Differences in the expression of cathepsin B in B16 melanoma metastatic variants depend on transcription factor Sp1.

Cathepsin B contributes to the invasiveness of B16 melanoma cells in mice, with the highly metastatic B16a melanoma producing six- to eightfold more cathepsin B mRNA and protein than the less metastatic B16F1 variant. The proximal promoter region of the cathepsin B (Ctsb) gene (-149 to +94) was previously found to be capable of reproducing this pattern of differential gene activation in B16 melanoma variants. The binding of B16 melanoma nuclear proteins to this promoter region has now been mapped to three GC-boxes (Sp1 transcription factor binding sites) and a potential X-box [tax response element (TRE)/c-AMP responsive element (CRE) site].

Sp1 regulates cathepsin B transcription and invasiveness in murine B16 melanoma cells.

Background: Increased expression of cathepsin B contributes to extracellular matrix degradation and invasion in cancer. Cathepsin B expression is under transcriptional control in murine melanomas and the major promoter contains potential binding sites for the Sp1 transcription factor.

Materials And Methods: Murine melanoma cells transfected with an Sp1 expression plasmid or its control were used in Matrigel invasion and cell motility assays in the presence or absence of the cathepsin B inhibitor, CA-074Me.

An intracellular form of cathepsin B contributes to invasiveness in cancer.

Cathepsin B is a lysosomal cysteine proteinase whose expression and trafficking are frequently altered in cancer, and plasma membrane and secreted forms are thought to contribute to the invasive and metastatic properties of malignant tumors. We have manipulated the expression of cathepsin B in several tumor cell lines and measured their capacity to invade through a reconstituted extracellular (Matrigel) matrix. Transient expression of human cathepsin B in a poorly metastatic B16F1 murine melanoma variant produced a 3-5-fold increase in cathepsin B activity and a comparable increase in invasiveness.

Loss of insulin-like growth factor II receptor expression promotes growth in cancer by increasing intracellular signaling from both IGF-I and insulin receptors.

The insulin-like growth factor-II receptor (IGF-IIR) is frequently mutated or deleted in some malignant human tumors, suggesting that the IGF-IIR is a tumor suppressor. However, the exact mechanism by which IGF-IIR suppresses growth in tumors has not been definitively established. We demonstrate that IGF-IIR-deficient murine L cells (D9) have higher growth rates than IGF-IIR-positive L cells (Cc2) in response to IGF-II.

In vivo expression of an alternatively spliced human tumor message that encodes a truncated form of cathepsin B. Subcellular distribution of the truncated enzyme in COS cells.

Cathepsin B is a lysosomal cysteine protease whose increased expression is believed to be linked to the malignant progression of tumors. Alternative splicing and the use of alternative transcription initiation sites in humans produce cathepsin B mRNAs that differ in their 5'- and 3'-untranslated ends. Some human tumors also contain cathepsin B-related transcripts that lack exon 3 which encodes the N-terminal signal peptide and 34 of the 62-amino acid inhibitory propeptide.

Physiological and biochemical response of glomerular epithelial cells to exogenous epidermal growth factor.

Epidermal growth factor (EGF) is a growth-promoting cytokine which acts in a paracrine and autocrine fashion on epithelial cells of various tissues. Although previously demonstrated, we have now confirmed the presence of EGF receptors in cultured glomerular epithelial cells (GEC) using radioligand binding studies. Further, the biochemical consequences of EGF receptor activation in this cell type were investigated.

A Continuous Culture Model To Examine Factors That Affect Transduction among Pseudomonas aeruginosa Strains in Freshwater Environments.

Transduction among Pseudomonas aeruginosa strains was observed in continuous cultures operated under environmentally relevant generation times, cell densities, and phage-to-bacterium ratios, suggesting its importance as a natural mechanism of gene transfer. Transduction was quantified by the transfer of the Tra(sup-) Mob(sup-) plasmid Rms149 from a plasmid-bearing strain to an F116 lysogen that served as both the recipient and source of transducing phages. In control experiments in which transduction was prevented, there was a reduction in the phenotype of the mock transductant over time.

Transcriptional regulation of cathepsin B expression in B16 melanomas of varying metastatic potential.

The highly metastatic B16a melanoma has been shown to express higher levels of cathepsin B (CB) mRNA when compared to the less metastatic variants, B16-F1 and B16-F10, and with normal mouse tissues. This increased expression is now shown to be due to increased gene transcription by nuclear run-off assays and measurements of mRNA stability. Transient expression assays, using promoter fragments from the mouse and human CB genes, demonstrated that both promoters were more active in B16a than in the less metastatic melanomas, B16-F1 and B16-F10.

Characterization of the cathepsin B gene and multiple mRNAs in human tissues: evidence for alternative splicing of cathepsin B pre-mRNA.

We have cloned and characterized multiple messages for cathepsin B that differ in their 5' and 3' untranslated regions (UTRs) from human kidney and the hepatoma cell line HepG2. A comparison of these messages with the cloned human cathepsin B gene reveals that they arise by alternative splicing of a single gene. Processing at a cryptic intron donor site in exon 11 and splicing to exon 12 produces a 4.

Characterization of multiple cathepsin B mRNAs in murine B16a melanoma.

We have previously shown that the highly metastatic murine B16a melanoma expresses a high level of cathepsin B mRNA which is associated with three transcripts of 2.2, 4.0 and 5.

The structure of the mouse cathepsin B gene and its putative promoter.

The mouse cathepsin B gene and its flanking regions were cloned and characterized. The gene contains 10 exons and 9 introns spanning about 20 kb. Although the exon-intron organization of the mouse cathepsin B gene showed some similarity to the rat cathepsin H and L genes, significant differences were found.

The expression of cathepsin B and other lysosomal proteinases in normal tissues and in tumors.

The mRNA for the lysosomal proteinases cathepsins B, D, H, L, and S are broadly distributed in normal rodent tissues. Although total cathepsin mRNA levels generally parallel the protein catabolic activity of the tissues, the expressions of the individual enzymes do not appear to be linked. Thus, the relative proportions of the individual messages are found to vary from tissue to tissue.

Differences in targeting and secretion of cathepsins B and L by BALB/3T3 fibroblasts and Moloney murine sarcoma virus-transformed BALB/3T3 fibroblasts.

BALB/3T3 fibroblasts (3T3) were observed to secrete latent, pepsin-activatable forms of cathepsin B and cathepsin L as well as an active form of beta-glucuronidase when cultured in the absence of serum. The secretion of these proteins was stimulated by the cation ionophore monensin: cathepsin B, 4.3-fold; cathepsin L, 7.

Molecular cloning of rat precursor cathepsin H and the expression of five lysosomal cathepsins in normal tissues and in a rat carcinosarcoma.

1. A rat cathepsin H cDNA was isolated from a rat liver cDNA library with synthetic oligonucleotide probes. 2.

Expression of five cathepsins in murine melanomas of varying metastatic potential and normal tissues.

The relative levels of mRNAs for cathepsins B, D, H, L, and S in eight normal murine tissues and three murine melanoma variants, B16-F1, B16-F10, and B16a, have been analyzed by RNA dot blot and densitometry. A direct correlation was observed between the levels of cathepsin B mRNA and the metastatic potentials of these three melanoma variants. The relative amount of cathepsin B mRNA in B16a, which is the melanoma variant with the highest metastatic potential, was at least 3 times greater than that found in any of the normal murine tissues surveyed.

Active and inactive forms of the transition-state analog protease inhibitor leupeptin: explanation of the observed slow binding of leupeptin to cathepsin B and papain.

Leupeptin and similar peptide argininal (arginine aldehyde) transition-state analog protease inhibitors exist in three covalent forms in aqueous solution, the leupeptin hydrate (IH), a cyclic carbinolamine form (IC) generated by the addition of the guanidino epsilon N to the aldehydic carbon, and the free aldehyde form (IA). 1H NMR in D2O show their equilibrium concentrations to be 42, 56, and 2% for IH, IC (R and S enantiomers), and IA. The rates of conversion of (formula; see text) were determined by 1H NMR in D2O by trapping IA with semicarbazide.

Effect of subunit size and conformation on the rate of lysosomal degradation of extracellular proteins in cultured mouse peritoneal macrophages.

The degradation of nine well-defined proteins was studied in cultured mouse peritoneal macrophages following their uptake by fluid phase pinocytosis. After uptake, approximately one-third of the radioactivity was released into the medium in the form of trichloroacetic acid/phosphotungstic acid-insoluble material. When the time courses for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and -insoluble radioactivities were independently analyzed, identical observed rate constants (kobs) were obtained.

Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible.

The uptake and degradation of bovine serum albumin (BSA), bovine liver catalase, and rabbit muscle enolase have been studied in cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK cells). Rates constant for the uptake of the three proteins by MPM were similar. In addition, BSA accumulation was independent of BSA concentration in the uptake medium and was not inhibited by a large excess of serum, suggesting that protein accumulation was by fluid phase pinocytosis.

Steady state kinetic evidence for an acyl-enzyme intermediate in reactions catalyzed by bovine spleen cathepsin B.

Cathepsin B from bovine spleen was shown to catalyze transacylation reactions between esters of N-substituted amino acids and nucleophiles. These reactions appeared to proceed through an intermediate between cathepsin B and the acyl portion of the substrate. Of the various nucleophiles tested, dipeptides were found to be the most effective acyl group acceptors.

The pH dependency of bovine spleen cathepsin B-catalyzed transfer of N alpha-benzyloxycarbonyl-L-lysine from p-nitrophenol to water and dipeptide nucleophiles. Comparisons with papain.

Cathepsin B has been shown to catalyze the transfer of the N alpha-benzyloxycarbonyl-L-lysyl residue from the corresponding p-nitrophenyl ester substrate to water and dipeptide nucleophiles. These reactions occurred through the formation of an acyl-enzyme intermediate. The pH dependency of the acylation and deacylation steps were determined from the increases in the maximum rate of appearance of p-nitrophenol on addition of glycylglycine or L-leucylglycine to the reaction.

Regional distribution of acetylcholinesterase in the right atria of humans and dogs.

The regional distribution of acetylcholinesterase in the right atrium was determined by quantitative chemical measurements on hearts obtained from 14 infant and 9 adult humans of autopsy, and 9 adult dogs after termination of acute animal experiments. The atrium and interatrial septum were dissected, and the appendage was cut along its fold from the ventricular border to the superior vena cava. The atrium was cut into 20 consecutive sections.

Studies on the distribution of cholinesterases: activity in the human and dog heart.

The distribution and postnatal variation of cholinesterase (ChE) activity were studied in 25 human and 25 dog hearts. The observed distribution pattern is remarkably constant, In dog hearts, the pattern is as follows: sinus node (SN) greater than left atrium (LA) greater than right atrium (RA) greater than right ventricle (RV) congruent to left ventricle (LV). The average acetylcholinesterase (AcChE) activities as expressed in international units per g wet tissue are: 1.

Gel filtration studies on oxyhemerythrin. II. Effect of temperature and ionic strength on the association-dissociation equilibria.

The effects of temperature and ionic strength on the association of oxyhemerythrin have been studied. deltaH degrees and deltaS degrees for association at pH 7.0 are -2.

Gel filtration studies of oxyhemerythrin. I. Effects of pH on the association-dissociation equilibria.

The pH dependency of the dissociation of oxyhemerythrin has been studied by frontal gel chromatography on Sephadex G-75. The extent of dissociation is markedly pH dependent increasing below pH 6.0 and above pH 8.