherent to basement membrane are capable of forming a digestive microenvironment in which lysosomal proteinases such as cathepsin B function optimally, a microenvironment similar to that formed between adherent osteoclasts and bone. One of the endogenous cysteine proteinase inhibitors, stefin A, also is affected by malignancy. Reduced expression (mRNA and protein) of stefin A is found as well as a reduction in its inhibitory capacity against cysteine proteinases. The data to date at both the molecular and protein levels supporting a functional role(s) for cathepsin B and its endogenous inhibitors in cancer progression are only correlative. Experimental approaches utilizing well-defined model systems in conjunction with genetic manipulation of cathepsin B and its endogenous inhibitors are needed to provide convincing evidence that cathepsin B has an important role in cancer.

Animals↗
PubMed1990

Cathepsin B and cystatins: evidence for a role in cancer progression.

The cysteine proteinase cathepsin B has been implicated in the progression of tumors from a premalignant to a malignant state. Activity of cathepsin B has been shown to be elevated in parallel with malignancy or metastatic potential of human and rodent tumors. These increases in cathepsin B activity correspond in part to increases in mRNA for cathepsin B and in part to reduced regulation by endogenous low Mr cysteine proteinase inhibitors. Most properties of tumor cathepsin B appear to be similar to those of cathepsin B from normal tissues. However, the subcellular distribution of cathepsin B is altered in tumors, resulting in association of cathepsin B with plasma membrane fractions or in release of high Mr forms of cathepsin B into the extracellular milieu. Since cathepsin B can degrade laminin, fibronectin and type IV collagen, we speculate that the presence of cathepsin B at the surface of tumor cells may contribute to the local dissolution of basement membrane observed during tumor cell extravasation. Direct evidence that cathepsin B plays a role in cancer progression awaits studies in which upregulation or downregulation of the expression of cathepsin B and its endogenous inhibitors is found to alter tumorigenesis, metastatic potential, etc.

Animals↗
PubMed1989

Bacterial expression of human cysteine proteinase inhibitor stefin A.

Stefin A, a cysteine proteinase inhibitor of the cystatin superfamily, has been found to be most abundant in epidermal cells. In order to determine its cellular function, we have expressed human stefin A in Escherichia coli using plasmid expression vectors under the control of bacteriophage T7 RNA polymerase. The heat-stable, antibody-positive bacterial product was isolated using a papain-Sepharose affinity column and was shown to inhibit two cysteine proteinases, papain and human cathepsin B. Recombinant stefin A may have commercial and therapeutic potential in situations requiring inhibition of cysteine proteinase activities, and in cosmetics, as an ingredient in skin creams.

Blotting, Western↗
PubMed1989

Membrane-associated cathepsin L: a role in metastasis of melanomas.

Subcellular distribution of cathepsin L, the major protein released by transformed or ras transfected fibroblasts, was examined in murine liver, murine B16 amelanotic melanoma and human A2058 melanoma after sequential differential and Percoll density gradient centrifugation. In both murine and human melanomas, cathepsin L activity was found to be enriched in plasma membrane fractions; cathepsin L in these fractions was in both native and acid activatable forms. Plasma membrane fractions from B16 melanoma subpopulations of "low" and "high" metastatic potential were assayed for activity of cathepsin L and of heat stable endogenous inhibitors. The relative specific activity of cathepsin L was 7-fold greater in the subpopulation of "high" metastatic potential, whereas cysteine proteinase inhibitory activity was 5-fold less. Since cathepsin L can degrade intact basement membrane, this membrane-associated cathepsin L may well contribute to metastatic spread of melanomas.

Animals↗
PubMed1989

Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma.

Elevated activities of cysteine proteinases such as cathepsins B and L and cancer procoagulant have been linked to tumor malignancy. In the present study we examined the hypothesis that these elevated activities could be due to impaired regulation by the endogenous low molecular mass cysteine proteinase inhibitors (cystatins). Inhibitors from human sarcoma were compared to those from human liver, a normal tissue in which the inhibitors had been characterized previously. An extract of cystatins from sarcoma was less effective against papain and cathepsin B (liver or tumor) than was an extract from liver. This reduced inhibitory capacity in sarcoma was not due to a reduction in either the concentrations or specific activities of the cystatins or an absence of any family or isoform of cystatins. We purified two members of the cystatin superfamily (stefin A and stefin B) to homogeneity and determined their individual inhibitory properties. Stefins B from liver and sarcoma exhibited comparable inhibition of papain and cathepsin B. In contrast, stefin A from sarcoma exhibited a reduced ability to inhibit papain, human liver cathepsins B, H and L and human and murine tumor cathepsin B. The Ki for inhibition of liver cathepsin B by sarcoma stefin A was 10-fold higher than that for inhibition of liver cathepsin B by liver stefin A, reflecting a reduction in the rate constant for association and an increase in the rate constant for dissociation. Cancer is now the third pathologic condition reported to be associated with alterations in cystatins, the other two being amyloidosis and muscular dystrophy.

Chromatography, Affinity↗
PubMed1989

Enhanced levels of cathepsin B mRNA in murine tumors.

Relative amounts of mRNA for cathepsin B were measured in normal murine liver and three murine tumors, an invasive liver tumor (hepatoma, Hepa cl 9) and two melanoma variants (B16-F1 and B16 amelanotic melanoma, B16a). Using a human cDNA to the cathepsin B coding region as a hybridization probe, we detected two species of cathepsin B specific RNA transcripts (2.2 and 4.1 kb) in total RNA preparations of all four tissues. The concentrations of the 2.2 and 4.1 kb species were 3.6 and 2.7-fold greater in the highly metastatic B16a melanoma than in normal liver. The concentration of the 2.2 kb species in the invasive hepatoma was 1.7-fold greater than in normal liver. The increased levels of the 2.2 kb message were reflected in increases in activity of cathepsin B in both Hepa cl 9 and B16a.

Animals↗
PubMed1988

Role of the coagulation system in tumor-cell-induced platelet aggregation and metastasis.

The ability of tumor cells to initiate coagulation and subsequent platelet aggregation is believed to facilitate the metastatic process. The mechanism by which tumor cells initiate thrombotic alterations is unclear. We have purified a plasma membrane protein platelet aggregating activity/procoagulant activity (PAA/PCA) from several rodent tumors which initiates the coagulation of homologous plasma and aggregation of homologous platelets by a mechanism independent of factor VII. This protein does not possess any proteinase activity; however, its activity is dependent upon the presence of factor X. In addition, PAA/PCA requires reconstitution with phospholipid for expression of activity. These results suggest that tumor cells express a unique protein which possesses procoagulant activity resulting in thrombin generation. Thrombin is responsible for subsequent tumor-cell-induced platelet aggregation.

Amino Acids↗
PubMed1987

Properties of a plasma membrane-associated cathepsin B-like cysteine proteinase in metastatic B16 melanoma variants.

Activities of a cathepsin B-like cysteine proteinase have previously been observed to correlate with the malignancy of several animal and human tumors. Plasma membrane fractions of some of these tumors have been found to be enriched in cathepsin B-like activity. We have determined the subcellular distribution of this enzyme and three additional lysosomal hydrolases (cathepsin H, beta-hexosaminidase, and beta-glucuronidase) in normal murine liver and six metastatic variants of the B16 melanoma. The tissues were fractionated initially by differential centrifugation followed by Percoll density gradient centrifugation of the light mitochondrial fraction. Two fractions were obtained: an L-2 fraction enriched in all four lysosomal hydrolases; and an L-1 fraction enriched in a marker enzyme for the plasma membrane. Cathepsin B-like and beta-hexosaminidase activities, but not the other hydrolase activities, were also found to be enriched in the L-1 fractions of the metastatic B16 tumors. We explored the nature of the association of the cathepsin B-like activity with the plasma membrane using fractions from the spontaneously metastatic B16 amelanotic melanoma. Activity could not be dissociated from the plasma membrane fraction by washing with a physiological salt solution suggesting that it was not adsorbed to this fraction nonspecifically, nor could it be displaced by mannose 6-phosphate or other sugars which compete for binding to the known lysosomal receptors. High salt concentrations, low concentrations of the mild detergent saponin, mild acidification, or phosphatidylinositol-specific phospholipase C did not elute the cathepsin B-like activity. However, activity was eluted by exposure to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a detergent used in the purification of integral membrane proteins. The B16 amelanotic melanoma plasma membrane-associated cathepsin B-like activity had a slightly higher pH optimum and was resistant to inactivation by neutral pH and to inhibition by three low molecular weight inhibitors of cysteine proteinases. The Ki values for inhibition by leupeptin and stefin A were 20-fold higher. The presence of a cathepsin B-like cysteine proteinase at the surface of metastatic tumor cells, particularly in a form which can retain activity at physiological pH and retain activity in the presence of extracellular proteinase inhibitors, may contribute to the focal dissolution of the extracellular matrix observed at sites of contact with invading tumor cells.

Animals↗
PubMed1987

Role of platelet membrane in enhancement of tumor cell adhesion to endothelial cell extracellular matrix.

Tumor cell adhesion to subendothelial matrix in the presence of platelets and plasma has been examined in vitro using an entirely homologous system of rat Walker 256 carcinosarcoma cells, matrix laid down by rat aortic endothelial cells and rat platelets and plasma. In the presence of platelets or platelets plus plasma, tumor cell adhesion was significantly enhanced when compared to adhesion in the absence of platelets. In the presence of plasma alone (0.1%), we observed no significant increase in tumor cell adhesion. In order to determine which platelet factors contribute to the enhancement of tumor cell adhesion by platelets, we subjected washed rat platelets to mechanical lysis or thrombin stimulation followed by centrifugation. The membrane fractions and supernatant fractions containing platelet attachment proteins were compared for their abilities to support tumor cell adhesion to subendothelial matrix. Platelet membranes were also recombined with platelet supernatant fractions to determine if platelet attachment proteins or platelet membranes required the presence of the other to enhance tumor cell adhesion. Platelet supernatant fractions which contained release reaction proteins (confirmed by polyacrylamide gel electrophoresis) did not enhance tumor cell adhesion. Purified thrombospondin, fibronectin, beta-thromboglobulin, platelet derived growth factor, and serotonin had no effect on tumor cell adhesion. Platelet membrane containing fractions affected tumor cell adhesion to subendothelial matrix as follows: (a) platelets formed an adhesive bridge between tumor cells and the subendothelial matrix as demonstrated by scanning electron microscopy; (b) intact platelets and thrombin stimulated platelets were the most effective at facilitating tumor cell adhesion; (c) preparations containing partially lysed platelet ghosts were more effective in supporting tumor cell adhesion to subendothelial matrix than were preparations containing completely lysed platelet membrane fragments; (d) recombination of platelet supernatant fractions with mechanically lysed platelets did not enhance their ability to support adhesion; (e) fixed platelets, either alone or in combination with platelet supernatant fractions, failed to enhance adhesion. These data indicate that platelet enhanced tumor cell adhesion appears to be dependent on platelet membrane factors including receptor mobility, rather than intraplatelet components.

Animals↗
PubMed1987

The role of platelet cyclooxygenase and lipoxygenase pathways in tumor cell induced platelet aggregation.

Walker 256 carcinosarcoma cells induce the aggregation of rat platelets and concomitant production of eicosanoid metabolites (e.g., 12-hydroxyeicosatetraenoic acid, thromboxane A2). Cyclooxygenase inhibitors, but not lipoxygenase inhibitors, were able to inhibit platelet aggregation induced in vitro by low concentrations of agonists. At high agonist concentrations, neither cyclooxygenase nor lipoxygenase inhibitors affected platelet aggregation; however the combination of both inhibitors resulted in inhibition of aggregation. Also, a low concentration of agonist induced minimal eicosanoid metabolism, whereas a high concentration resulted in increased eicosanoid metabolism. These inhibitors, at the doses tested, did not inhibit protein kinase C activity.

Animals↗
PubMed1987

A new in vitro model for investigation of tumor cell-platelet-endothelial cell interactions and concomitant eicosanoid biosynthesis.

We have developed a new in vitro model system to examine tumor cell-platelet-endothelial cell interactions under dynamic conditions. Using the same model, we can determine endogenous eicosanoid metabolism and alterations in the prostacyclin-thromboxane A2 balance associated with interactions among tumor cells, platelets, and endothelial cells. The model consisted of cloned rat aortic endothelial cells grown on gelatin microcarrier beads under dynamic conditions (i.e., spinner culture). Interactions of these endothelial cells with platelets (heparinized rat platelet rich plasma) and/or tumor cells (rat Walker 256 carcinosarcoma) were assessed in an aggregometer. Gelatin beads alone or microcarrier grown endothelial cells did not elicit spontaneous aggregation of platelet rich plasma over a time period of 30 min. Microcarrier grown endothelial cells inhibited tumor cell induced platelet aggregation in a dose dependent fashion (i.e., depending on endothelial cell number). The ability of microcarrier grown endothelial cells to inhibit tumor cell induced platelet aggregation depended on endogenous production of prostacyclin. This conclusion is based on the following results: an increased number of microcarrier grown endothelial cells caused a prolongation of the aggregation lag time; an increased number of microcarrier grown endothelial cells caused a proportionate increase in 6-keto-prostaglandin F1 alpha concentration; an increased number of microcarrier grown endothelial cells was inversely correlated with thromboxane A2 production by platelets; indomethacin pretreatment of microcarrier grown endothelial cells caused a decrease in prostacyclin production and therefore overcame the associated inhibition of tumor cell induced platelet aggregation; and the inhibition of tumor cell induced platelet aggregation in the presence of endogenous prostacyclin produced by microcarrier grown endothelial cells was the same as that observed in the presence of exogenous prostacyclin. Scanning electron microscopy of aggregometry samples revealed: little or no platelet or tumor cell adhesion to gelatin beads alone, a low basal adhesion of tumor cells to microcarrier grown endothelial cells, and large aggregates of platelets and tumor cells located primarily at gaps in the monolayer of indomethacin treated microcarrier grown endothelial cells. This new in vitro model provides a method for examining the effects of eicosanoid metabolism by endothelial cells on tumor cell-platelet-endothelial cell interactions under dynamic conditions.

Blood Platelets↗
PubMed1987

Plasma membrane-associated cysteine proteinases in human and animal tumors.

The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.

Animals↗
PubMed1987

Inhibition of human tumor cell induced platelet aggregation by antibodies to platelet glycoproteins Ib and IIb/IIIa.

Tumor cell induced platelet aggregation was shown to be inhibited in a dose dependent manner by preincubation of human platelets with antibodies to platelet glycoprotein Ib and the IIb/IIIa complex. Combination of antibody to Ib and antibody to the IIb/IIIa complex at concentrations which produced half maximal inhibition of platelet aggregation alone caused complete inhibition of tumor cell induced platelet aggregation. Antibodies to platelet glycoproteins Ib and the IIb/IIIa complex also inhibited platelet synthesis of thromboxane A2, but not synthesis of 12-hydroxyeicosatrienoic acid. Inhibition of tumor cell induced platelet aggregation with antibodies against platelet glycoproteins suggests a role for these glycoproteins in tumor cell-platelet interactions and possibly platelet facilitated tumor cell metastasis.

Antibodies↗
PubMed1987

Tumor cell-platelet-endothelial cell interactions and prostaglandin metabolism.

We have proposed that tumor cell induction of platelet aggregation facilitates tumor cell attachment to endothelium and that this attachment is limited by endogenous PGI2. We have developed an in vitro model to study tumor cell-platelet-endothelial cell interactions in which endothelial cells are grown on microcarrier beads. Untreated endothelial cells inhibit tumor cell-induced platelet aggregation, as well as tumor cell attachment to endothelial cells. Inhibition of endothelial cell PGI2 production by indomethacin obviates this inhibition.

Animals↗