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B F Sloane

Publications and source records attributed to B F Sloane.

At least 37 records · Page 2Linked to original sources
PubMed1998

Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells.

AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-->Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.

Animals↗
PubMed1998

Differential localization of cysteine protease inhibitors and a target cysteine protease, cathepsin B, by immuno-confocal microscopy.

The cystatin superfamily of cysteine protease inhibitors and target cysteine proteases such as cathepsin B have been implicated in malignant progression. The respective cellular/extracellular localization of cystatins and cysteine proteases in tumors may be critical in regulating activity of the enzymes. Confocal microscopy has enabled us to demonstrate the differential localization of cystatins and cathepsin B in an embryonic liver cell line and an invasive hepatoma cell line. In both, stefins A and B were distributed diffusely throughout the cytoplasm, whereas cystatin C was distributed in juxtanuclear vesicles. Stefin A and cystatin C, but not stefin B, were present on the cell surface. Cystatin C was found on the top surfaces of both cell lines, whereas stefin A was found only on the top surface of the embryonic liver cells. Cathepsin B staining was concentrated in perinuclear vesicles in the embryonic liver cells. In the hepatoma cells, staining for cathepsin B was also present in vesicles adjacent to the cell membrane and on localized regions of the bottom surface. Such a disparate distribution of cathepsin B and its endogenous inhibitors may facilitate proteolysis by the hepatoma cells and thereby contribute to their invasive phenotype.

Animals↗
PubMed1998

Cathepsin B on invasion and metastasis of gastric carcinoma.

OBJECTIVE: To investigate the expression of cathepsin B (CB) in human gastric carcinoma tissue. METHODS: The expression of CB in human gastric tissue was studied by using monospecific polyclonal rabbit antibody raised against human liver CB for immunohistochemistry, and full length cDNA of CB for in situ hybridization and dot blot. RESULTS: CB overexpression in gastric carcinoma was found when compared with non-neoplastic gastric tissue at both mRNA and protein levels. Diffuse cytoplasmic CB staining of mRNA and protein were identified in malignant cells of 53.3% and 69.1% of gastric adenocarcinoma respectively. The increased staining of CB in malignant cells was associated with the depth of the invasiveness and growth pattern as well as metastasis of lymph nodes, but not with the histological classification. It was also found that there were the expression of CB in stromal cells of the tumor and the expression localized mainly in the endothelial cells of the microvessels which correlated with angiogenesis. CONCLUSION: These results indicate that the expression of CB in gastric carcinoma is related to tumor progression, and leads to development of the invasive phenotype.

Adenocarcinoma↗
PubMed1998

Cathepsin B and human tumor progression.

Cathepsin B has been implicated in progression of various human tumors. Overexpression of cathepsin B mRNA, increased cathepsin B staining and elevated cathepsin B activity have been found in different human cancers. These occur especially at the invasive edges of cancers, suggesting a role for cathepsin B in tumor invasion. In some tumors, mRNA expression and protein staining for cathepsin B correlate with clinical progression. Cathepsin B can facilitate tumor progression directly through degradation of components of the basement membrane and extracellular matrix. In vitro studies show that cathepsin B may exert its degradative effects intracellularly or extracellularly, depending on the cell type and location of cathepsin B. Cathepsin B can also facilitate tumor progression indirectly through activation of other latent proteases and/or degradation of protein inhibitors of other proteases. Thus cathepsin B may be an integral component of the proteolytic cascade linked to malignant progression of human tumors.

Animals↗
PubMed1997

Exon 2 of human cathepsin B derives from an Alu element.

Transcripts for the cysteine protease cathepsin B are alternatively spliced in the untranslated regions (UTRs). We show that a cathepsin B probe containing 5'-UTR sequences hybridized to an RNA of approximately 300 nt in addition to the typical 2.2 and 4.0 kbp mRNAs. Within this 5'-UTR, exon 2 was found to be homologous to Alu repetitive elements. Specifically, exon 2 was part of an Alu element interspersed with the cathepsin B gene. The approximately 300 nt band that hybridized to our cathepsin B probe likely corresponds to Alu transcripts, which are known to accumulate in human cells. Indeed, a similarly migrating band was detected with an authentic Alu probe. Thus, we suggest that primary transcripts for cathepsin B contain Alu sequences which are preserved as exon 2 in some fully spliced mRNAs.

Alternative Splicing↗
PubMed1997

Oncogenic c-Ki-ras but not oncogenic c-Ha-ras up-regulates CEA expression and disrupts basolateral polarity in colon epithelial cells.

Colon carcinomas commonly contain mutations in Ki-ras4B, but very rarely in Ha-ras, suggesting that different Ras isoforms may have distinct functions in colon epithelial cell biology. In an earlier study we had demonstrated that oncogenic Ki-ras4BVal-12, but not oncogenic Ha-rasVal-12, blocks the apicobasal polarization of colon epithelial cells by preventing normal glycosylation of the integrin beta1 chain of the collagen receptor. As a result, only the Ki-ras mutated cells exhibited altered cell to substratum attachment, whereas mutation of either Ras isoform activated mitogen-activated protein kinases. We have now asked whether intercellular adhesion proteins implicated in establishing basolateral polarity in colon epithelial cells are modulated by oncogenic Ki-Ras4BVal-12 proteins but not oncogenic Ha-RasVal-12 proteins. The embryonic adhesion protein carcinoembryonic antigen (CEA) was up-regulated on the mRNA and protein levels in each of three stable Ki-rasVal-12 transfectant lines but in none of three stable Ha-rasVal-12 transfectant lines. The elevated protein levels of CEA in Ki-ras4BVal-12 transfectant cells were decreased by blocking expression of Ki-ras4BVal-12 with antisense oligonucleotides. N-cadherin levels were decreased in only the Ki-ras transfectants, whereas E-cadherin levels were unchanged. Immunohistochemical analysis demonstrated that Ki-ras4BVal-12 transfectant cells did not polarize into cells with discrete apical and basal regions and so could not restrict expression of CEA to the apical region. These unpolarized cells displayed elevated levels of CEA all along their surface membrane where CEA mediated random, multilayered associations of tumor cells. This aggregation was both calcium-independent and blocked by Fab' fragments of anti-CEA monoclonal antibody col-1. Trafficking of the lysosomal cysteine protease cathepsin B may also be altered when cell polarity cannot be established. Ki-ras4BVal-12 transfectant cells expressed 2-fold elevated protein levels of the lysosomal cysteine protease cathepsin B but did not up-regulate cathepsin B mRNA expression. One function of oncogenic c-Ki-Ras proteins in colon cancer progression may be to up-regulate CEA and thus to prevent the lateral adhesion of adjacent colon epithelial cells that normally form a monolayer in vivo.

Carcinoembryonic Antigen↗
PubMed1996

Expression of functional recombinant human procathepsin B in mammalian cells.

Cathepsin B has been implicated in numerous pathobiological processes. In order to study its interactions with other proteins implicated in these processes, quantities of functional recombinant cathepsin B are needed. Therefore, we expressed recombinant human procathepsin B in mammalian cells (BSC-1 monkey kidney cells and HeLa human cervical carcinoma cells) using a vaccinia virus expression system. The recombinant human procathepsin B appeared to be authentic and expressed in its native conformation as indicated by: (1) N-terminal sequencing; (2) molecular size; (3) processing intracellularly to mature double-chain cathepsin B; (4) in vitro cleavage by pepsin to mature cathepsin B coincident with appearance of activity against a selective synthetic substrate; and (5) substrate/inhibitor profiles. This is the first report of the expression of functional recombinant human procathepsin B in mammalian cells. We also report a single-step immunoaffinity purification procedure for the isolation of electrophoretically pure proenzyme. By the methodologies described, human procathepsin B can now be obtained in high yield. This should facilitate studies of its interactions with protease inhibitors, other proteases, extracellular matrices, cell-surface proteins and biological substrates that may be of relevance to the pathobiological functions of this enzyme.

Adenocarcinoma↗
PubMed1996

Cathepsin B expression in human tumors.

Cathepsin B has been linked to tumor progression through observations that its activity, secretion or membrane association are increased. The most malignant tumors, and specifically the cells at the invasive edge of those tumors, express the highest activity. Cathepsin B may facilitate invasion directly by dissolving extracellular matrix barriers like the basement membrane, or indirectly by activating other proteases capable of digesting the extracellular matrix. Cathepsin B also might play a role in tumor growth and angiogenesis. Cathepsin B activity is the result of several levels of regulation: transcription, post-transcription processing, translation and glycosylation, maturation and trafficking, and inhibition. The majority of reports on cathepsin B expression in tumors have focused on measurements of activity or protein staining. In some tumors, e.g. gliomas, a correlation between the amounts of cathepsin B mRNA, protein and activity and tumor progression has been established. Regulation of cathepsin B at the transcriptional and post-transcriptional levels is still poorly understood. Although the putative promoter regions have characteristics of housekeeping-type promoters, cathepsin B mRNA expression varies depending on the cell type and state of differentiation. We have evidence that more than one promoter could direct expression of human cathepsin B. Multiple transcript species have been detected, resulting from alternative splicing in the 5'- or 3'-untranslated regions, and possibly the use of alternative promoter regions. The existence of transcript variants indicates a potential for post-transcriptional control of expression. In support of this, ras-transformation of MCF-10A human breast epithelial cells results in an increase in protein levels without a concomitant increase in mRNA levels. Cathepsin B mRNA species with distinct 5'- or 3'-untranslated regions may differ in their stability and translatability. Variations in the coding region may also alter cathepsin B properties. We and Frankfater's group have observed transcript species that would encode a truncated protein, lacking the prepeptide and about half of the propeptide. This truncated protein, if synthesized in cells, would be expected to be cytosolic; therefore its function is unclear. Once the several mechanisms of regulation of cathepsin B expression and activity are better understood, they could provide us with new strategies to specifically reduce cathepsin B activity in tumors.

Biomarkers, Tumor↗
PubMed1996

Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

Animals↗
PubMed1996

Tumor progression and angiogenesis: cathepsin B & Co.

Experimental and clinical evidence reveals that the growth of solid tumors is dependent on angiogenesis. Proteolytic enzymes such as plasminogen activators and matrix metalloproteinases have been implicated in this neovascularization. The role of lysosomal proteases in this process has yet to be explored. Increased expression of the lysosomal cysteine protease cathepsin B has been observed in many etiologically different tumors, including human brain, prostate, breast, and gastrointestinal cancers. Immunohistochemical and in situ histochemical studies have demonstrated expression of cathepsin B in neovessels induced during malignant progression of human glioblastoma and prostate carcinomas. In these two tumor types, neovessels stain strongly for cathepsin B compared with the normal microvasculature. As an initial point to elucidate whether cathepsin B is an important component of the angiogenic response in tumours, we analyzed expression of cathepsin B in endothelial cells during neovessel formation. We present evidence for strong immunostaining of cathepsin B in rat brain microvascular endothelial cells as they form capillary tubes in vitro. This finding is discussed within the general framework of the role of proteolytic enzymes in tumor invasion and angiogenesis.

Animals↗
PubMed1996

Cathepsin B: multiple enzyme forms from a single gene and their relation to cancer.

Overexpression and altered trafficking of cathepsin B characterize the malignant phenotype of tumor cells. Human cathepsin B is encoded by a single-copy gene located on chromosome 8p22. With its 13 exons, the gene encompasses at least 27 kb of DNA. Expression of cathepsin B can be regulated at transcriptional and posttranscriptional levels. Multiple cathepsin B mRNA species arising from alternative splicing may be related to tissue- and tumor-specific differences in expression. There is selective overexpression, increased activity, membrane association and secretion of cathepsin B in many etiologically different cancers. This suggests that cathepsin B may play a functional role in malignant progression. Recent clinical studies provide confirmatory evidence in that cathepsin B expression is a prognostic indicator in colon carcinoma.

Alternative Splicing↗
PubMed1995

Identification of two new exons and multiple transcription start points in the 5'-untranslated region of the human cathepsin-B-encoding gene.

Transcripts for cysteine protease cathepsin B (CTSB) were found to be highly variable in the 5'-UTR (untranslated region). In cDNA clones from a human gastric adenocarcinoma cDNA library, we have identified two new exons (designated 2a and 2b) between exons 2 and 3 in the 5'-UTR of the gene. All of the exons of the 5'-UTR could be alternatively spliced to produce several transcript species. In addition, transcription was initiated from more than one promoter region. Using RT-PCR (reverse transcription-polymerase chain reaction) and primer extension assays, CTSB mRNA species were found to differ among tissues and between a glioblastoma sample and a cell line derived from it. Exons 2a and 2b were detected more frequently in tumor samples than in matched normal samples. Thus, factors related to the cell differentiation and environment seem likely to determine the types of transcripts that are expressed which in turn could influence the overall steady-state level of CTSB mRNAs and their rate of translation. Interestingly, at least three upstream translation initiation codons were observed and could constitute a means of controlling translation initiation.

Adenocarcinoma↗
PubMed1995

Cathepsin B in angiogenesis of human prostate: an immunohistochemical and immunoelectron microscopic analysis.

BACKGROUND: Angiogenesis (or neovascularization) is required for the growth of solid organ tumors and precedes invasion of the adjacent stroma by neoplastic cells. We investigated the relative density and distribution of cathepsin B (CB) immunostained microvessels (i.e., small blood vessels and capillaries) in benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and prostatic adenocarcinoma (CAP) by immunocytochemical localization of an antibody directed against a cathepsin B-derived synthetic peptide (Syn-CB). METHODS: We studied 16 formalin-fixed, prostatectomy specimens that were embedded in paraffin/paraplast for histological examination by hematoxylin and eosin and immuno-localization of the Syn-CB antibody. Selected paraformaldehyde-fixed specimens were embedded in K4M Lowicryl or LRWhite resins. We localized the antibody in thin sections using immunoelectron microscopy techniques. RESULTS: Eight patients had BPH [4 patients with BPH alone, 2 with BPH and PIN, and 2 with BPH and CAP]. Ten cancer cases included one with Gleason histologic score 4, two with score 6, four with score 7, and three with score 8. In CAP cases, Gleason score 6 and 7 tumors had more microvessels than the score 4 or 8 tumors. In both BPH and CAP cases, the antibody was localized chiefly in the endothelial cells of microvessels, but occasionally in ductal and glandular epithelial cells. Ultrastructurally, CB-immunoreactive gold particles were markedly increased at the luminal and basal plasma membrane surfaces and folds of endothelial cells in neoplastic prostate, but not in the endothelial cells of BPH. Furthermore, the presence of CB localizing gold particles in collagen and smooth muscle fibers near the microvessels indicated leakage of the enzyme in prostatic stroma of neoplastic prostate. Similar leakage was not observed in BPH. Morphometric analysis showed that the relative density of microvessels increased two to three times in cancer patients when compared to patients with BPH alone. Our study also indicated that BPH associated with PIN or CAP had an increased density of microvessels when compared to BPH alone. CONCLUSIONS: Our study showed that the relative density and distribution of microvessels are the most important features of neovascularization in prostatic tumors. The relative density of microvessels increased in PIN and CAP when compared to BPH alone. Although the localization of CB is associated with lysosomes of endothelial cells in both BPH and CAP, there is a greater association of CB with the plasma membranes of endothelial cells in CAP than BPH. Immunoelectron microscopy provided evidence that CB might be involved in dissolution of basement membranes in neoplastic tumors during angiogenesis. CB localization has the potential of defining a role for this protease in degradation of extracellular matrix constituents during early steps of angiogenesis.

Adenocarcinoma↗
PubMed1995

Immunohistochemical localization of cathepsin B in neoplastic human prostate.

Cathepsin B (CB) has been shown to degrade extracellular matrix (ECM) proteins, and has been reported to be involved in invasion and metastasis of several types of solid organ tumors in human and animals, but CB has not been studied in human prostate cancer (CAP). Our objective was to determine the CB protein immunostaining pattern in CAP and to correlate the immunostaining with the degree of malignancy as reflected in the Gleason grading system. We used two types of CB antibodies (namely, monospecific, polyclonal antibodies to human liver CB prepared in rabbits, and polyclonal antibody produced in sheep) to establish CB localization patterns in neoplastic prostate. Our analysis showed a heterogeneous CB immunostaining pattern in the neoplastic human prostate. CB immunostaining occurred in many, but not all, of the neoplastic columnar/cuboidal cells of acini and isolated cells, i.e., in small ragged glands and clusters (groups) of invasive cells in the prostatic stroma. We have shown that, in general, there was a positive correlation of the intensity of CB immunostaining with the Gleason histologic score (or Gleason grade sum) tumors, i.e., from the lowest scores through score 8, but many of the tumors with scores 9 and 10 showed little CB immunostaining. Our study indicated that the increased CB immunostaining in the Gleason grade sum 5-8 tumors may be associated with increased degradation of ECM, but not in 9 and 10 despite the fact that the latter tumors are more malignant clinically. In well-differentiated tumors, fewer CB immunostaining cells were present than the moderately-differentiated tumors. In other words, most of the stromal invasion of the prostatic ECM occurred in tumors of Gleason grade sums 5-8. We suggest that CB immunostaining might be a useful method to assess stromal invasion of prostatic carcinoma, especially in the higher grade tumors.

Animals↗
PubMed1995

Microglial cathepsin B: an immunological examination of cellular and secreted species.

The cysteine proteinase cathepsin B (CB) was isolated from immortalized murine BV-2 microglial cells and examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to establish physicochemical properties of CB from what is generally considered the resident CNS macrophage. Microglial proteases have been implicated in several pathological processes occurring in the CNS, including neurodegeneration. Murine microglial CB was observed to consist of two major single-chain species of 32 and 34 kDa, with pls of 5.5-5.2 and 5.1-4.5, respectively. In addition, a minor 24-kDa CB species was also observed in some microglial preparations. The major CB isozymes in microglia differed from those observed in murine liver and brain, which consisted of both single- and double-chain CB variants of 31 and 24-25 kDa/5 kDa, respectively, with pl values of 5.5-4.5. A microglial pro-CB of 37 kDa was also isolated, which could be processed to the 34-kDa single-chain CB species. Cystatin was observed to prevent pro-CB processing, whereas E-64 and leupeptin were only partially inhibitory. The 37-kDa pro-CB species was observed to undergo processing into the 34-kDa CB species when incubated at pH 5.5 but remained stable with respect to molecular mass when incubated at pH 7.0. In contrast, the 34-kDa single-chain CB species was observed to autodegrade when incubated at pH 7.0, whereas incubation at pH 5.5 did not affect the integrity of the species as monitored by immunoblotting. Both pro-CB and 32-kDa single-chain CB species were observed extracellularly following lipopolysaccharide activation of BV-2 microglial cells.

Animals↗
PubMed1995

Immunolocalization of cathepsin B in human glioma: implications for tumor invasion and angiogenesis.

The poor prognosis of patients with malignant gliomas is at least partially due to the invasive nature of these tumors. In this study, the authors investigated the possibility that the cysteine protease cathepsin B (CB) is a participant in the process of glial tumor cell invasion. To accomplish this, an immunohistochemical analysis was made of the localization of antibodies to CB in biopsies of five specimens of normal brain, 16 astrocytomas, 33 anaplastic astrocytomas, and 33 glioblastomas multiforme. Staining was scored according to the percentage of positive cells and the intensity of the stain, graded from 0 to 3+. Staining for CB was not seen in any of five samples of normal brain except for occasional neuronal cell bodies and microglia. Only five (31%) of 16 astrocytomas showed a small percentage of positive cells (0.01%-3%) that were stained in a light, diffuse cytoplasmic pattern (1+). Twenty-nine (87.8%) of 33 anaplastic astrocytomas showed positive light, granular staining in 2% to 40% of cells. In anaplastic astrocytoma, the staining within a tumor was heterogeneous with intensities of 1+ (17%), 1+ to 2+ (29%), or 2+ (55%). In contrast, all 33 (100%) glioblastomas were positive in 10% to 90% of cells. The staining was present in a coarse, granular pattern with an intensity of 2+ (12%) or 3+ (88%). Tumor cells infiltrating into brain adjacent to malignant gliomas stained positively in 26 cases that could be evaluated for glioblastoma multiforme; these invading cells frequently followed penetrating blood vessels as typical "secondary structures of Scherer." Moderate to intense CB staining associated with endothelial proliferation in high-grade tumors was also observed, especially in regions of tumor infiltration into adjacent normal brain. These results provide evidence consistent with the hypothesis that CB is functionally significant in the process of tumor invasion and angiogenesis in the clinical progression of the malignant phenotype in astrocytes.

Antibodies, Monoclonal↗