Prognostic significance of cysteine proteinases cathepsins B and L and their endogenous inhibitors stefins A and B in patients with squamous cell carcinoma of the head and neck.
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Cysteine proteinases cathepsins (Cats) B and L and their endogenous inhibitors stefins (Stefs) A and B are implicated in the processes of local and metastatic tumor spread. They were identified as potential prognosticators in various malignant diseases, particularly in breast cancer. The aim of the present study was to determine the concentrations of Cats B and L and Stefs A and B in the tumor and adjacent normal tissue samples collected from 49 patients (the present group) with squamous cell carcinoma of the head and neck (SCCHN), using quantitative immunosorbent assays (ELISA; KRKA d.d., Novo mesto, Slovenia). Their clinical significance was compared with that from a previous study (the reference group, 45 patients; Budihna et al., Biol. Chem. Hoppe-Seyler, 377: 385-390, 1996). The follow-up of patients from the latter report was updated for this purpose. In the present group, significantly higher concentrations of Cat B (P < 0.0001), Cat L (P < 0.0001) and Stef A (P = 0.006) were found in tumors compared with concentrations in their normal tissue counterparts. Cat concentrations in normal laryngeal tissue were significantly/marginally elevated compared with nonlaryngeal tissue (Cat B, P = 0.02; Cat L, P = 0.06). The tumor concentration of Cat L was found to correlate with pT classification (P = 0.005) and tumor-node-metastasis stage (P = 0.05), whereas the concentrations of Stefs A and B correlated with pN classification (P = 0.007 and P = 0.03, respectively) and tumor-node-metastasis stage of the disease (P = 0.02 and P = 0.03, respectively). There was no statistically significant difference between low and high Cat B or Cat L groups, regarding either disease-free survival or disease-specific survival, using a minimum P approach to determine cutoff concentrations. The risk of disease recurrence and SCCHN-related death was significantly higher in patients with low Stef A (P = 0.0006 and P = 0.0005, respectively) and Stef B (P = 0.0009 and P = 0.0007, respectively) tumors, compared with those with high-Stef A and Stef B tumors. These results remained significant even after Ps were adjusted for a possible bias in the estimated effect on survival. The survival analysis in the reference group also confirmed these findings (Stef A: P = 0.0009 and P = 0.002, respectively; Stef B: P = 0.03 and P = 0.009, respectively). To avoid any possible bias arising from the differences between the laboratories that performed the biochemical analysis, the concentrations of both Stefs in the present group and in the reference group were standardized and coupled together to form a uniform group. In univariate survival analysis, standardized values of Stef A and Stef B correlated inversely with the rate of relapse (P = 0.0000) and mortality rate (P = 0.0000). Multivariate regression analysis showed that the standardized value of Stef A is the strongest independent prognostic factor for both disease-free survival and disease-specific survival. These findings show the specific role of Cats B and L and Stefs A and B in the invasive behavior of SCCHN. Furthermore, Stef A proved to be a reliable prognosticator of the risk of relapse and death in patients with this type of cancer. (+info)
Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene.
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AIMS: Increased or altered activities of cysteine proteases have been implicated in serious human disorders such as cancer, rheumatoid arthritis, sepsis, and osteoporosis. To improve the current knowledge of the regulatory role of a major mammalian cysteine protease inhibitor, cystatin C, in such disease processes, a cystatin C deficient mouse was generated and characterized. METHODS: The mouse cystatin C gene was inactivated by insertion of a bacterial neo gene through homologous recombination in 129/Sv embryonic stem cells. Embryonic stem cell clones were injected into C57BL/6J blastocysts followed by injection of the blastocysts into pseudopregnant female mice. F1 offspring with agouti coat colour after mating of chimaeric males with C57BL/6J females were examined by DNA analysis, and mice carrying the targeted mutation were intercrossed to obtain homozygous cystatin C deficient (CysC-/-) mice. To study the role of cysteine proteases and their inhibitors in metastasis, the spread of B16-F10 melanoma cells in CysC-/- and wild-type mice was compared. Analysis of the formation of remote metastases was carried out by intravenous injection of beta-galactosidase transfected B16-F10 cells and subsequent determination of cancer cell colonies in the lungs. RESULTS: Cystatin C deficient mice were fertile and showed no gross pathological abnormality up to 6 months of age. Compared with wild-type mice, seven times fewer large metastatic colonies were counted by means of a dissecting microscope in CysC-/- mice two weeks after tail vein injection of B16-F10 cells. At all of eight time points from 15 minutes to two weeks after intravenous injection of tumour cells, the CysC-/- mice had significantly fewer lung metastases. The observed differences were smaller when beta-galactosidase transfected cells were used to allow counting of small colonies. Subcutaneous and intracerebral tumour growth was not different in the CysC-/- mice. CONCLUSIONS: Cystatin C concentrations in vivo might influence metastasis in some tissues. The decreased metastatic spread of B16-F10 cells in CysC-/- mice is the result of both reduced seeding and reduced growth of tumour cells in their lungs. (+info)
A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera).
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Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN. (+info)
Comparative studies on the cysteine proteinase inhibitory capacity of mammalian blood.
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The inhibition of cysteine proteinase (papain) by human, bovine, horse, rabbit, guinea pig, rat, hamster, and mouse plasma, and pig, sheep, goat, and dog sera was investigated. The rat and mouse plasma and the pig serum showed such high inhibitory capacity as 813, 380, and 508%, respectively, of the human plasma. On the other hand, the horse, guinea pig, and rabbit plasma indicated 36, 40, and 54%, respectively, of the human plasma. A cysteine proteinase inhibitor (s) was separated from alpha-macroglobulin by Sephacryl S-300, and the inhibitory activity of all of the mammalian plasma/sera except for the guinea pig plasma appeared at around the 50-150-kDa region. The guinea pig plasma exhibited well-resolved two peaks of 100 and 200 kDa. (+info)
The major transition state in folding need not involve the immobilization of side chains.
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During protein folding in which few, if any, definable kinetic intermediates are observable, the nature of the transition state is central to understanding the course of the reaction. Current experimental data does not distinguish the relative contributions of side chain immobilization and dehydration phenomena to the major rate-limiting transition state whereas this distinction is central to theoretical models that attempt to simulate the behavior of proteins during folding. Renaturation of the small proteinase inhibitor cystatin under oxidizing versus reducing conditions is the first experimental case in which these processes can be studied independently. Using this example, we show that sidechain immobilization occurs downstream of the major folding transition state. A consequence of this is the existence of states with disordered side chains, which are distinct from kinetic protein folding intermediates and which lie within the folded state free energy well. (+info)
Does adjustment of GFR to extracellular fluid volume improve the clinical utility of cystatin C?
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BACKGROUND: Cystatin C measurement has been proposed as a replacement for creatinine as a serum measure of glomerular filtration rate (GFR). It has also been suggested that GFR itself should be adjusted to the extracellular fluid volume (ECV) of a child rather than the body surface area (BSA). AIMS: To assess the potential of cystatin C compared to serum creatinine in assessing GFR and to establish whether adjustment of GFR to ECV rather than BSA affects the potential usefulness of cystatin C. METHODS: Cystatin C and plasma creatinine were measured in 64 paediatric patients undergoing 77 (51)Cr-EDTA GFR measurements over a six month period. RESULTS: 1/cystatin C concentrations were more closely related to GFR (median 98 ml/min/1.73 m(2), range 8-172) after adjustment for patient BSA (r = 0.81 versus r = 0.44). 1/Creatinine concentrations appeared to be an inferior estimate of BSA adjusted GFR (r = 0.41), even following the use of the Schwartz formula (r = 0.37). Bland Altman statistics showed cystatin C could still only predict 95% of GFR values to within +/-41 ml/min/1.73 m(2) of the (51)Cr-EDTA method. The relation between GFR and 1/cystatin C was not improved by adjusting (51)Cr-EDTA GFR to ECV rather than BSA (r = 0.76 versus r = 0.81). CONCLUSIONS: Cystatin C appears superior to serum creatinine in paediatric subjects although its performance is unlikely to supplant (51)Cr-EDTA GFR measurement. This performance is not being underestimated because of adjusting GFR to BSA rather than ECV. (+info)
A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. Cystatin with a urokinase receptor binding site.
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Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors. (+info)
Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds.
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Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins. (+info)