Charles Schick

Squamous Cell Carcinoma Antigen 2 Is a Novel Serpin That Inhibits the Chymotrypsin-like Proteinases Cathepsin G and Mast Cell Chymase

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for more advanced squamous cell tumors. Molecular cloning of the SCCA genomic region revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. Analysis of the primary amino acid sequences shows that both genes are members of the high molecular weight serpin superfamily of serine proteinase inhibitors. Although SCCA1 and SCCA2 are nearly identical in primary structure, the reactive site loop of each inhibitor suggests that they may differ in their specificity for target proteinases. SCCA1 has been shown to be effective against papain-like cysteine proteinases. The purpose of this study was to determine whether SCCA2 inhibited a different family of proteolytic enzymes. Using recombinant DNA techniques, we prepared a fusion protein of glutathione S-transferase and full-length SCCA2. The recombinant SCCA2 was most effective against two chymotrypsin-like proteinases from inflammatory cells, but was ineffective against papain-like cysteine proteinases. Serpin-like inhibition was observed for both human neutrophil cathepsin G and human mast cell chymase. The second order rate constants for these associations were on the order of ∼1 × 105 M−1 s−1 and ∼3 × 104 M−1 s−1 for cathepsin G and mast cell chymase, respectively. Moreover, SCCA2 formed SDS-stable complexes with these proteinases at a stoichiometry of near 1:1. These data showed that SCCA2 is a novel inhibitor of two physiologically important chymotrypsin-like serine proteinases.

Co-expression of the Squamous Cell Carcinoma Antigens 1 and 2 in Normal Adult Human Tissues and Squamous Cell Carcinomas

Squamous cell carcinoma antigen (SCCA) serves as a serological marker for advanced squamous cell carcinomas (SCCs) and as an indicator of therapeutic response. Recent molecular studies show that the SCCA is transcribed by two almost identical tandemly arrayed genes, SCCA1 and SCCA2. These genes are members of the high molecular weight serine proteinase inhibitor (serpin) superfamily. Although SCCA1 and SCCA2 are 92% identical at the amino acid level, they have distinct biochemical properties. Paradoxically, SCCA1 is an inhibitor of papain-like cysteine proteinases, such as cathepsins L, S, and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, cathepsin G, and mast cell chymase. Using a new set of discriminatory monoclonal antibodies (MAbs) and polymerase chain reaction (PCR) assay, we showed that SCCA1 and SCCA2 were co-expressed in the suprabasal layers of the stratified squamous epithelium of the tongue, tonsil, esophagus, uterine cervix and vagina, Hassalls corpuscles of the thymus, and some areas of the skin. SCCA1 and SCCA2 also were detected in the pseudo-stratified columnar epithelium of the conducting airways. Examination of squamous cell carcinomas of the lung and head and neck showed that SCCA1 and SCCA2 were co-expressed in moderately and well-differentiated tumors. Moreover, there was no differential expression between these SCCA “isoforms” in normal or malignant tissues. In contrast to previous studies, these data indicated that the expression of SCCA1 and SCCA2 was not restricted to the squamous epithelium and that these serpins may coordinately regulate cysteine and serine proteinase activity in both normal and transformed tissues.

Circulating serpin tumor markers SCCA1 and SCCA2 are not actively secreted but reside in the cytosol of squamous carcinoma cells

An elevation in the circulating level of the squamous‐cell carcinoma antigen (SCCA) can be a poor prognostic indicator in certain types of squamous‐cell cancers. Total SCCA in the circulation comprises 2 nearly identical, ∼45 kDa proteins, SCCA1 and SCCA2. Both proteins are members of the high‐molecular weight serine proteinase inhibitor (serpin) family with SCCA1 paradoxically inhibiting lysosomal cysteine proteinases and SCCA2 inhibiting chymotrypsin‐like serine proteinases. Although SCCA1 and SCCA2 are detected in the cytoplasm of normal squamous epithelial cells, neither serpin is detected normally in the serum. Thus, their presence in the circulation at relatively high concentrations suggests that malignant epithelial cells are re‐directing serpin activity to the fluid phase via an active secretory process. Because serpins typically inhibit their targets by binding at 1:1 stoichiometry, a change in the distribution pattern of SCCA1 and SCCA2 (i.e., intracellular to extracellular) could indicate the need of tumor cells to neutralize harmful extracellular proteinases. The purpose of our study was to determine experimentally the fate of SCCA1 and SCCA2 in squamous carcinoma cells. Using subcellular fractionation, SCCA‐green fluorescent fusion protein expression and confocal microscopy, SCCA1 and SCCA2 were found exclusively in the cytosol and were not associated with nuclei, mitochondria, lysosomes, microtubules, actin or the Golgi. In contrast to previous reports, metabolic labeling and pulse‐chase experiments showed that neither non‐stimulated nor TNFα/PMA‐stimulated squamous carcinoma cells appreciably secreted these ov‐serpins into the medium. Collectively, these data suggest that the major site of SCCA1 and SCCA2 inhibitory activity remains within the cytosol and that their presence in the sera of patients with advanced squamous‐cell carcinomas may be due to their passive release into the circulation. Int. J. Cancer 89:368–377, 2000.

Development of specific monoclonal antibodies and a sensitive discriminatory immunoassay for the circulating tumor markers SCCA1 and SCCA2

The squamous cell carcinoma antigen (SCCA) serves as a serologic marker for advanced squamous cell carcinomas (SCC) of the uterine cervix, lung, esophagus, head and neck and vulva. Elevations in serum levels of SCCA following treatment for SCC correlate with tumor relapse or metastasis. Recent molecular studies show that SCCA is transcribed by two nearly identical genes (SCCA1 and SCCA2) that encode for members of the high molecular weight serine proteinase inhibitor (serpin) family. Despite a high degree of similarity in their amino acid sequences, SCCA1 and SCCA2 have distinct biochemical properties: SCCA1 is an inhibitor of papain like cysteine proteinases, such as cathepsins (cat) L, S and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, catG and mast cell chymase. In this paper, we report the generation and characterization of anti-SCCA1 and anti-SCCA2 specific monoclonal antibodies (MAbs). Using these MAbs, we developed an enzyme-linked immunoassay (ELISA) that discriminated between SCCA1 and SCCA2 without any cross-reaction. This assay measured both the native and complexed forms of SCCA1 and SCCA2. The sensitivity of detection of SCCA1 and SCCA2 assays were 0.17 ngml(-1) and 0.19 ngml(-1), respectively. Mean inter- and intra-assay coefficients of variation were 12.1% and 9.9% for SCCA1 assay and 12% and 8.8% for SCCA2 assay, respectively. Recovery and parallellism studies indicated that SCCA1 and SCCA2 were detected in the plasma and amniotic fluids without any major interference by the biologic fluid components. This assay provides a simple and accurate procedure for the quantitation of total SCCA1 and SCCA2.

SCCA1 and SCCA2 Are Proteinase Inhibitors That Map to the Serpin Cluster at 18q21.3

The genes for the squamous cell carcinoma antigen (SCCA) were found flanking a deletion breakpoint from a patient with the 18q-syndrome. The genes are <10 kb apart, tandemly arrayed in a head-to-tail fashion, and∼10 kb in size. Both genes also contain 8 exons and identical intron-exon boundaries. The cDNAs encode for proteins that are 92% identical and 95% similar. Amino acid comparisons show that SCCA1 and SCCA2 are members of the high-molecular weight serine proteinase inhibitor (serpin) family. Physical mapping studies show that the genes reside within the 500-kb region of 18q21.3 that contains at least four other serpin genes. The gene order is cen-maspin (PI5), SCCA2, SCCA1, PAI2, bomapin (PI10), PI8-tel. Biochemical analysis of recombinant SCCA1 and SCCA2 proteins shows that SCCA1 is a potent cross-class inhibitor of papain-like cysteine proteinases such as cathepsins L, S and K, whereas SCCA2 is an inhibitor of chymotrypsin-like serine proteinases such as cathepsin G and mast cell chymase. These findings suggest that SCCA1 and SCCA2 are capable of regulating proteolytic events involved in both normal (e.g., tissue remodeling, protein processing) and pathologic processes (e.g., tumor progression).

Gene expression in activated brain microglia: identification of a proteinase inhibitor that increases microglial cell number.

Microglia, the intrinsic immune cells of the central nervous system, are activated in a variety of inflammatory brain diseases in which they play a pathogenetic role. However, mechanisms underlying activation are largely unknown. To begin elucidating molecular mechanisms associated with activation, we characterized the pattern of gene expression in virtually pure dissociated microglial cultures, using RT-PCR differential display. Microglia were activated with bacterial lipopolysaccharide (LPS), a traditional stimulant, and the profile of gene expression was compared to that in basal, control cultures. Activation resulted in altered expression of six genes. The cDNAs were isolated, sequenced and characterized. Homology searches identified three novel genes, and two that exhibited very high sequence similarity to the gene encoding squamous cell carcinoma antigen (SCCA). SCCA (1 and 2) are tandemly arranged genes that encode two serine proteinase inhibitors (serpins). SCCA has been detected exclusively in cancer cells, and is a plasma marker for squamous cell carcinoma. Immunoblot analysis indicated that gene expression was accompanied by a 5-fold increase in the synthesis of SCCA protein in LPS-activated microglia. To assess potential biological actions of the SCCA serpins, SCCA1 protein was added to cultures. SCCA1 altered microglial morphology, and elicited a dramatic, 5-fold increase in cell number within 72 h. The effects appeared to be cell-specific, since the protein had no effect on other cell types: cortical astrocytes and neurons from cortex or basal forebrain were unaffected. We tentatively conclude that SCCA1 may play a cell-specific role in increasing cell number, a critical early step in microglial activation and brain inflammation. More generally, differential display of genes in the microglial model system may help define patterns of expression associated with CNS disease, thereby identifying pathogenetic mechanisms and new therapeutic targets.

Cross-Class Inhibition of Elastolytic Cysteine Proteinases by the Human Serpin SCCA1. |[bull]| 1824

In disorders such as asthma, cystic fibrosis and bronchitis, the bronchial mucosa is injured by neutrophil-derived degradative enzymes such as cathepsin G (catG) and elastase. Damaged epithelial cells exacerbate the injury by releasing lysosomal cysteine proteinases (LCPs) capable of degrading extracellular matrix. The LCPs catK and S are of particular concern, since they are potent elastolytic agents and are active at both acidic and neutral pH. We hypothesize that the bronchial mucosa has evolved an antiproteinase defense system to protect against excessive LCP activity. We identified two high-molecular-weight serine proteinase inhibitors (serpins), SCCA1 and SCCA2. These molecules are 92% identical at the amino acid level and are highly expressed in cells of the bronchial epithelium and glands. Kinetic analysis shows that SCCA2 is a potent inhibitor of the chymotrypsin-like serine proteinases, catG and mast cell chymase. In contrast, SCCA1 inhibited members of the papain-like LCP family. To confirm that SCCA1 was a specific inhibitor and not just a preferred substrate for LCPs, we performed a kinetic analysis. Typical of serpins, SCCA1 formed tight complexes with catS, L, and K at a stoichiometry of 1:1 and with second order rate constants(kass)=5, 3, and 1 × 105 M1 sec-1, respectively. These rate constants are comparable to those measured for serpin-serine proteinase interactions. To determine whether SCCA1 could inhibit degradation of complex substrates, we measured the inhibition of3 H-elastin degradation by catK. SCCA1 inhibited elastolytic activity by 80% and on a molar basis was more potent than the archetypal cysteine proteinase inhibitor, cystatin C (cysC). Finally, we compared the first order rate constants(kdiss) and kass for the interactions of catS with either cysC vs. SCCA1 by progress curves. The catS-cysC complex association and dissociation = 4 × 105M1 sec-1 and 2 × 10-4 sec, respectively. The complex half-life (t1/2) was 55 minutes. For the catS-SCCA1 interactions, the kass=1 × 105M1 sec-1, but the kdiss was not measured since no dissociation of the complex was detected. Thus, a minimal estimate of the catS-SCCA1 t1/2 was 1155 minutes. These results show that relative to cysC, SCCA1 was slightly slower to form complexes with catS, but once formed were extremely long lived. We conclude that SCCA1 is a bona fide cross-class inhibitor of elastolytic LCPs and that its location in the bronchial mucosa may serve to limit mucosal injury mediated the leakage of endogenous proteinases.

A NEW FAMILY OF SERINE PROTEINASE INHIBITORS (SERPINS) EXPRESSED IN THE LUNG INHIBIT BOTH SERINE AND CYSTEINE PROTEINASES. |[dagger]| 1950

Lung injury can result from an imbalance between proteinases (e.g. neutrophil elastase, cathepsin G, cathepsin L, cathepsin S) and their specific inhibitors. Neutrophil elastase and cathepsin G are elastolytic serine proteinases that are released from the azurophilic granules of activated neutrophils. In addition, the cysteine proteinases cathepsin L and S are contained in the lysosomal compartment of human lung macrophages. Cathepsin S and L are two of the most potent elastolytic enzymes found in humans. Alpha 1-antitrypsin and secretory leukoproteinase inhibitor are serine proteinase inhibitors that partially protect the lung from proteolytic degradation, however these enzymes are easily inactivated by the highly oxidative environment at the mucosal surface.

SYNDECANS, CELL SURFACE HEPARAN SULFATE PROTEOGLYCANS, ARE PRESENT IN AND CAN MODIFY THE PROTEOLYTIC BALANCE OF TRACHEAL ASPIRATES. |[dagger]| 1996

An imbalance between proteinases and antiproteinases is thought to mediate, in part, the inflammatory injury that leads to chronic lung disease in mechanically ventilated pre-term infants. The activities of some proteinases and proteinase inhibitors found in inflammatory fluids can be modifiedin vitro by heparin, a mast cell-derived glycosaminoglycan (GAG). The major cellular source of heparin-like GAG is the syndecan family of transmembrane heparan sulfate proteoglycans, found on the surface of several cell types in the lung.