No abstract

CatB and catL levels measured in routinely prepared cytosols are strong parameters to predict the rate of relapse and the length of survival after treatment of the primary breast tumor.

Inactivation ofhuman eystatin C and kininogen by human cat, hepsin D Univcrslt, v th~sptml, S.221 85 l.und, 3"wcdcn Received i 6 Janultry 1991 A p~p~ln tnltibitor of 22 kDa was isolated from human pt=te=nta and shown to ~ identical to residues Cy~146.keu:t?.~ of the third domain of human klninol~en. This kinino~en dam;tin anti reeon~binan~ hun~an cystatin C were tmtettvated by pcptlde bond eleavalles at hydrophobic amino acid residues due to Ihe etClion of cathepsin D, Tlie~e results ftmher support 11~c prop=seal role ofcalhepsin D in the regulation orcysteinc proteinus~ activity. In this report we show that cathepsin D is able to degrade human cystatin C and the third domain of kininogen, both extracell.ular protein inhibitors of cysteine proteinases. This finding might be important for elucidation of additional e×tracellular actions of cathepsin D. MATERIALS AND METHODS 2,1 MaterialsRecombinant human cystatin C was prepared as described [13]. Human cathepsin D was isolated from spleen [14], Papain (type 111) (EC 3,4,22,2) and sttbstrate BANA were from Sigma, Bovine haemoglobin was prepared as described in [15]. Pepstatin was from Peptide Research Foundation (Osaka, Japan). Acetonitrile and t rifluoroacetic acid were from Merck and BDH Chemicals, respectively. All other chemicals were of analytical grade, Isnlatlan of thv third don~ain af kinhlogc, nThe isolation was carried out followinj tile procedure as described in [161 with some modirietttions. Briefly, human placenta (1000 g) was homogenized in 0.9~, NaCI (I :1.5, w/v) sohnlon and centrifuged for 60 rain a= 14 000 x /~. The supernatant war adjusted to pet 10,$ with 5 M NaOH, and after standing I h at roan1 temperature, to pH ?.S with 3 M HCI, After ¢entrifugation the supernatant was applied to an affinity column (7.5 × $.0 era) of Cm-papain-Sepharose 4B prepared as in [1"7], et|uilibrated with 10 mM Tris-HCI buffer (pH 7.5). The coluntn was washed with the same t~uffer and the bomtd proteins eluted tl~en with 10 mM NaOH (pH I 1.01, Papain.inhibitlng fractions were pooled, adjusted to pH g.O with 3 M HCI and concentrated by ultrafiltration on an YM-5 membrane (Amicon). The concentrated sample war dialyzed against 10 mM Tris-HCI buffer (pH 7,5) containing 0,1 M NaCI and applied to a column (120 x 5 cm) of Sephadex G-75 equilibrated in the same buffer, Papain.inhihiting fractions were resolved as three peaks corresponding to M, of about 12 25 attd 6S kDa, The 25 kDa.M, protein peak was dialyzed against 10 mM Tris-HCI buffer (pH 8,5) at~d subsequently cllromatographed on DEAESephacel eq uilibrated with the same buffer, The inhibitor was eluted in a single symmetrical peak with linear gradient of NaCI (0,05-0,15 M) in the same buffer, The sample was concentrated, assayed for its purity by SDS-PAGE and amino acid sequence analysis, and stored at -25"C until use, 2,3. Assays of enzymes and inhibitorsThe inhibitory activity of cystatin C and the third domain of kininogen was determined by measuring the inhibition of papain using BANA as substrate [18], The c...

To estimate the prognostic value of cathepsins B, H, L, D and stefins A and B in head and neck carcinoma, their concentrations in cytosols of primary tumours and adjacent normal tissue were measured (cathepsins B, D stefins A, B in 45, cathepsin L in 24 and cathepsin H in 21 patients). Median concentrations of cathepsins B, L, and D were significantly higher in tumour than in the adjacent normal tissue (B and D: p < 0.0001; L: p = 0.004); cathepsin H concentration was higher in normal tissue (p = 0.001). Concentrations of either stefin did not differ significantly between normal and tumour tissue. Concentrations of cathepsins B, H, L, and D were higher in laryngeal than in non-laryngeal normal and tumour tissues. The difference was statistically significant for cathepsin B in tumour tissue (p = 0.045), and marginally significant in normal tissue (p = 0.07). Early tumours had lower concentrations of stefins A and B than locally advanced tumours (stefin A: p = 0.04; stefin B: p = 0.07). Disease-free and disease-specific survival rates were better in patients with concentrations of cathepsin L in tumour tissue below or equal to the cut-off values (p = 0.035; p = 0.05), whereas for cathepsin B the difference was established only for disease-free survival (p = 0.07). The opposite was true for stefin A (p = 0.0002; p = 0.002) and stefin B (p = 0.009; p = 0.003), and in disease-free survival also for cathepsin H (p = 0.055). The concentration of cathepsin D did not correlate with survival. Our data indicate that cathepsins B, H, L and stefins A and B might have prognostic value in head and neck carcinoma.

Summary In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its protein levels were determined in 148 pairs of lung tumour tissue and adjacent non-tumourous lung parenchyma using the enzyme-linked immunosorbent assay technique. Additionally, Cath H levels were determined in sera of 171 patients with malignant tumours, 34 patients with benign lung diseases and 47 healthy controls. The median level of Cath H in tumour tissue was 0.64 times that in the corresponding lung parenchyma. Relating tumour levels with histological type we found higher Cath H levels in small-cell and adenocarcinomas and lower levels in squamous cell carcinoma, large-cell carcinoma and secondary tumours. A significant difference in Cath H level between lung tumour tissue and non-tumourous lung parenchyma was associated with the group of cigarette smokers (156 vs 263 ng mg -1 protein, P < 0.001). For this group of patients Cath H tumour levels correlated with the survival rate, while for the entire patient population this was not the case. Smokers with high tumour levels of Cath H experienced poor survival. Cath H was significantly higher in sera of patients with malignant and benign lung diseases than in control sera (P < 0.001). The increase was significant for all histological types, being the highest in small-cell and squamous cell carcinomas. Our study reveals that in lung tumours there is different behaviour of Cath H compared with other cysteine peptidases, e.g. cathepsin B and cathepsin L. Variations between tissue and serum levels of Cath H indicate either reduced expression or enhanced secretion of this enzyme in lung tumours.