Antisense RNA inhibition of cathepsin L expression reduces tumorigenicity of malignant cells.

Several tumour-forming cell lines are known to overproduce the lysosomal cysteine peptidase cathepsin L. We have used an antisense approach to investigate whether inhibition of cathepsin L overexpression in two malignant cell lines (myeloma SP cells and L cells) reduces their tumorigenic potential. Two different cDNA fragments of murine cathepsin L were inserted in the antisense direction into the pcDNA3 vector, and SP and L cells were stably transfected with these plasmid constructs. Several of the selected clones expressing the antisense transcript showed specific reduction of the mRNA level and the intracellular activity of cathepsin L, and a greatly diminished amount of secreted procathepsin L. When tested in Balb/c nu/nu mice, the cell lines with low cathepsin L activity exhibited a significantly decreased potential for tumour growth when compared with control cells expressing wild-type levels of cathepsin L activity. This observation suggests that cathepsin L is a critical factor in tumour growth.

Expression of acid cysteine proteinase inhibitor (ACPI) in the normal human prostate, benign prostatic hyperplasia and adenocarcinoma.

Acid cysteine proteinase inhibitor (ACPI or cystatin A) is a protein (12 kDa) which inhibits the action of several cysteine proteinases, e.g. cathepsins B, H, L and S. In this study the cellular location of ACPI has been immunohistochemically investigated in the normal human prostate, in benign prostatic hyperplasia (BPH) and in adenocarcinoma. ACPI was found in the basal epithelial cells of the normal prostate. The secretory epithelial cells did not express ACPI. In the hyperplastic prostate, the expression of ACPI was decreased and it was also expressed more focally in the basal cells. Hyperplastic basal cells also expressed ACPI. In prostatic adenocarcinoma, no ACPI expression was found. The absence of ACPI expression was obvious and if the sections contained both benign and malignant cells, only the benign glandular structures always expressed ACPI. The results suggest that expression of ACPI might be related to prostatic epithelial cell proliferation and differentiation. Possibly the detection of ACPI in tissue sections might be helpful in identifying prostatic adenocarcinoma, especially in cases with small carcinomatous foci.

Acid cysteine proteinase inhibitor in cutaneous lymphocytic infiltrates.

Acid cysteine proteinase inhibitor (ACPI, cystatin A) is normally present in squamous epithelium and dendritic cells of lymphoid follicles. Its expression is altered both in proliferative and malignant squamous epithelium and in neoplastic lymphoid follicles. The expression of ACPI in the lymphoid infiltrates of cutaneous psuedolymphomas and B-cell lymphomas was studied. Eighteen pseudolymphomas from 15 patients were divided into three groups according to the proportion of B and T lymphocytes. The B-cell-type lesions with well-developed follicles and germinal centers showed a pronounced ACPI expression in dendritic cells. Varying amounts of ACPI-positive cells were present in the mixed B- and T-cell-type and also in the T-cell-type lesions. The labeled cell population was distinct from the factor XIIIa-positive dermal dendrocytes, S-100-positive histiocytes, and HAM 56-positive histiocytes. Malignant lymphomas contained a few haphazardly arranged ACPI-positive cells with short dendrites and granular cytoplasm. It was concluded that follicular dendritic cells can be reliably labeled with ACPI antiserum in cutaneous pseudolymphomas. The structure and distribution of ACPI-containing cells in malignant cutaneous B-cell lymphomas is altered when compared with pseudolymphomas.

Identification of acid cysteine proteinase inhibitor (cystatin A) in the human thymus.

BACKGROUND: Acid cysteine proteinase inhibitor (ACPI, also called cystatin A) is a protein that is present in the epithelial cells of the skin and in the dendritic reticulum cells of lymphoid tissues. In this study the presence and cellular localization of ACPI in the thymus was investigated. METHODS: The cellular and topographical location of ACPI was immunohistochemically demonstrated in the normal thymus of man. RESULTS: ACPI was found in the cells of the Hassall's corpuscles and in many medullary cells. Most of these cells were epithelial cells, as shown by the results of immunohistochemical cytokeratin and epithelial membrane antigen stainings. Also, some individual cytokeratin negative but S-100 positive medullary reticular dendritic cells were stained with ACPI. CONCLUSIONS: The finding that ACPI is constantly present in the thymus at restricted and specific cellular locations leads to the suggestion that protease inhibitors may play a role in specific thymic functions.

An inactive cathepsin B-like enzyme and cysteine proteinase inhibitors in colon cancer ascites.

The ascitic fluid of a patient with colon cancer was found to contain an inactive cathepsin B-like enzyme. The inactive enzyme with a molecular weight of 40 kDa was converted by pepsin treatment into an active form with a molecular weight of 28 kDa as revealed by Sephadex G-75 gel chromatography. The inactive cathepsin B-like enzyme was considered to represent a precursor form and not an enzyme-inhibitor complex. The activated cathepsin B-like enzyme resembled human liver cathepsin B in its enzymatic characteristics. Cysteine proteinase inhibitor activity was also detected in the same ascitic fluid, and it was separated into two main forms by Sephadex G-75 gel chromatography. The high molecular weight inhibitor fractions reacted with antiserum against alpha-CPI and the low molecular weight fractions reacted with antiserum against cystatin B.

Inactive cathepsin B-like enzyme in human melanoma culture medium.

An inactive cathepsin B-like enzyme with a molecular mass of 40 kD was found in a human melanoma culture medium. The inactive form of this enzyme was converted into an active form with a molecular mass of 28 kD by pepsin treatment. This activated cathepsin B-like enzyme had almost the same characteristics regarding molecular size, substrate specificity, dependence on chemical reagents, and Km values as intracellular cathepsin B. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by electroblotting with an antiserum against cathepsin B yielded inactive cathepsin B-like enzyme fractions which showed two immunoreactive bands with molecular masses of 40 and 28 kD, respectively. On the other hand, alkali treatment of the inactive cathepsin B-like enzyme fractions released a cysteine proteinase inhibitor with a molecular mass of 12 kD. These data suggest that these inactive cathepsin B-like enzymes in melanoma culture medium are present not only in the precursor form, but that they are also present as enzyme-inhibitor complexes, both of which can be activated enzymatically in vitro.

Purification and characterization of a cystatin-type cysteine proteinase inhibitor in the human hair shaft.

We found a cysteine proteinase inhibitor in human hair shaft extract treated with 0.01 M Tris HCl buffer, pH 8.0. A yield of 0.2 mg of purified cysteine proteinase inhibitor was obtained from 86 g of hair shaft. The cysteine proteinase inhibitor had a molecular mass of 13 kDa as determined by high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was more stable to heat and pH than most proteins and had a pI of 4.7. Immunologically, its antigenicity was the same as that of cystatin A, but differed from that of cystatin B and C, and kininogen. The amino-acid sequence of the first 30 residues from the NH terminus of the inhibitor was identical to that of cystatin A from human epidermis. Hair shaft cysteine proteinase inhibitor is thus considered to be identical to epidermal cystatin A.

Immunolocalization of cystatin A in neoplastic, virus and inflammatory lesions of the uterine cervix.

Cystatin A was immunohistochemically demonstrated in the normal squamous epithelium of the uterine cervix, particularly in the parabasal and superficial cell layers whereas it was absent or scanty in the basal cells and in areas with parakeratosis. Cystatin A was also found in neoplastic lesions (dysplasia, carcinoma in situ and squamous cell carcinoma), but less abundant than in normal squamous epithelium. The immunoreaction in intraepithelial neoplasia was closely related to the degree of morphological maturation of the squamous cells with more abundant cystatin A in low grade dysplasia and less in high grade dysplasia and carcinoma in situ. In squamous cell carcinoma, cystatin A was often abundant in highly differentiated areas and almost absent in poorly differentiated ones. Cystatin A was found in the squamous epithelium in herpes and in condylomatous lesions. It was also found in the cytoplasm of neutrophils, but not in lymphocytes and plasma cells. In unspecific cervicitis, cystatin A was found extracytoplasmatically as small vesicles in the epithelial-stromal junction. The implications of cystatin A in neoplastic, virus, and inflammatory processes are discussed.

Ultrastructural signs of altered intracellular metabolism in acral persistent papular mucinosis.

An asymptomatic 24-year-old woman presented with multiple discrete papules on the extensor surfaces of the hands and wrists. Light microscopy revealed focal increase in the amount of dermal fibroblasts as well as deposition of hyaluronidase-labile mucoid substance. The collagen and elastin were decreased. The changes were consistent with acral persistent papular mucinosis (APPM). In electron microscopy, the intercellular glycosaminoglycans showed small ruthenium red-positive granules and thin filaments indicating normal morphology. The fibroblastic cells, however, were conspicuously altered. Endoplasmic reticulum was dilated, cytoplasm contained large amounts of osmiophilic, concentric lysosomal structures, and there was distinct fibrous lamina in the nuclear membrane. It was concluded that the primary event in APPM probably affects the intracellular metabolism of the dermal fibroblast. The accumulation of lysosomal structures may be a distinct feature of APPM differentiating it from the other reactive cutaneous mucinoses, or it may only reflect nonspecific degeneration in a long-standing lesion.

Characterization of a cathepsin-H-like enzyme from a human melanoma cell line.

A cathepsin-H-like enzyme has been isolated from cultured human melanoma cells (G 361 cell line). The enzyme is similar to cathepsin H(s) of normal tissues in molecular weight, enzymatic characteristics (substrates, inhibitors, pH optima, Km values), and immunoreactivity. The inactive form of the enzyme with a molecular mass of 40 kDa has been found in the culture medium. The inactive enzyme is activated by acid pH, pepsin, and cathepsin-D-like enzyme treatments and converted into a form with a molecular mass of 28 kDa. The activated extracellular cathepsin-H-like enzyme and the active intracellular enzyme exhibit the same characteristics. The melanoma-derived cathepsin-H-like enzyme degrade fibrinogen and fibronectin, but not laminin or type-IV collagen. We conclude that the extracellular cathepsin-H-like enzyme may have important functions, together with other proteinases, in the destruction of extracellular matrix components, thus enabling proliferation, migration, and metastasis to occur.

Expression of the 43 kDa papain inhibitor during human fetal skin development.

The presence of the 43 kDa papain inhibitor protein in the skin of 40 fetuses with the gestational age varying from 9 to 40 weeks was studied. Immunohistochemistry showed the first evidence of the 43 kDa papain inhibitor in the acral parts, mostly in the nail bed, at the 14th week when the epidermis in all other sites was not stained. Staining of the follicular infundibulum as well as some parts of the uppermost epidermis and keratin layer was observed from 17 to 40 weeks. The staining was most intensive at 20-30 weeks gestational age, and later the intensity gradually decreased so that in the full-term newborn the 43 kDa papain inhibitor was hardly detectable or absent. It was concluded that the 43 kDa papain inhibitor is present in fetal skin at the stage of stratification (9-14 weeks) only in the nail region and its volar surroundings. It appears at the stage of interfollicular keratinization (14-24 weeks) in the uppermost epidermis, especially in the appendical openings and its amount seems the decrease during the stage of interfollicular keratinization (24 weeks--full-term). The expression of 43 kDa inhibitor protein is temporally related to that of filaggrin.

A chloride activated alanine aminopeptidase from a melanoma cell line.

Alanine aminopeptidase was partially purified from cultured human melanoma cells (Bowes) by gel filtration on Sephadex G-200 and DEAE Sepharose column chromatography. The molecular weight of the enzyme was about 52,000 as determined by gel filtration on Sephadex G-100. The enzyme hydrolyzed L-alanine beta-naphthylamide (NA), but not or slightly L-methionine-NA, L-leucine-NA, and L-arginine-NA. The Km value for L-alanine-NA was 0.17 mmol/l, pH optimum was 7.4. The enzyme was stable at 50 degrees C for 20 min, but lost about 50% of its activity at 60 degrees C within 20 min. It was markedly stimulated by chloride ions, and was inhibited by sulfhydryl blocking agents and EDTA. The activity was restored by the addition of Co2+ or Zn2+ after EDTA treatment. The enzyme is a metallo- and thiol-dependent and chloride-activated, low-molecular weight aminopeptidase.

Low molecular weight alanine aminopeptidase of human serum: separation and some characteristics.

An alanine aminopeptidase, which has characteristics different from those of known alanine cleaving aminopeptidases, was partially purified from human serum by Sephadex G-200 gel chromatography and DEAE Sepharose column chromatography. The enzyme exhibited a molecular weight of 58,000 by gel chromatography. The pI of the enzyme was 5.0, and it was inactivated at 60 degrees C in 20 min. The enzyme readily hydrolyzed L-alanine beta-naphthylamide, but hardly hydrolyzed the other tested beta-naphthylamides. The Km value for L-alanine beta-naphthylamide was 0.29 mM, the pH optimum 7.5. The activity of the enzyme was enhanced by chloride ions and by sulfhydryl ethylenediaminetetraacetic compounds, and was inhibited by sulfhydryl blocking ethylenediaminetetraacetic agents, and bestatin. Furthermore, 1.10 phenanthroline and ethylenediaminetetraacetic acid were inhibitory, and the activity was restored by CoCl2 and ZnCl2. The enzyme is a chloride-enhanced thiol dependent metalloaminopeptidase.

Cysteine proteinase inhibitors in human squamous cell carcinoma.

Cysteine proteinase inhibitors in Tris extract of human skin squamous cell carcinoma tissue were studied. From 17.2g of the tissue, approximately equal to 113.5 U inhibitor was obtained. With Sephadex G-75 gel chromatography, 3 papain inhibitor peaks of Mr approximately equal to 100,000, 38,000, and 12,000 were separated. In the immunodiffusion studies, Mr approximately equal to 100,000 fractions reacted with antisera made against human kininogen (alpha-cysteine proteinase inhibitor) and cystatin B. By using alkali treatment, free cystatin B was dissociated from the Mr approximately equal to 100,000 fractions. It is suggested that Mr approximately equal to 100,000 fractions contain cystatin B and alpha-cysteine proteinase inhibitor complexed with tissue proteinases. The Mr approximately equal to 38,000 fractions reacted with antiserum made against the papain inhibitor of Mr approximately equal to 43,000. The inhibitors in the Mr approximately equal to 12,000 fractions were identified as cystatin A and B on the basis of their reactions with antisera made against these inhibitors purified from human tissues.

[Precipitating keratin antibodies in psoriasis vulgaris]. / Präzipitierende Keratin-Antikörper bei Psoriasis vulgaris.

A microprecipitation method was used to test sera of psoriasis patients and control persons for precipitating keratin interfilament antibody (KIF-Ab). Precipitating KIF-Ab were detected in 83% of the psoriasis patients. The sera of only 20.5% of controls without dermatological diseases and 40% of nonpsoriatic patients contained KIF-Ab. The mean KIF-Ab titer of the control and psoriasis group did not differ significantly. The different therapy had different effects on the detectability of precipitating KIF-Ab. Upon completion of dithranol treatment and clinical healing, all sera reacted with KIF from psoriasis scales (pso-sc). PUVA treatment lowered the Ab-titer as well as the number of seropositive sera. These results were confirmed by means of immunoblot and immunodot techniques. Sera from psoriasis patients contained Ab of the IgG and IgM-types against 65, 55 and 45 kD proteins. KIF-IgA-Ab were found frequently in the cases of severe forms of psoriasis.

The 43 kDa papain inhibitor in normal and diseased skin.

The presence of 43 kDa papain inhibitor in 43 different skin diseases was immunohistochemically studied by using both poly- and monoclonal antibodies. Psoriasis and various eczematoid reactions as well as viral infections showed the most pronounced staining in the squamous cells of the epidermis. The antigen was also present in benign tumours or precancerous lesions which showed keratinization. Cells of poorly differentiated squamous cell carcinomas, basal cell carcinomas and melanocytic tumours were negative. The antigen seems to be related to disturbed keratinization and benign proliferation in non-neoplastic dermatoses and it is also present in differentiating squamous neoplasms.

Cysteine proteinase inhibitors in human melanoma transplanted into nude mice.

Cysteine proteinase inhibitors in the human melanoma tissue transplanted into nude mice were found to increase in concentration during tumor growth. The activity (unit/g) at 8 weeks was about 3 times higher than the activity at 4 weeks after transplantation. The inhibitors were separated into two main forms (Mr about 76,000 and 10,000) with Sephacryl S-200 and/or Sephadex G-75 gel chromatography. The activities of the inhibitors of both molecular masses increased parallely during tumor growth. The high molecular mass inhibitor fractions reacted with antisera made against alpha-cysteine proteinase inhibitor (alpha-CPI, human kininogen) and against neutral low-molecular mass proteinase inhibitor (cystatin B). Free cystatin B appeared to be liberated in SDS-polyacrylamide gel electrophoresis following electroimmunoblotting with an antiserum to cystatin B. Similarly, free cystatin B was detected in gel chromatography on Sephadex G-75 after alkali treatment at pH 11.5. It may thus represent a cystatin B--cysteine proteinase complex mixed with alpha-CPI. The low molecular mass inhibitor fractions reacted with antisera made against cystatin A and cystatin B. When the low-molecular mass inhibitor fraction was subjected to isoelectric focusing, it was separated into three peaks with pIs 8.0, 7.4, and 6.0. The inhibitors with pI 8.0 and 7.4 reacted with antisera made against cystatin B, while the inhibitor with pI 6.0 reacted with antisera made against cystatin B and cystatin A.