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B F Sloane

Publications and source records attributed to B F Sloane.

At least 55 records · Page 3Linked to original sources
PubMed1995

Mammalian cysteine protease inhibitors: biochemical properties and possible roles in tumor progression.

The endogenous cysteine protease inhibitors represent the final level at which cysteine protease activity can be regulated. These inhibitors are subdivided into three families (stefins, cystatins and kininogens) which belong to the protein superfamily, cystatins. Cystatins do not form a covalent bond with cysteine proteases, but instead cover the active site cleft blocking access to the active site. The most important biochemical characteristics of the cystatins are described in the first part of this review. Alterations in the balance between endogenous cysteine protease inhibitors and cysteine proteases have been postulated to contribute to malignant progression. A few studies have demonstrated the enrichment of cysteine protease inhibitor activity in the membrane fraction of tumors/cells. Evidence is accumulating that an inverse correlation exists between the level of stefin A, one of the cysteine protease inhibitors, and malignant progression. Stefin A has even been hypothesized to be a tumor suppressor. However, additional studies are necessary in order to prove functional roles for the individual cysteine protease inhibitors in tumor growth and progression.

Animals↗
PubMed1994

Pericellular pH affects distribution and secretion of cathepsin B in malignant cells.

Redistribution of lysosomes to the cell surface and secretion of lysosomal proteases appear to be general phenomena in cells that participate in local proteolysis. In the present study, we have determined whether malignant progression affects the intracellular distribution and secretion of the lysosomal protease cathepsin B in three model systems, each of which consists of cell pairs that differ in their degree of malignancy. The intracellular distribution of vesicles staining for cathepsin B was evaluated by immunofluorescent microscopy and the secretion of cathepsin B was evaluated by two complementary techniques: stopped assays of activity secreted into culture media; and continuous assays of activity secreted from viable (> or = 95%) cells growing on coverslips. We observed that the intracellular distribution of cathepsin B+ vesicles was more peripheral in the cells of higher malignancy in all three model systems and that active cathepsin B was secreted constitutively from these cells. Because an acidic pericellular pH has been shown to induce translocation of lysosomes in macrophages and fibroblasts, we evaluated the intracellular distribution of cathepsin B+ vesicles and secretion of cathepsin B in cell pairs incubated at slightly acidic pH. Acidic pericellular pH induced a redistribution of cathepsin B+ vesicles toward the cell periphery. In the more malignant cells, this resulted with time in reduced intracellular staining for cathepsin B and enhanced secretion of active cathepsin B. Translocation and secretion of cathepsin B were dependent on a functional microtubular system. Both the redistribution of cathepsin B+ vesicles toward the cell surface induced by acidic pH and the constitutive and acidic pH-induced secretion of active cathepsin B could be inhibited by microtubule poisons and stabilizers. We suggest that the redistribution of active cathepsin B to the surface of malignant cells and its secretion may facilitate invasion of these cells.

Animals↗
PubMed1994

Cathepsin B expression and localization in glioma progression and invasion.

The poor prognosis of human malignant gliomas is due to their invasion and recurrence, the molecular mechanisms of which remain poorly characterized. We have accumulated substantial evidence implicating the cysteine protease cathepsin B in human glioma malignancy. Increases in cathepsin B expression were observed throughout progression. In primary brain tumor tissue, transcript abundance (Northern blot analysis) increased in low-grade astrocytoma to high-grade glioblastoma from 3- to 6-fold, respectively, above normal brain levels. This increase correlated with increases in protein abundance (from + to ) as measured by immunohistochemistry. Furthermore, in glioblastoma cell lines increases in transcript abundance (ranging from 3- to 12-fold) were accompanied by increases in enzyme activity (44-133 nmol/min x mg protein). Altered subcellular localization was observed both immunohistochemically and by indirect immunofluorescence confocal microscopy and was found to correlate with increased grade. In addition, this increase in cathepsin B expression and altered subcellular localization correlated with histomorphological invasion and clinical evidence of invasion as detected by magnetic resonance imaging. These data support the hypothesis that cathepsin B plays a role in human glioma progression and invasion.

Animals↗
PubMed1994

Human gastric adenocarcinoma cathepsin B: isolation and sequencing of full-length cDNAs and polymorphisms of the gene.

Four full-length cDNA clones coding for preprocathepsin B were isolated from a human gastric adenocarcinoma cDNA library (AGS 1-6-30-1) and analyzed for possible sequence modifications that might be linked to altered intracellular trafficking and secretion of cathepsin B (CTSB) in malignant tumors. Comparison of AGS 1-6-30-1 cDNAs with human kidney/hepatoma cDNAs revealed: (1) three potential N-glycosylation sites instead of two, (2) a nucleotide (nt) substitution in the coding region for the propeptide from GTG to CTG which would result in a Val26-->Leu change, (3) three silent nt replacements in the coding region for the mature protein, (4) five single-nt differences in the 5'- and 3'-UTR (untranslated regions), (5) heterogeneity in the 5'-UTR, and (6) a 10-bp insertion in the 3'-UTR. The 10-bp insertion in the 3'-UTR may alter the stability of CTSB mRNA transcripts and thereby the expression of CTSB. These clones should be useful for expressing human tumor CTSB and analyzing the function of this enzyme in malignant progression. Two restriction-fragment length polymorphisms (RFLPs), EcoRI and TaqI, were detected by Southern blot analysis of genomic DNA from 36 unrelated Caucasians. Inheritance and distribution of the EcoRI alleles (13.0 and 11.0 kb) and the TaqI alleles (5.7 and 4.6 kb) indicated they were independent polymorphisms. In contrast to the EcoRI alleles of 13.0 and 11.0 kb observed in the population survey, genomic DNA from two AGS gastric adenocarcinoma subclones revealed two EcoRI alleles of 13.0 and 7.8 kb.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1994

A lipoxygenase metabolite, 12-(S)-HETE, stimulates protein kinase C-mediated release of cathepsin B from malignant cells.

The process of tumor cell invasion of the basement membrane is proposed to consist of three steps: attachment, local proteolysis and migration. 12-(S)-HETE, a 12-lipoxygenase metabolite of arachidonic acid, upregulates surface expression of integrin cytoadhesins and an autocrine motility factor receptor, suggesting that this metabolite may play an important regulatory function in tumor cell invasion. In the present study, we determined whether 12-(S)-HETE affects surface expression and/or release of cathepsin B, a cysteine protease that has been implicated in focal degradation of basement membrane. Secretion and distribution of cathepsin B was evaluated in two model systems for various stages of neoplastic progression: (i) murine B16 melanoma lines of low (B16-F1) and high (B16a) lung colonization potential, and (ii) immortalized and ras-transfected MCF-10 human breast epithelial cells that differ in their invasive capacities in vitro. In the B16a cells, 12-(S)-HETE induced release of native and latent cathepsin B activity and concomitantly reduced cell-associated cathepsin B immunoreactivity. In contrast, 12-(S)-HETE did not induce the release of cathepsin B from B16-F1 cells, suggesting that there may be an enhanced response to 12-(S)-HETE in more malignant cells. This was confirmed in the MCF-10 system, in which 12-(S)-HETE was able to induce the release of cathepsin B from the ras-transfected cells, but not from the immortal cells. A simultaneous reduction in staining for cathepsin B was observed in the ras-transfected cells, but not in their immortal counterparts. The release of cathepsin B may be mediated by PKC as pretreatment of B16a cells with the selective PKC inhibitor calphostin C, but not with the PKA inhibitor H8, prevented the stimulated release of cathepsin B. In B16a cells, the release of cathepsin B was accompanied by a translocation toward the cell periphery of vesicles staining for cathepsin B, resulting in focal areas of accumulation of cathepsin B. After 12-(S)-HETE stimulation of the ras-transfected MCF-10 cells, cathepsin B was distributed homogeneously on the apical surface. Thus, 12-(S)-HETE can upregulate the surface expression on tumor cells of proteins able to mediate each of the three steps of tumor cell invasion: adhesion, degradation, and migration.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1994

Immunohistochemical analysis of cathepsins D, B, and L in human breast cancer.

Cathepsins D, B, and L are acidic lysosomal proteinases involved in intracellular protein turnover. Increased levels of these enzymes have been reported to be indicators of aggressive tumor behavior in human and rodent tumors. In breast cancer increased levels of cathepsin D have been reported to be an independent prognostic factor in women with stage I disease. We used standard immunohistochemical techniques on formalin-fixed, paraffin-embedded tissue to examine the levels of cathepsins D, B, and L in 80 carcinomas of the breast and compared that with other indicators of aggressive tumor behavior, including stage of disease, tumor size, nuclear grade, estrogen receptor status, disease recurrence, and 5-year survival rates. Positive granular cytoplasmic staining was detected for cathepsin D in 90% of the tumors, for cathepsin B in two thirds of the tumors, and for cathepsin L in approximately one half of the tumors. Positive staining also was seen in normal breast epithelium, areas of apocrine metaplasia, stromal fibroblasts, and macrophages. Our results did not show a correlation between the expression of cathepsins D, B, and L and other indicators of aggressive tumor behavior. We conclude that the results obtained using polyclonal anticathepsin antibodies do not support the prognostic usefulness of immunohistochemical analysis of these three proteinases in tumor cells in human breast cancer.

Adult↗
PubMed1994

Membrane association of cathepsin B can be induced by transfection of human breast epithelial cells with c-Ha-ras oncogene.

Alterations in trafficking and increases in expression of the lysosomal proteases cathepsins B, D and L have been observed in transformed cells and malignant tumors, including human breast carcinoma. ras and the related rab proteins participate in the vesicular transport processes required for normal trafficking of lysosomal enzymes. In addition, transfection of murine fibroblasts with the ras oncogene has been shown to increase the expression of cathepsins L and B. As human cancers are primarily epithelial in origin, we have investigated whether there are alterations in the trafficking and expression of cathepsin B in MCF-10 human breast epithelial cells transfected with wild-type and mutated ras. In all cells examined, i.e. mortal MCF-10M cells, immortal MCF-10A or MCF-10F cells, and transfected MCF-10A cells (transfected with the neomycin resistance gene (MCF-10Aneo) or cotransfected with wild-type proto-oncogenic ras (MCF-10AneoN) or mutated oncogenic ras (MCF-10AneoT)), levels of mRNA transcripts for cathepsin B were similar. However, alterations in trafficking of cathepsin B were observed in the cells transfected with oncogenic ras. In these cells there was an increased association of cathepsin B activity and cathepsin B protein with plasma membrane/endosomal fractions and a more peripheral distribution of immunofluorescent staining for cathepsin B. At the electron microscopic level, immunogold labeling for cathepsin B was localized to the cell membrane as well as to vesicles in the microvilli and adjacent to the cell membrane. In the parental MCF-10A cells, in contrast, cathepsin B was localized to vesicles in the perinuclear region. The cathepsin B associated with plasma membrane/endosomal fractions in the cells transfected with oncogenic ras was mature cathepsin B as demonstrated by immunoblot analysis. This was confirmed further by showing an absence of peripheral immunofluorescent staining in these cells using an antibody specific for the propeptide of cathepsin B. Thus, we have demonstrated by multiple techniques that transfection of human breast epithelial cells with oncogenic ras results in alterations in the trafficking of cathepsin B similar to those observed previously in human and animal tumors of both epithelial and mesenchymal origin.

Breast↗
PubMed1994

Cathepsin B expression in colorectal carcinomas correlates with tumor progression and shortened patient survival.

Cathepsin B is a lysosomal cysteine proteinase that has the ability to degrade several extracellular matrix components at both neutral and acidic pH and has been implicated in the progression of several human and rodent tumors. We have studied the expression of cathepsin B in human colorectal tissues using a monospecific polyclonal rabbit antibody raised against human liver cathepsin B. In immunoblots of normal and neoplastic colorectal tissues this antibody specifically recognized only cathepsin B. We studied 101 cases of formalin-fixed, paraffin-embedded tissue (15 normal mucosa, 17 adenomas, and 69 carcinomas). Epithelial cells of normal mucosa and adenomas were either negative or showed a weak granular reactivity located in the paranuclear and apical cytoplasm of superficial cells. Small clusters of histiocytes were also positive in the region of the superficial area of the lamina propria. In carcinomas, increased expression of cathepsin B correlated with advanced stage of the disease. Increased immunoreactivity of cathepsin B in malignant cells was associated with either a diffuse cytoplasmic staining or was polarized to the basal pole of the cells. This is in contrast to the punctate paranuclear staining pattern observed in normal colonic mucosal cells. In tumor stromal cells, increased expression of the enzyme correlated with neoplastic progression. Expression of high levels of cathepsin B in the tumor epithelial cells was associated with a significantly shorter survival of the patients. In conclusion, our results indicate that cathepsin B expression is up-regulated in human colorectal carcinomas compared with normal mucosa and adenomas and correlates with tumor progression.

Adenoma↗
PubMed1994

Increased gelatinase A (MMP-2) and cathepsin B activity in invasive tumor regions of human colon cancer samples.

Gelatinase A (MMP-2) and cathepsin B are proteinases which have been proposed to participate in human tumor invasion and metastasis. Precise quantitation of the activity of these enzymes in invading tumors has not been previously described. We utilized a novel tissue microdissection technique to determine levels of enzyme activity in specific microscopic areas of invasive human colon cancer. Tissue specimens smaller than one high power field can be extracted from the samples and analyzed. Increased levels of pro-enzyme and active enzyme forms of gelatinase A (MMP-2) and increased cathepsin B activity were localized in regions of tumor invasion as compared with a matched number of normal epithelial cells from the same patient. Levels of progelatinase B (MMP-9) were also increased in the tumors; however, we did not observe activation of this enzyme. To investigate the mechanism of gelatinase A activation, we amplified DNA of specific microdissected tumor cell populations using polymerase chain reaction. We did not detect a mutation in the activation locus of the enzyme in any of the tumors studied, which suggests that activation may be due to up-regulation of a tumor-associated gelatinase A activating species. Microdissection of frozen tissue sections may prove valuable in the study of proteinases in human tumor invasion as well as in the detection of genetic alterations in human cancers.

Base Sequence↗
PubMed1994

Clinicopathologic significance of cathepsin B immunostaining in transitional neoplasia.

Fifty Stage heterogeneous urinary bladder carcinomas were immunostained for cathepsin B, a lysosomal endoproteinase putatively associated with tumor invasion. Neoplastic cell CB immunoreactivity was strongly correlated to both grade (I/II--42% positive versus III--68% positive, P = 0.01) and invasion beyond the lamina propria (Ta/T1--42% positive versus T2/T3--68% positive, P = 0.02). Most low grade, papillary tumors displayed a granular cytoplasmic staining pattern, compatible with lysosomal distribution, in contrast to high grade tumors, in which diffuse staining was present in the cytoplasm. Staining was also accentuated at the advancing front of invading tumors and in angiolymphatic tumor emboli. Non-neoplastic mononuclear inflammatory cells, particularly those at the host-tumor interface, displayed variable, sometimes intense staining. Strong tumor-cell CB was more frequent among recurrent TCC than in patients who remain free of disease (55% versus 29%, n = 18, T2-3, cystectomy, 5-yr min. follow-up). We conclude these observed staining patterns and grade/stage associations are compatible with an important biological role for CB in facilitating host invasion in some bladder tumors. Levels and/or distribution of CB may also be of potential value in defining clinically aggressive tumor subsets.

Carcinoma, Transitional Cell↗
PubMed1993

Localization of a biotinylated cathepsin B oligonucleotide probe in human prostate including invasive cells and invasive edges by in situ hybridization.

The cysteine endopeptidase cathepsin B (CB) can degrade basement membrane (BM) proteins (such as laminin, type IV collagen, and fibronectin) at both acid and neutral pHs suggesting that CB has a role in tumor invasion and distant metastasis. The distribution and intensity of CB protein localization vary in normal prostate, benign prostatic hyperplasia (BPH), and neoplastic prostate. These considerations have led us to examine whether the distribution of CB localization in malignant and normal cells is due to storage or active synthesis of CB. In the present study, we examined the localization patterns of CB at the mRNA level in normal prostate, BPH, and well to moderately differentiated neoplastic prostate, focusing on invasive groups of cells and invasive edges of malignant tumors. We used a 25-base biotinylated oligonucleotide CB cDNA "sense" probe to localize CB message in prostate samples obtained from radical prostatectomies. We have determined that CB is actively synthesized by the epithelia of normal, hyperplastic, and neoplastic prostate including some invasive cells in the invasive edges. In both normal and BPH, CB mRNA was localized predominantly in acinar basal cells with some localization in cuboidal/columnar cells. In contrast, in neoplastic prostate, CB mRNA was localized predominantly in columnar cells and in groups of invasive cells and invasive edges. Thus, in malignant prostate the predominant cell types expressing CB differed from those of the normal prostate and BPH. Analysis of CB mRNA localizations indicated a heterogeneity in staining distribution in prostate cancer with some invasive groups of cells and invasive edges exhibiting CB mRNA and others exhibiting little or no reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

A membrane-associated cysteine protease inhibitor from murine hepatoma.

A cysteine protease inhibitor was purified from total membrane fractions of an invasive murine hepatoma, Hepa cl 9. On gel filtration under non-reducing conditions the purified inhibitor was eluted in a single peak of M(r) 10-15 kDa, but resolved as two bands at 14 and 70 kDa on SDS-PAGE under reducing conditions. By isoelectric focusing, the inhibitor ran at an isoelectric point of 4.75. Immunoblotting studies using the enhanced chemiluminescence technique indicated no crossreactivity with monoclonal antibodies to stefin B and cystatin C or with a polyclonal antibody to low M(r) kininogen. In contrast, the 14 kDa and 70 kDa bands both crossreacted with a polyclonal antibody to stefin A, suggesting that the cysteine protease inhibitor associated with Hepa cl 9 membranes may be a modified form of stefin A.

Animals↗
PubMed1992

Human tumour cathepsin B. Comparison with normal liver cathepsin B.

Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.

Amino Acid Sequence↗
PubMed1992

Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues.

Our laboratory has previously demonstrated that increased malignancy of several histological types of human and animal tumours is associated with increases in their cathepsin B activity, particularly cathepsin B activity associated with plasma-membrane/endosomal vesicles or shed vesicles. Here we report that cathepsin B from normal or tumour tissues degrades purified extracellular-matrix components, type IV collagen, laminin and fibronectin, at both acid pH and neutral pH. The number and sizes of degradation products were analysed by SDS/PAGE. Cathepsin B from both sources exhibited similar activities towards, and similar patterns of cleavage of, the extracellular-matrix proteins. At neutral pH, cathepsin B from both sources appeared to undergo autodegradation, a process that was decreased in the presence of alternative substrates such as the extracellular-matrix proteins. Cathepsin B readily degraded type IV collagen at 25 degrees C, indicating activity towards native type IV collagen. Fibronectin degradation products of 100-200 kDa and of 18 and 22 kDa were observed. A single 70 kDa fragment was released from laminin under non-reducing conditions and multiple fragments ranging from 45 to 200 kDa under reducing conditions. These results suggest that cathepsin B at or near the surface of malignant tumour cells may play a functional role in the focal dissolution of extracellular matrices.

Amino Acid Sequence↗
PubMed1992

Cysteine endopeptidases and their inhibitors in malignant progression of rat embryo fibroblasts.

Cathepsins B and L and their endogenous inhibitors were evaluated in rat embryo fibroblast lines which have been developed as a model system for the study of malignant progression and metastatic capability. Three groups of lines were analyzed: 1) immortalized/non-tumorigenic, 2) tumorigenic/metastatic lines transfected with c-Ha-ras, and 3) metastatic revertants transfected with c-Ha-ras+the E1A region of adenovirus type 2. The metastatic revertants are tumorigenic, but non-metastatic. No correlation was seen between tumorigenicity and metastatic potential and the level of expression of cathepsin B or the subcellular distribution of cathepsins B and L. However, cathepsin L activity was increased 2-fold in the 4R metastatic line. Although transfection of aneuploid 3T3 fibroblasts with ras has been shown to increase the expression of cathepsin L and cathepsin B, transfection of the diploid rat embryo fibroblasts with ras did not correlate with increased expression of cathepsin L or cathepsin B. However, ras transfection of the rat embryo fibroblasts was associated with a significant (4-15-fold) decrease in the activity of heat-stable cysteine endopeptidase inhibitors. Thus, in tumorigenic rat embryo fibroblast lines, regulation of the activities of cysteine endopeptidases by their endogenous inhibitors may be compromised, resulting in increased effective activities of the cysteine endopeptidases.

Adenoviridae↗
PubMed1991

Mapping of the gene for human cysteine proteinase inhibitor stefin A, STF1, to chromosome 3cen-q21.

The gene for the human cysteine proteinase inhibitor stefin A (STF1), alias cystatin A, has been mapped to chromosome 3, using the polymerase chain reaction to specifically amplify the human stefin A sequence in human-hamster hybrid DNA. STF1 is further sublocalized to regions between centromere and 3q21 using a deletion mapping panel for this chromosome. This assignment shows that stefin A is not syntenic with cystatin C which has been localized to chromosome 20.

Animals↗
PubMed1991

The malignant phenotype and cysteine proteinases.

Expression, membrane association and secretion of both cathepsin B and cathepsin L have been associated with the malignant phenotype of murine B16 melanomas. Only native forms of the two enzymes were found in the lysosomal fractions, whereas both native and latent forms were found in the membrane fractions. Brief exposure to acid pH induced secretion of only native forms of both cathepsin B and cathepsin L and a concomitant reduction in membrane-associated activities. Thus, the pericellular acidification associated with malignant tumors may provide optimal conditions for proteolysis by the cysteine proteinases in terms of their enhanced stability and their induced release in native forms not requiring proteolytic activation.

Animals↗