ARTICLE |
Correspondence to: Gary A. Silverman, Children's Hospital, Harvard Medical School, 300 Longwood Ave., Enders 970, Boston, MA 02115-5737.
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Summary |
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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, Hassall's 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. (J Histochem Cytochem 48:113122, 2000)
Key Words: squamous cell carcinoma, antigen 1 (SCCA1), squamous cell carcinoma, antigen 2 (SCCA2), serine proteinase inhibitor, (serpin), tissue distribution
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Introduction |
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IN AN EFFORT to identify serological markers of cervical carcinoma,
Several studies show that increased serum SCCA levels correlate with the extent of disease in patients with cervical SCC (
In addition to squamous cell tumors, SCCA is also detected in the sera of patients with skin disorders, such as psoriasis and eczema (
Molecular cloning of the cDNA shows that SCCA belongs to the superfamily of high molecular weight serine proteinase inhibitors (serpins) (
Although SCCA1 and SCCA2 are almost identical members of the serpin superfamily, significant differences in their reactive site loops (i.e., that part of the molecule that is bound by the active site of the proteinase) suggest that they inhibit different classes of proteinases. Biochemical analysis of recombinant proteins shows that SCCA1 is a cross-class 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 (
Previous descriptions of the expression pattern of the SCCA isoforms in normal and malignant tissues were deduced from studies using nondiscriminatory monoclonal reagents or an antibody specific for the acidic isoform (SCCA2) only (
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Materials and Methods |
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Selection of Primers and PCR Conditions
PCR assays were performed in 10-µl volumes containing 0.10.2 ng first-strand cDNA, 200 µM of each dNTP, 0.5 µM of each primer, 1.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl and 0.5 U of Thermus aquaticus (Taq) DNA polymerase. The reaction mixtures were cycled 35 times at 94C for 30 sec, 58C for 30 sec, and 72C for 30 sec. Genomic DNA-free first-strand cDNA was purchased from Clontech Laboratories (Palo Alto, CA). Negative controls (no cDNA) and positive controls [SCCA1 and SCCA2 cDNA in pBluescript plasmid vectors (Stratagene; La Jolla, CA)] were included for each primer set. Discriminatory sense (S) primers for SCCA1 (18S, 5'-CGCGGTCTCGTGCTATCTGG-3' (MA39) and SCCA2 (2-8S, 5'-CACGGTCTCTCAGTATCTAA-3') were from exon 8 and correspond to nucleotides 10221041 of the published cDNAs of SCCA1 and SCCA2 [GenBank accession numbers
U19558 (SCCA1) and
U19569 (SCCA2) (
Generation of MAbs Specific for SCCA1 and SCCA2
BALB/c mice (Charles River Laboratories; Stockbridge, MA) were injected with whole yeast extract containing full-length recombinant SCCA1 or SCCA2. Antigens emulsified in RIBI adjuvant were injected IP into mice on Days 1 and 21. Responders received an intrasplenic immunization on Day 35. Spleen cells were harvested on Day 38 and fused to P3X63Ag8U.1 mouse myeloma cells (American Type Culture Collection; Rockville, MD) in the presence of polyethylene glycol 1500 (Boehringher Mannheim; Indianapolis, IN) Hybridomas were selected for growth in Dulbecco's modified Eagle's medium (DMEM, 4.5% glucose) (BioWhittaker; Walkersville, MD) supplemented with 100 µg/ml endothelial cell mitogen (Biomedical Technologies; Stoughton, MA), 10 mM sodium hypoxanthine, 40 µM aminopteridin, 1.6 mM thymidine, 20% fetal bovine serum, 1 mM nonessential amino acids solution, 2 mM L-glutamine, 1 mM sodium pyruvate solution, 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies; Grand Island, NY). Hybridomas were screened by an antibody capture ELISA using purified recombinant SCCA1 or SCCA2 as the antigen. Positive clones were subcloned by two or three rounds of limiting dilution (one cell/well).
Specificity of SCCA1 and SCCA2 MAbs by Immunoblotting
Immunoblots containing recombinant proteins GST-SCCA1, GST-SCCA2, and a related serpin, chicken ovalbumin, were prepared to test the specificity of the MAbs (Figure 1). Proteins were separated by SDS-PAGE and electroblotted at 100 V for 1 hr at 4C onto reinforced nitrocellulose (Nitroplus; Micron Separations, Westborough, MA). The transfer buffer was 25 mM Tris base, pH 8.0, 190 mM glycine (pH 8.3). After three rinses in washing buffer (PBS, pH 7.4, 0.1% Tween-20), the blot was incubated for 1 hr in I-Block (Tropix; Bedford, MA) blocking buffer (PBS, pH 7.4, 0.1% Tween-20, 0.2% I-Block) and then incubated for 1 hr with purified mouse monoclonal ascites as the primary antibody. The primary antibodies were used at the following dilutions: anti-SCCA1 (clone 8H11) 1:2000 (1.7 µg/ml); anti-SCCA2 (clone 10C12) 1:100,000 (0.03 µg/ml). The blot was washed three times in wash buffer and then incubated for 1 hr with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories; Westgrove, PA) as the secondary antibody. After three rinses in wash buffer and two rinses in chemiluminescent assay buffer (0.1 M diethanolamine-HCl, pH 9.8, 1.0 mM MgCl2, 5% Nitroblock), the blot was incubated for 5 min with chemiluminescent substrate (CDP-Star; Tropix). Chemiluminescence was detected by autoradiography.
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Cell Transfections and Immunocytochemistry
The MDA-MB-435 breast carcinoma cell line (kindly provided by Dr. Ruth Sager), which expresses no SCCA by immunoblotting or RT-PCR (unpublished data) was transfected stably with plasmids containing recombinant SCCA1 or SCCA2 cDNA under control of the EF-1 promoter (
For immunostaining, SCCA1, SCCA2, and mock-transfected MDA-MB-435 cells were grown to 50% confluency on 4-chambered slides (Nalge Nunc International; Naperville, IL). The cells were washed in PBS (0.01 M phosphate buffer, 27 mM KCl, 137 mM NaCl, pH 7.4), fixed by incubating in 10% neutral-buffered formalin for 15 min, and permeabilized by incubating in 0.2% Triton X-100 in PBS for 4 min. The immunocytochemistry was performed as described below, except that the peroxidase blocking step was omitted, undiluted hybridoma supernatants served as the primary antibody, and hematoxylin was used as the counterstain.
Immunohistochemistry
Most of the tissues used for immunohistochemical analysis were obtained from surgical resection and diagnostic biopsy material from Beth Israel Deaconess Medical Center. All heart specimens (3/3), and some pancreas (1/4), liver (1/4), spleen (1/4), and adrenal (1/3) tissues were obtained from human adult autopsies performed within 10 hr of expiration. The normal tissue survey included specimens from three or more patients (except for tongue and urethra; see Table 1) and was confined to non-neuronal tissues. Three or more specimens of lung (squamous cell, adeno-, large-cell, and small-cell) and head and neck (tongue) SCCs of various histologic types and differentiation states were also examined (Table 2). All tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin according to conventional procedures. Four-µm-thick tissue sections placed on polysine-coated slides (Fisher; Atlanta, GA) were deparaffinized in xylenes (two 5-min washes) and rehydrated in graded ethanol solutions (100%, two 5-min washes; 95% and 70%, two 1-min washes) and distilled water (one 5-min wash). Between the absolute and 95% ethanol washes, samples were incubated for 10 min in a 0.3% H2O2 (v/v) methanol solution to quench endogenous peroxidase activity. Antigen retrieval methods employing enzymatic digestion (0.050.25% trypsin; 0.1% pepsin at 37C) and microwave/steamer heating (10 mM citrate buffer, pH 6.0) were tested to optimize SCCA1 and SCCA2 immunostaining (
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Immunostaining was performed at room temperature in a humidified chamber. The standard incubation volume for each reagent was 200 µl. Sections were circled with a PAP pen (Zymed Laboratories; San Francisco, CA) and blocked by incubating with 10% normal goat serum diluted in PBS with 1% (w/v) bovine serum albumin (Sigma Chemical; St Louis, MO) for 30 min. After washing in PBS, the sections were incubated with the primary antibody (purified mouse monoclonal ascites) for 1 hr at the following dilutions: anti-SCCA1 (clone 8H11) at 1:1000 (final concentration 3.4 µg/ml); anti-SCCA2 (clone 10C12) at 1:500 (6.0 µg/ml). Optimal hair follicle staining in skin required a 1:500 dilution of anti-SCCA1 MAb (6.8 µg/ml). For certain normal tissues and for all neoplastic tissues, it was necessary to block endogenous biotin activity, especially in antigen-retrieved sections (e.g., salivary gland ducts, bronchial mucous glands, liver, kidney, testis, and endocrine tissues). A biotin blocking kit (SP-2001: Vector Laboratories; Burlingame, CA) was used as follows: 4 drops of Avidin D solution (70 µg/ml) was added per ml normal goat serum before incubation on sections. Sections were washed briefly in PBS, then incubated with 4 drops of biotin solution (20 µg/ml) per ml diluted primary antibody. For all tissues tested, the primary antibody was replaced with mouse monoclonal anti-glutathione-S-transferase IgG (Sigma) diluted 1:1000 (10 µg/ml) as a negative control. As a positive control, each tissue type was stained with a rabbit anti-SCCA heteroantiserum that does not discriminate between SCCA1 and SCCA2 (
After the primary antibody incubation, sections were washed for 10 min in PBS, then incubated with a biotinylated goat anti-mouse IgG (Jackson Immunoresearch Laboratories) diluted 1:10001:2000 (1.0 0.5 µg/ml) as the secondary antibody. After a 10-min PBS wash, the secondary antibodies were detected by incubation for 30 min with an avidinbiotinperoxidase complex (Vectastain Elite ABC horseradish peroxidase kit; Vector Laboratories), followed by a 10-min PBS wash, then a 5-min incubation with a 3,3'-diaminobenzidine substrate/chromogen solution (Liquid DAB-Plus substrate kit; Zymed Laboratories). Sections were washed in several changes of distilled water and counterstained with methyl green.
To control for fixation artifacts, immunostaining was also performed on fresh frozen vagina (positive control), skin, breast, fetal membranes, and testis. These tissues were embedded in Tissue-Tek OCT Compound (Miles; Elkhart, IN), sectioned to 4 µm using a cryostat and fixed by dipping in 25C acetone. Slides were stored at -80C until use. Before staining, sections were equilibrated to -20C in a cryostat, fixed in acetone for 10 min at -20C, washed with cold (4C) PBS, and blocked for endogenous peroxidase as above. Immunohistochemistry was performed as above with 1:100 dilutions of anti-SCCA MAb 8H11 (34 µg/ml) and anti-SCCA2 MAb 10C12 (30 µg/ml).
Immunostaining results were assessed in a blinded fashion by two independent observers. In normal tissues, immunoreactivity was reported as present or absent with qualitative comments about the intensity of staining, such as weak, moderate, or strong. In addition, immunostaining in tumor tissues was assessed as the percentage of positively stained cells.
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Results |
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Survey of SCCA1 and SCCA2 Expression by PCR
First-strand cDNA libraries were assayed to determine whether SCCA1 and SCCA2 were expressed in different tissues (Figure 2A and Figure 2B). SCCA1- and SCCA2-specific primers amplified the correct-sized fragments from lung, lymph node, thymus, tonsil, placenta, prostate, and testis cDNAs. Neither SCCA1 nor SCCA2 was detected in samples from brain, heart, kidney, liver, pancreas, skeletal muscle, bone marrow, fetal liver, peripheral blood leukocytes, spleen, colon, ovary, and small intestine. All PCR assays were performed at least three times and the positive reactions for SCCA1 and SCCA2 were repeated by using the primer set from exons 6 and 8 (not shown). This result confirmed that the positive signals were not derived from contaminating genomic DNA sequences. The control ß-actin fragment was amplified from all of the cDNA samples tested (Figure 2C).
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Specificity of SCCA1 and SCCA2 MAbs Using Immunocytochemistry on Transfected Cell Lines
To confirm the specificity of these antibodies as an immunohistochemical reagent, we studied their reactivity against transfected MDA-MB-435 cell lines expressing either SCCA1 or SCCA2. A mock-transfected (non-SCCA-expressing) MDA-MB-435 cell line served as a negative control. Anti-SCCA1 MAb 8H11 showed immunoreactivity only with SCCA1-transfected MDA-MB-435 cells and not with the SCCA2 or mock-transfected MDA-MB-435 cells (Figure 3B, Figure 3E, and Figure 3H). Similarly, anti-SCCA2 MAb 10C12 reacted against only the SCCA2-transfected MDA-MB-435 cells (Figure 3C, Figure 3F, and Figure 3I). These studies confirmed that the MAbs 8H11 and 10C12 could discriminate between SCCA1 and SCCA2 when used as an immunohistochemical reagent.
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Immunohistochemical Analysis of Human Tissues Using SCCA1- and SCCA2-specific MAbs
The results are summarized in Table 1 and Table 2 and representative sections are illustrated in Figure 3J3U and Figure 4.
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Adipose Tissue. No SCCA1 or SCCA2 immunoreactivity was observed in adipocytes.
Musculoskeletal System. No SCCA1 or SCCA2 immunoreactivity was observed in skeletal muscle, myofibrils, chondrocytes, or osteocytes.
Skin. In normal adult skin, weak to moderate focal SCCA1 and SCCA2 cytoplasmic immunoreactivity was detected in the external root sheath of selected hair follicles. In some cases, the epidermis adjacent to positively stained hair follicles exhibited weak to moderate suprabasal staining for SCCA1 and SCCA2. However, normal epidermis away from these structures was negative for SCCA1 and SCCA2 immunoreactivity (Figure 3K and Figure 3L). Moderate to strong cytosolic staining was noted in the suprabasal epidermal keratinocytes (strata granulosum and spinosum) over areas of inflammation. Often these areas were marked by epidermal thickening (acanthosis).
Cardiovascular System. No SCCA1 or SCCA2 immunoreactivity was detected in cardiomyocytes, fibroblasts, endothelial cells, or arterial smooth muscle cells.
Respiratory System. The pseudostratified columnar epithelial cells lining the nasopharynx, trachea, bronchi, and bronchioles exhibited strong primarily cytosolic immunoreactivity for SCCA1. SCCA2 staining was moderate to strong (Figure 3N and Figure 3O). In the alveoli, both Type I and Type II pneumocytes were nonreactive.
Digestive System. Strong cytosolic SCCA1 immunoreactivity was observed in the suprabasal squamous epithelium of the tongue, and SCCA2 staining was moderate to strong. Weak to moderate cytosolic immunoreactivity for both SCCA1 and SCCA2 was detected in the suprabasal squamous epithelium lining the tonsils. Parotid and minor salivary glands were negative for SCCA1 and SCCA2, and submandibular gland ducts showed weak to moderate immunoreactivity only in areas of squamous metaplasia (not shown). Strong cytosolic immunoreactivity was detected for SCCA1 in the suprabasal keratinocytes of the stratified squamous epithelia in both esophageal and anal mucosa. SCCA2 staining was moderate to strong in these structures. No staining was detected in any cell type of the stomach, small or large intestine, pancreas, liver, or gallbladder.
Hematolymphoid System. Moderate to strong staining was observed for both SCCA1 and SCCA2 in the thymic epithelial cells in Hassall's corpuscles (Figure 3Q and Figure 3R). Hematopoietic cells of the bone marrow, medullary and cortical thymocytes, and lymphoid cells of lymph nodes, tonsils, and spleen were all nonreactive.
Urinary System. No SCCA1 or SCCA2 immunoreactivity was detected in kidney, ureter, urethra, or bladder.
Male Reproductive System. No SCCA1 or SCCA2 immunoreactivity was observed in normal prostate ducts or acinar tissue. However, selected areas of prostate showed expression of SCCA1 and SCCA2. These areas were focal and were marked by atrophy and/or chronic inflammation as well as squamous metaplasia (not shown). No SCCA1 or SCCA2 immunoreactivity was observed in testis, epididymis, vas deferens, seminal vesicles, or rete testis.
Female Reproductive System/Mammary Gland. Strong cytosolic SCCA1 immunoreactivity was detected in the intermediate, suprabasal stratified squamous epithelia of the uterine cervix and vagina. SCCA2 staining was moderate to strong in the former and strong in the latter (Figure 3T and Figure 3U). All cells in the uterus, Fallopian tubes, ovaries, and pre-/postmenopausal mammary gland were nonreactive for SCCA1 and SCCA2.
Female Reproductive System/Placenta. No SCCA1 or SCCA2 immunoreactivity was observed in any cell type within the fetal membranes, placental disk, or umbilical cord.
Endocrine System. In addition to negative results observed in the reproductive endocrine tissues, neither antigen was detected in thyroid, parathyroid, adrenal gland, or pancreatic islets.
Head and Neck SCCs. Weak to moderate cytosolic SCCA1 immunoreactivity was observed in four of the seven primary tongue SCC cases (Table 2). In general, SCCA1 immunoreactivity was observed in the more keratinized and better-differentiated cells within the tumors and was confined to less than 50% of the population (Figure 4B). Similarly, cytosolic SCCA2 immunoreactivity was detected in five of the seven primary tongue SCCs, and the intensity was moderate to strong in well-differentiated tumors compared to the less intense staining of poorly differentiated tumors (Figure 4C). In almost all cases, the antigens co-localized to selected keratinizing tumor nests, with no appreciable difference in intensity of the staining between SCCA1 and SCCA2. In contrast to the homogeneous staining pattern in suprabasal layers of normal stratified squamous epithelia, SCCA1 and SCCA2 immunoreactivity in SCCs was heterogeneous and sparse, with some predilection for the superficial portion of the tumors.
Lung Carcinomas. Of the four major forms of lung carcinoma (squamous cell, adeno-, large-cell, and small-cell), SCCA1 and SCCA2 immunoreactivity was detected only in SCCs (Table 2). In the moderately and well-differentiated tumors, the intensity and extent of SCCA1 and SCCA2 staining were comparable and were correlated with their state of differentiation of the SCCs. Focal moderate to intense cytoplasmic staining was observed in selected nonbasal keratinizing cells (Figure 4E and Figure 4F). In two of the three poorly differentiated lung SCCs, weak SCCA2 immunoreactivity was observed in a small percentage of cells. None of the poorly differentiated cases showed SCCA1 staining.
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Discussion |
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In this study we showed that SCCA1 and SCCA2 co-localized to several normal adult human tissues at both the mRNA and the protein level. This co-localization pattern was also observed in most cases of head and neck SCCs and in lung SCCs.
The RNA transcripts of both SCCA1 and SCCA2 genes were detected by RT-PCR (reverse transcription-PCR) in human lung, tonsil, thymus, lymph node, placenta, prostate, and testis. Using discriminatory MAbs whose specificities were confirmed by immunocytochemistry on transfected cell lines, SCCA1 and SCCA2 proteins co-localized to the skin, esophagus, tonsil, tongue, thymus, trachea, bronchi, bronchiole, vagina, and uterine cervix. Overall, SCCA1 and SCCA2 showed a restricted expression pattern in normal tissues, implying tissue-specific control of coordinated gene expression for these serpins. In four tissues (placenta, lymph node, prostate, and testis) in which SCCA1 and SCCA2 transcripts were detected by RT-PCR, immunohistochemistry failed to localize the corresponding proteins. This discrepancy probably reflects the greater sensitivity of the former technique. Consistent with the RT-PCR data, dbEST also contains SCCA cDNA sequences that were cloned from normal placenta and testis libraries.
In normal adult skin, we detected weak and focal SCCA1 and SCCA2 immunoreactivity only in the outer root sheath of selected vellus and terminal hair follicles and, in some of these cases, in the epidermis immediately adjacent to these structures. SCCA immunoreactivity in normal squamous epithelium of the skin was reported in several studies (
In a previous study, TA-4 immunoreactivity was detected in the intermediate layer of the squamous epithelium in the tongue, pharynx, and esophagus and in the ductal epithelia of the submandibular glands (
Interestingly, we noted that both SCCA1 and SCCA2 were present in areas of squamous metaplasia in the ductal epithelium of the submandibular glands and the prostate gland. Other investigators report the presence of TA-4 in areas of squamous metaplasia within the lung (
SCCA1 and SCCA2 expression was not restricted to squamous epithelial cells. SCCA1 and SCCA2 were detected in the pseudo-stratified columnar epithelium of the conducting airways. Although previous reports indicated the presence of SCCA in respiratory secretions, its source was not identified (1-proteinase inhibitor). Because SCCA1 and SCCA2 are inhibitors of serine and cysteine proteinases, their location in the bronchial mucosa would place them in an ideal position to protect the airways from proteinases derived from inflammatory cells, epithelial cells, and microorganisms.
Another novel finding of this survey was the detection of both SCCA1 and SCCA2 in Hassall's corpuscles of the thymus. Hassall's corpuscles are composed of terminally differentiated keratinized epithelial cells and resemble the suprabasal squamous epithelium in their cytokeratin contents (
In the female genitourinary system, we detected comparable SCCA1 and SCCA2 staining in the suprabasal layers of the stratified squamous epithelium of the uterine cervix and vagina.
In head, neck, and lung SCCs, we detected SCCA1 and SCCA2 immunoreactivity in well-differentiated squamous cell components of the tumor tissues. This pattern of expression was similar to that observed in several studies using nondiscriminatory antibodies raised against the TA-4 complex (
The co-expression of SCCA1 and SCCA2 in the squamous epithelium of mucous membranes, some areas of the skin, lung, thymus, and in moderately and well-differentiated SCCs suggests an important role for these serpins in the coordinate regulation of certain serine and cysteine proteinases associated with both normal and transformed cells. Further analysis of SCC with the discriminatory MAbs described in this report should determine whether perturbations of the coordinate expression of SCCA1 and SCCA2 alter the proteinaseinhibitor balance and contribute to the transformed phenotype associated with different types of squamous cell carcinomas.
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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Supported by grants from the Smokeless Tobacco Research Council, Inc. (GAS), the Nell and Nancy Fund, a Cancer Prevention Fund supported by AFLAC, Inc. (MPU), the Pine Mountain Benevolent Association (MPU), the Beth Israel Pathology Foundation, Inc., Boston, MA (MPU), and the National Institutes of Health grants HD28475 (GAS), CA69331 (GAS), and CA73031 (CS).
We wish to thank Drs Theodore Kwan, Larry Brown, Stephen Pak, and Cliff Luke for helpful discussions, and Ellie Manseau, Doina Mandrila, and John Prioleau for technical advice.
Received for publication March 3, 1999; accepted August 18, 1999.
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