Alterations in glycoproteins and lipids in azaserine-induced acinar cell carcinoma of rat pancreas

Glycoproteins and lipids of rat pancreatic acinar cell carcinomas maintained in nude mice and in cell culture, were analyzed. The tumor contained significantly elevated levels of glycoproteins when compared with their normal counterparts. SDS-PAGE of tumor glycoproteins revealed that there were increased amounts of small molecular weight glycoproteins and the tumor also contained a 51,000 dalton glycoprotein which was not detected in the pancreas, liver or the sera of the control animals. The tumor in nude mice and cancer cells in culture had decreased lecithins and triglycerides, and increased amounts of free fatty acids, and both free and esterified cholesterols. The results indicate that altered glycoprotein and lipid compositions represent some of the characteristic features of the acinar cell carcinoma.     

Immunological reactivity of mucinous and serous ovarian adenocarcinomas

Comparison of immunological reactivity of glycoprotein antigens extracted from individual cases of mucinous and serous ovarian adenocarcinomas was performed taking into account the immunological relationship with carcinoembryonic antigen (CEA), nonspecific cross-reacting antigen (NCA), alpha-1-antichymotrypsin, and alpha-1-acid glycoprotein. In all immunological tests, the specific immune sera against perchloric acid extracts of ovarian mucinous and serous cystadenocarcinomas and antisera against the reference antigens mentioned above were used. It was established that: 1) ovarian mucinous and serous adenocarcinomas are immunologically different and possess various tumor-associated antigens, 2) ovarian mucinous adenocarcinomas contain considerable amounts of CEA and NCA, whereas serous type neoplasms show negligible amounts or lack of these antigens; and 3) in both types of tumors, alpha-1-antichymotrypsin and alpha-1-acid glycoprotein activities are found. Immunological data indicate that ovarian mucinous and serous adenocarcinomas derive from separate lineages of epithelium.     

Analysis of normal neoplastic human tissues for the tumor-associated protein p97

We have used immunoprecipitation to test tumor biopsies and normal adult and fetal human tissues for p97, a tumor-associated protein. Five of nine melanoma biopsies contained p97 in low to very high levels. Three of seven non-melanoma tumors contained p97, but in smaller amounts. No p97 was detected in any of the normal adult tissues examined. The protein was, however, observed in samples of fetal colon and umbilical cord, and in one sample of fetal lung. One of two benign nevi contained high levels of p97, whole on benign angiofibroma was negative. We conclude that the presence of p97, in levels detectable by our method, appears to be characteristic of certain neoplastic and fetal tissues.     

Antitumor activity of SS(dsFv)PE38 and SS1(dsFv)PE38, recombinant antimesothelin immunotoxins against human gynecologic cancers grown in organotypic culture in vitro.

PURPOSE: Mesothelin, a cell surface glycoprotein overexpressed in ovarian cancer, mesotheliomas, and some squamous cell carcinomas, is an attractive candidate for targeted therapy because it is not shed in significant amounts into the bloodstream and is not present in significant amounts on normal human tissues except for mesothelial cells. The objective of this study was to determine the antitumor activity of SS1(dsFv)PE38, a recombinant antimesothelin immunotoxin, against human gynecologic tumors grown in short-term culture in vitro. EXPERIMENTAL DESIGN: Tumor cells obtained from primary cultures of five ovarian and one cervical tumor were mixed with an equal proportion of NIH-3T3 fibroblasts and plated inside collagen gels in tissue culture plates. After 4-7 days of growth, these organotypic cultures were treated with media alone, SS1(dsFv)PE38, and a control immunotoxin RFB4(dsFv)PE38, which targets the CD22 antigen not present on gynecologic tumors, every other day x 3. The organotypic culture gels were then formalin fixed, paraffin embedded, and evaluated for immunotoxin sensitivity using light microscopic examination of H&E-stained slides and also evaluated for apoptosis using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. RESULTS: Tumors expressing mesothelin showed a significant dose-dependent sensitivity to SS1(dsFv)PE38 even at concentrations as low as 1 ng/ml, whereas no antitumor activity was seen at 100 ng/ml in tumors that did not express mesothelin. This activity was specifically attributable to mesothelin targeting because RFB4 (dsFv)-PE38 had no activity against mesothelin-expressing tumors. CONCLUSIONS: These results demonstrate that ovarian and cervical tumor cells obtained from patients can be grown in short-term culture using an organotypic culture model. Our results also show low concentrations of an immunotoxin targeting mesothelin is cytotoxic to mesothelin-expressing human tumors by inducing apoptosis.     

Elastosis in benign and malignant salivary gland tumors. A histochemical and ultrastructural study

One hundred sixteen cases of various types of salivary gland tumors were examined for the presence of elastic tissue. Almost all the pleomorphic adenomas (97%) and all the malignant pleomorphic adenomas contained elastic tissue in varying amounts. A high percentage (82%) of adenoid cystic carcinomas also contained elastic tissue but the overall quantity was significantly less than in pleomorphic adenomas. All other salivary gland tumors studied, i.e. adenolymphomas, oxyphilic adenomas, mucoepidermoid tumor, and various variants of monomorphic adenomas, were devoid of significant elastic tissue. At the ultrastructural level, the elastic fibers were mainly seen close to neoplastic myoepithelial-like cells, and all stages of elastogenesis were present, ranging from young elastic fibers with a high microfibrilelastin ratio, usually associated with basal-membrane-like material, to mature fibers consisting mainly of an amorphous electron lucent central elastin component. It is postulated that elastic tissue in the salivary gland tumors is produced by the myoepithelial-like tumor cells rather than by tumor stromal induction as has been described in some types of breast carcinomas.     

Structures of the oligosaccharide chains of two forms of alpha 1-acid glycoprotein purified from liver metastases of lung, colon, and breast tumors

Two forms of alpha 1-acid glycoprotein with common immunological determinants and almost identical amino acid compositions but different amounts of carbohydrate were isolated from liver metastases of primary colon, lung, and breast tumors by extraction with perchloric acid, gel filtration on Sepharose CL-6B and Sephadex G-200, and affinity chromatography on concanavalin A:agarose and Ricinus communis agglutinin l:agarose. Both forms of the antigen yielded single bands which stained for protein and carbohydrate when examined by disc gel electrophoresis and immunodiffusion. The molecular weights of the two forms were 45,000 and 37,000 respectively. The larger form contained about five to six oligosaccharide chains, whereas the smaller form had only three to four chains. The composition and structures of the oligosaccharide chains in the two forms of this glycoprotein were very similar. Each contained di-, tri-, and tetraantennary complex-type oligosaccharide chains. The diantennary oligosaccharide chains caused both forms of alpha 1-acid glycoprotein to be retained by concanavalin A-agarose columns. The lower-molecular-weight form contained fewer chains and correspondingly fewer terminal galactosyl residues. This resulted in the separation of this species from the higher-molecular-weight form on columns containing R. communis agglutinin I. Three types of reduced oligosaccharides were released from the light and heavy forms of alpha 1-acid glycoprotein by treatment with alkaline borohydride or by hydrazinolysis. These chains were isolated by chromatography on concanavalin A:agarose and Bio-Gel P-6 columns. The arrangement and linkage of sugars in the purified oligosaccharides were determined by periodate oxidation, sequential hydrolysis with glycosidases, and methylation analysis. The major oligosaccharide chain, comprising 50 to 55% of the carbohydrate, had a triantennary structure as shown in the structure: (formula; see text) in which NeuNAc is N-acetylneuraminic acid, Gal is galactose, GlcNAc is N-acetylglucosamine, Man is mannose, GlcNAcol is N-acetylglucosaminitol, and Fuc is fucose. Tetraantennary chains comprised about 25 to 30% of the carbohydrate, and the additional outer chain was attached to the alpha 1,6-mannosyl residue through a beta 1,6-linked GlcNAc unit. The remaining 15 to 20% of the oligosaccharide chains had a diantennary structure. The extent of sialylation of these chains varied in samples isolated from tumors of the same histological type from different individuals. However, a relatively constant proportion of the three types of chains was present in different forms of the glycoprotein isolated from liver metastases.     

Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and peripheral primitive neuroectodermal tumor.

Ewing's Sarcoma (ES), the second most frequent bone tumor in childhood and adolescence, and the probably closely related peripheral primitive neuroectodermal tumor (pPNET) share a unique cytogenetic translocation between chromosomes 11 and 22. Both of them expose high amounts of a glycoprotein on their cell surface, which can be specifically detected by the mAb HBA-71. The cDNA coding for the HBA-71 antigen was isolated by screening a cDNA expression library constructed from a pPNET-derived cell line. Nucleotide sequencing revealed the HBA-71 antigen to be the product of the pseudoautosomal gene MIC2 previously identified by the mAb 12E7 in haematopoietic cells. This antigen is a glycoprotein with a molecular weight of about 29,000 and is expressed in low amounts in most human cell lines and probably normal tissues and tumors with only a few exceptions. In T-cells the antigen is involved in cell adhesion processes. In ES- and pPNET-derived cell lines MIC2 expression is significantly enhanced. No gross changes in posttranslational modification could be observed. The high expression results in easy and specific detection of the antigen in immunocytochemical analysis of paraffin embedded tissue sections making HBA-71 a useful tool in tumor diagnosis.     

Production of calcitonin, adrenocorticotropic hormone, and beta-melanocyte-stimulating hormone in tumors derived from amine precursor uptake and decarboxylation cells.

The tumor production of human calcitonin (CT) was examined by radioimmunoassay, and it was found that 50 of 85 (59%) tumor tissues collected at random contained immunoreactive CT. These tumors were grouped as to whether they were derived from the amine precursor uptake and decarboxylation (APUD) series. The group that was derived from APUD cells showed appreciable amounts of CT in 30 of 31 (97%) of these tumors or in 20 of 21 (95%) when the medullary carcinomas of the thyroid were excluded. However, of the non-APUD group of tumors only 20 of 54 (37%) were found to contain CT, so that the difference between these two groups was highly significant (p less than 0.001). Of the tumors with ectopic adrenocorticotropic hormone-melanocyte-stimulating hormone production, 12 of 14 were shown to contain CT. These data indicate that CT is a common product of the APUD tumors and that tumor production of CT is often associated with that of adrenocorticotropic hormone and beta-melanocyte-stimulating hormone.     

Expression of glutathione S-transferases in normal gastric mucosa and in gastric tumors

Glutathione S-transferases from both normal gastric mucosa and its matched gastric tumors from 10 different patients were investigated. The transferases were purified and subsequently the isoenzyme composition was studied. Glutathione S-transferase (GST)-pi was present in all specimens in large amounts. Class alpha GSTs were present in 9 out of 10 normal specimens and in six tumors. In malignant tissue, expression of GST-pi was increased at the expense of class alpha GST. In six patients, the ratio GST-pi/GST-alpha was higher in tumorous versus normal tissue. On a Western blot, using a monoclonal antibody, GST-mu was shown to be present in both normal and malignant tissue from four patients, the other six patients completely missed the enzyme in their gastric tissue. When present, GST-mu amounts to only a few per cent of total GST protein. GST-pi was quantified by densitometric analysis of Western blots, treated with a monoclonal antibody against GST-pi. Both total GST enzyme activity as well as the absolute amounts of GST-pi protein were significantly higher in the tumors, as compared to its matched normal mucosa. The importance of this overexpression of GST-pi was previously unknown. However, the frequent occurrence of this phenomenon in many refractory tumors, and as shown now also in gastric cancers, suggests a role for GST-pi in the mechanism of anti-cancer drug resistance.     

Melanoma-associated antigen p97 continues to be expressed after prolonged exposure of cells to specific antibody

p97, a 97,000 molecular weight cell surface glycoprotein associated with human melanoma, has attracted attention as a possible target for tumor therapy. Melanoma cells were grown overnight in the presence of IgG2a or IgGI monoclonal antibodies specific for p97, and then exposed to complement and also tested for binding of monoclonal antibodies specific for other epitopes of p97. The results indicated that melanoma cells continued to express p97 after prolonged exposure to specific antibody, a prerequisite for many therapeutic applications.     

Human tumor cells synthesize and secrete alpha-2-macroglobulin in vitro

In previous studies we showed that human sarcoma and melanoma cell lines synthesize and secrete into culture medium a glycoprotein, migrating in urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 140,000. It is not detected in cultures of the corresponding normal cells. Conditioned medium of the melanoma cell line HMB-2, producing among the cell lines tested the largest amounts of this glycoprotein, has now been used as a source for purification of the protein. NH2-terminal amino-acid sequence determination of the purified glycoprotein showed that it is identical to human alpha 2-macroglobulin (alpha 2M). Rabbit antibodies raised against the glycoprotein specifically reacted in immunoblotting and immunodiffusion tests with alpha 2M present in human plasma. Likewise, these antibodies immunoprecipitated from the conditioned media of 35S-methionine-labelled melanoma and osteosarcoma cell lines the protein which had a molecular weight corresponding to alpha 2M. alpha 2M was also synthesized and secreted by 2 strains of fetal lung fibroblasts but not by fetal skin fibroblasts or adult skin fibroblasts autologous to the osteosarcoma cell line.     

Nephrocalcin: biosynthesis by human renal carcinoma cells in vitro and in vivo.

Renal carcinoma cells removed surgically from two patients (one primary tumor and one bone metastasis) were maintained in short-term culture. Media conditioned by these cells contained calcium oxalate monohydrate crystal growth inhibitor, a glycoprotein named nephrocalcin (NC). NC was also detected in both cell lines by an enzyme-linked immunosorbent assay using anti-NC antibody raised in rabbits. The glycoprotein was purified from the culture medium and found to have an amino acid composition similar to that of normal human urinary NC. However, NC from the renal carcinoma cells, isolated in multiple forms by DEAE-cellulose column chromatography, contained larger amounts of carbohydrate residues than normal NC. Purified NCs showed a dissociation constant of 10(-6) to 10(-8) M toward calcium oxalate monohydrate crystal. Three renal carcinoma cell lines maintained in long-term culture failed to produce NC. Our study demonstrates that NC is produced by renal cell carcinoma cells (in vitro) from primary and metastatic tumors. Preliminary data suggest that urinary levels of NC corresponded with disease progression in patients with metastatic disease, suggesting that NC may be useful clinically as a tumor marker.     

Human chorionic gonadotropin and related glycoprotein hormones in lung cancer cell lines.

Twenty-five non-small cell lung cancer (NSCLC), 42 small cell lung carcinoma (SCLC), one extrapulmonary small cell carcinoma, 4 carcinoid, and 13 non-lung cancer cell lines were analyzed for human chorionic gonadotropin (HCG) and related glycoprotein hormones. HCG or its subunits were present in 72% of NSCLC, 10% of SCLC, one extrapulmonary small cell carcinoma, 3/4 carcinoids and 2/13 non-lung cancer cell lines. Related glycoprotein hormones were undetectable. These data indicate a frequent production of HCG or its subunits by NSCLC cell lines and cell lines from tumors with carcinoid features. They confirm the clinical inclusion of carcinoids under the broad category of NSCLC rather than SCLC despite their neuroendocrine features. Clinicians should not assume that undifferentiated NSCLC with HCG production represent germ cell tumors.     

Glycoproteins distinguishing non-small cell from small cell human lung carcinoma recognized by monoclonal antibody 43-9F

Cell lines derived from human squamous lung carcinoma release large amounts of a soluble glycoprotein into the culture media, having very high molecular weight (greater than 2 X 10(6] and mucin-like properties. A monoclonal antibody called 43-9F has been generated that recognizes a carbohydrate epitope on the glycoconjugate. The epitope is also present on a diverse set of smaller glycoproteins (Mr 50,000-200,000) distributed primarily on the surface of the squamous lung carcinoma cells. A sensitive assay using the 43-9F antibody in a dot blot procedure has been devised that is able to detect an amount of antigen less than that possessed by a single squamous lung carcinoma cell. This assay, and also conventional immunofluorescence and immunohistochemical assay procedures, have been used to screen different normal cells, normal tissues, cancer cells, and tumor biopsy specimens for the antigen. In the normal lung the 43-9F antigen is found only on cells of some of the seromucous glands. In the normal digestive system it is associated in certain organs only with a limited population of mucosal epithelial cells. Other organ systems lack any reactive cells. The cells of most human non-small cell lung carcinomas and their released glycoconjugates have large amounts of the 43-9F epitope, while small cell lung carcinomas and the glycoconjugates released by small cell lung cancer cells lack the epitope. The oligosaccharide recognized by the 43-9F antibody may therefore provide a useful marker to distinguish the different lung carcinomas and for investigating the different cells of origin of these tumors.     

Glutathione, glutathione-s-transferase and p-170 glycoprotein in metastases of malignant melanomas

P-170 glycoprotein, glutathione and glutathione S-transferases are important in in vitro drug resistance, but their clinical relevance is unclear. Therefore glutathione content, glutathione S-transferase enzyme activity, isoenzyme composition as well as P-170 glycoprotein level were studied in metastases of malignant melanomas of thirteen patients. P-170 glycoprotein and glutathione S-transferases were quantified by immunoblotting with monoclonal antibodies, glutathione S-transferase enzyme activity was measured with 1-chloro-2,4-dinitrobenzene as substrate, and glutathione was assayed by HPLC. Glutathione and glutathione S-transferase enzyme activity were measurable in all samples and mean values were 40+/-7 nmol/mg protein (mean+/-SEM; range: 13-98) and 310+/-72 nmol/min mg protein (range: 15-819), respectively. Glutathione S-transferases present were mainly of class pi (2817+/-402 ng/mg protein); class alpha enzymes were detectable only in one case in low amounts (71 ng/mg protein), and class mu transferases were present in 5 out of the 13 samples (38%; 391+/-206 ng/mg protein). The P-170 glycoprotein plasma membrane located drug efflux pump was found in 8 out of 12 samples (67%). In three samples values were much higher as compared to the other specimens. In the metastatic melanoma of one patient, both high levels of glutathione S-transferase and P-170 glycoprotein were found. Further studies are necessary to reveal whether melanoma tissues containing high levels of P-170 glycoprotein, glutathione S-transferases or a combination of both systems do respond differently towards anti-cancer drug treatment.     

Induction of the ceruloplasmin mRNA in Balb/C 3T3 cells after Rous-Sarcoma virus transfection

Ceruloplasmin is a copper containing serum glycoprotein. It is an acute-phase protein and its levels are increased in inflammation and in a number of experimental and human tumors. It is normally synthesized in the liver and not in fibroblasts. In this paper we present evidence that ceruloplasmin mRNA is synthesized in Balb/C 3T3 cells and its levels are increased about 3-fold in Rous-Sarcoma virus (RSV) transformed cells. The ceruloplasmin mRNAs from rat liver and RSV 3T3 cells have the same electrophoretic mobility.     

Comparative analysis of the expression of the extracellular matrix protein tenascin in normal human fetal, adult and tumor tissues.

Tenascin (TN) is a high-molecular-mass oligomeric glycoprotein of the extracellular matrix (ECM) endowed with a programmed expression in embryonic life and a neo-expression in the interstitium of some malignancies. Using monoclonal antibodies (MAbs) which identify human tenascin, we have conducted an extensive immunohistochemical analysis of TN expression in normal fetal and adult human tissues as well as in a wide variety of human tumors. Results of this study demonstrate that TN (1) is detectable in embryonic and fetal tissues at least from the 10th week of gestation; (2) is present in the interstitium of a variety of adult tissues of different embryonic origin; (3) may be neo-expressed in the stroma of benign and malignant tumors; (4) has the ability to accumulate in a highly variable manner in the ECM of tumors of the same and of different histotypes.     

Methylation analysis of the carbohydrate portion of carcinoembryonic antigen.

The carbohydrate structural units of carcinoembryonic antigen samples isolated from four different tumors were quantitated using gas chromatography-mass spectrometery after methylation and subsequent conversion to their alditol acetates. Different carcinoembryonic antigen preparations showed some quantitative but no qualitative differences in the structural units present. The results indicate that a large portion of the fucose residues in the glycoprotein were linked to N-acetylglucosamine and that most of the branching mannose residues were probably linked to three N-acetylglucosamine residues.