Variations in the relative amounts of biotin-containing enzymes present in both tumorigenic and non-tumorigenic hybrid cells and other cell lines.

The observation that radioactively labelled streptavidin binds to several biotin-containing enzymes in mammalian cells has led to the finding that there is considerable variation in the proportion of these enzymes present (namely beta-methyl crotonyl CoA; propionyl CoA; pyruvate and acetyl CoA, carboxylases). This is particularly striking when certain tumorigenic and non-tumorigenic hybrid cells are compared. It is found that there is a consistently higher proportion of pyruvate carboxylase in the tumorigenic hybrid cells. However, not all tumorigenic cell lines show this same characteristic and reasons for this are discussed. It is also shown that whilst the proportions of the four enzymes are apparently constant for a given cell type, there is a substantial degree of clonal variation and this is particularly so in tumorigenic cells in vitro. However, the more tumorigenic cells in a given population do show a higher proportion of pyruvate carboxylase. Also a range of cells derived from lymphoid tissue has been compared with normal human lymphocytes and considerable differences are again observed. The significance of these findings is considered in relation to other phenotypic properties of hybrid cells.

Heterogeneity of the glucose transporter in malignant and suppressed hybrid cells.

Previous work has demonstrated unequivocally that the kinetics of glucose transport of tumorigenic and suppressed hybrid cells show a consistent difference. This lies in an increased affinity for glucose in the tumorigenic cell lines (i.e., a reduced Km). Evidence has also been presented that the degree of glycosylation of the transporter may affect the Km. When a suitable antiserum to the transporter present in these cells became available, it was of interest to examine the patterns of binding to immunoblots of extracts of the hybrid cell pairs. It became apparent that there was a clear difference between the two parental cell lines and that this difference was reflected in the hybrid cells. Both the tumorigenic parent and the tumorigenic hybrid cell presented a much more heterogeneous distribution of apparent molecular weights than the nontumorigenic parent or suppressed cell line. That this was probably due to differences in glycosylation was indicated by the effect of tunicamycin on the cells; this gave rise to a more homogeneous band of lower molecular weight. The significance of these differences is discussed in relation to results obtained with other tumorigenic cell lines and to the published structure of the human erythrocyte glucose transporter.

Immunoreactive determinants of CA 125 in women with endometriosis.

Among 10 patients with endometriosis CA 125 was increased (greater than 35 U/ml) in endometriotic cyst fluid in all the patients, but only two had increased serum concentrations. Gel electrophoresis of serum, endometriotic cyst fluid, and endometriotic tissue resolved the CA 125 immunoreactive fragments from the three sources into bands of similar electrophoretic mobilities. Electrophoresis under reducing and non-reducing conditions showed immunoreactive fragments of apparent masses of 55,000 and 140,000 daltons, respectively. Analysis under reducing conditions did not result in loss of activity. CA 125 antigen is thought to be a high molecular weight glycoprotein complex. As far as is known, this is the first report describing lower molecular weight immunoreactive determinants of CA 125.

Further characterization of the antigen defined by the monoclonal antibody M27.

Using immunoblotting techniques, the antigen that binds the monoclonal antibody M27 has been clearly defined in terms of apparent molecular mass and distribution. In reducing conditions it has an apparent mass of 178K (K = 10(3) Mr) and is present in the cytoplasm and membranes of all mammalian tissue culture cells so far examined. It is absent from lines derived from avian, piscine and amphibian sources. It is also absent from foetal liver of both rat and mouse, but subsequently appears after cultivation in vitro. Similarly, it can be detected on rat lymphocytes only after mitogenic stimulation. However, it is found on both hepatoma and lymphoma cells in vitro, and on in vivo tumours from murine sources. It thus appears to be associated with cell proliferation.

Purification and characterization of the epitectin from human laryngeal carcinoma cells.

The purification and partial characterization of epitectin (previously called Ca antigen) from a human cancer cell line is described. This glycoprotein, which is expressed on a wide range of human tumors and certain specialized normal epithelia, can be detected using monoclonal antibodies, Ca1, Ca2, and Ca3. The purified glycoprotein had a high density (1.40 g/ml) on isopycnic centrifugation indicating a high carbohydrate content. The molecular mass of epitectin as determined by size-exclusion chromatography ranged from 1.0 to 1.5 x 10(6) daltons. However, the purified epitectin gave two bands of apparent molecular weight 390,000 and 350,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of epitectin and asialoepitectin were found to be 5.3-5.4 and 6.8, respectively. The oligosaccharides were isolated from metabolically labeled epitectin by alkaline borohydride treatment and their structures established based on high performance liquid chromatography and paper electrophoretic migration, sugar composition, the results of sequential exoglycosidase treatment, periodate oxidation, and methylation analysis. The structures of the three major fractions, which together account for about 80% of the radioactivity, were assigned as NeuNAc alpha 2----3Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----3Gal beta 1----3GalNAc(OH), and Gal beta 1----3 GalNAc(OH). The structures of the minor fractions were tentatively assigned as NeuNAc----Gal(NeuNAc----Gal----GlcNAc)----GalNAc(OH), Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----6GalNAc(OH), and GalNAc(OH). It is proposed that the protein sequence and/or the distribution of the saccharides on the protein core are the determinants on epitectin that are recognized by the Ca antibodies.

The breast tumour-associated epithelial mucins and the peanut lectin binding urinary mucins are coded by a single highly polymorphic gene locus 'PUM'.

A family of mucin-type glycoproteins, present in human urine, is coded by a single highly polymorphic gene locus PUM. We have previously shown that these glycoproteins carry epitopes recognized by a series of monoclonal antibodies, many of which were raised to the human milk-fat globule membrane, and which bind to a wide variety of carcinomas and certain normal epithelia. Here we show that in the normal human mammary gland, and in breast cancers the epitopes are present on the same family of molecules as that found in urine. Thus the genetically determined variation at the PUM locus accounts for much of the electrophoretic heterogeneity of the mucin-type glycoproteins present in breast cancer and serum from breast cancer patients that has been reported previously. Knowledge of this normal inherited polymorphism is essential to the interpretation of possible changes to these molecules in malignancy.

Characterization of biotinylated proteins in mammalian cells using 125I-streptavidin.

The presence of biotinylated proteins in detergent extracts of cells is demonstrated using 125I-streptavidin. Four components with apparent molecular masses (Mr) ranging from 70 to 220 kDa have been detected. These probably represent the biotinylated subunits of the carboxylase enzymes. The proportions of the four components were found to vary from cell type to cell type and also the Mr varied from species to species.

A glycoprotein rapidly labelled with [14C]glucose present in a murine melanoma cell line.

If a murine melanoma cell line PG19 is metabolically labelled with [14C]glucose for a short time, one particular membrane component incorporates more radioactivity than the rest. This is a glycoprotein of about Mr 100,000, which is not preferentially labelled in non-malignant variants of the melanoma or in suppressed hybrid cells formed between PG19 and mouse fibroblasts.

The human tumour-associated epithelial mucins are coded by an expressed hypervariable gene locus PUM.

A single highly-polymorphic autosomal gene locus PUM codes for a family of mucin-type glycoproteins, separable by SDS-gel electrophoresis, which we first identified in human urine. The locus also codes for glycoproteins which are abundant in several other normal epithelial tissues and body fluids, including milk, and in tumours of epithelial origin. These mucin-type glycoproteins seem to be very immunogenic in rodents and, in a search for epithelial specific or tumour-associated antigens, a large number of related antibodies have been isolated which bind to the PUM-coded mucins. Many of the antibodies show a pronounced tumour specificity on immunohistology and are being used widely in cancer diagnosis in vitro and in vivo and even in cancer therapy. To investigate the expression of these antigens in normal and malignant cells complementary DNA coding for the mammary mucin has been isolated. Here we present evidence obtained using this cDNA that the PUM locus is a hypervariable 'minisatellite' region of human DNA similar to those described by several groups, but which is novel in that it is transcribed and translated, and that the same polymorphism is demonstrable in the expressed gene product.

The suppression of malignancy by terminal differentiation: evidence from hybrids between tumour cells and keratinocytes.

When malignant human cells are crossed with diploid human keratinocytes, malignancy, as defined by progressive growth in vivo, is suppressed so long as the hybrid cells continue to produce involucrin, a protein that characterizes terminal differentiation in the keratinocyte. When, on continued cultivation in vitro, the cells lose the ability to produce involucrin, they reacquire the ability to grow progressively in the animal.

Electron-microscopic studies of the CA antigen, epitectin.

Epitectin, the mucin-like glycoprotein defined by the monoclonal antibodies CA1, CA2 and CA3, has been examined by electron microscopy to determine its shape and size. It appears to be a single extended strand with a mean length of about 270 nm. The antibodies CA1 and CA2 appear to bind preferentially to a terminal site on the epitectin molecule.

Ca2 and Ca3. New monoclonal antibodies evaluated as tumor markers in serous effusions.

Ca2 and Ca3 are new monoclonal antibodies of IgG1 class, directed against the Ca antigen, a mucus-type glycoprotein expressed on the surface of a wide range of malignant human cells and certain specialized normal epithelia. These antibodies were produced by immunization with purified preparations of the Ca antigen. They were tested to assess their value in the diagnosis of malignant effusions. Immuno-alkaline-phosphatase staining was used. Smears of pleural and peritoneal effusions were chosen to show: undoubted malignant cells of various types; and mesothelial cells in effusions from cases in which cancer was not in question. The Ca2 antibody, at 1 in 20 dilution of the culture supernatant, was the most specific, giving no reactions with benign mesothelial cells from any of the 35 cases tested. Malignant cells were clearly stained in 35 of 40 cases of carcinoma or mesothelioma. The staining was negative in two cases of oat cell bronchial carcinoma, and in three of four cases of carcinoma of the colon. Ca3 gave similar, but somewhat stronger, reactions with carcinoma cells, but was less specific, reacting weakly with mesothelial cells in 8 of 35 benign effusions. Because the false-negative reactions given by the Ca series of antibodies are to some extent complementary to those given by monoclonal antibodies directed against the carcinoembryonic antigen (CEA), a combination of Ca2 and anti-CEA is recommended as a most useful addition to the normal cytologic examination of effusions.

Alterations induced by glucose deprivation and tunicamycin in the kinetic parameters of hexose transport in hybrid cells.

Matched pairs of malignant and non-malignant hybrid cells were compared in their response to glucose deprivation and to tunicamycin. Glucose deprivation induced an increase in the maximum velocity in the malignant cells, but not in the non-malignant cells. The Michaelis constant of hexose uptake was largely unchanged by glucose deprivation except in the case of one melanoma derivative, PG19 G-, which showed a large increase in Michaelis constant when deprived of glucose. Tunicamycin increased the Michaelis constant of hexose uptake in both malignant and non-malignant cell lines. It is therefore possible that the Michaelis constant of hexose uptake is affected by the extent of glycosylation of one or more of the cell membrane glycoproteins.

Structure and function of the Ca antigen.

The Ca antigen, which can be detected in a wide range of malignant human tumours by means of the Cal antibody, is a glycoprotein of the mucin type. At least 95% of the carbohydrate is 0-glycosidically linked to the polypeptide which contains high proportions of glycine, serine and glutamic acid. The carbohydrate has a very simple structure: it is composed almost entirely of tetra- tri- and disaccharides having the general formula (NeuNac)n leads to [Gal leads to GalNac] alpha leads to, where n = 0, 1 or 2. In many malignant cell lines, the antigen is produced constitutively in vitro; but in one that has been examined, its synthesis can be induced by high concentrations of lactate. Evidence is presented for the view that a primary function of this glycoprotein is to shield the cells that produce it from hydrogen ion concentrations outside of the physiological range. The presence of the Ca antigen in malignant tumours may thus be a reflection of metabolic conditions that are known to be characteristics of such tumours.

Kinetic parameters of hexose transport in hybrids between malignant and nonmalignant cells.

Matched pairs of isogeneic hybrid cells, in which one member of the pair was malignant and the other not, were used to examine the linkage between malignancy and functional alterations in hexose transport. The kinetic parameters of uptake of 2-deoxy-D-glucose were measured in a range of such hybrids, both human and murine. Some other malignant cell lines were also examined and were compared with non-tumorigenic derivatives of tumour cells selected by exposure to the lectin, wheat-germ agglutinin. In every case, malignancy, as defined by the ability of cells to grow progressively in vivo, was found to be linked to a decrease in the Michaelis constant of hexose uptake. Independent measurement of the transport and phosphorylation reactions involved in hexose uptake revealed that this decrease was determined by the membrane transport system. The difference in Michaelis constant between malignant and non-malignant cells was observed with 3-O-methylglucose, a hexose that is transported into the cell but not further metabolized. The activity of hexokinase in cell homogenates was higher than the level that would be required to cope with transport and showed no correlation with tumorigenicity. Measurement of the uptake of D-glucose itself, by a rapid filtration centrifugation method, gave results similar to those obtained with 2-deoxy-D-glucose.

Cystic fibrosis: a detailed analysis of fibroblast membrane glycoproteins.

The binding properties of two lectins, concanavalin A and wheatgerm agglutinin to membrane glycoproteins extracted from cystic fibrosis fibroblasts have been examined. No differences were found between the binding patterns of either of these lectins to cystic fibrosis and normal fibroblast glycoproteins. The amount of concanavalin A binding to a glycoprotein of approximate Mr 150 000 has been shown to be related to the population doubling time of a cell culture at the time of analysis.

A new marker for human cancer cells. 1 The Ca antigen and the Ca1 antibody.

In a search for antibodies that might distinguish between malignant and non-malignant cells a panel of matched pairs of hybrid cells produced by fusion of diploid fibroblasts with malignant cells originating from a cervical carcinoma was used as a screen. Each pair consisted of a hybrid in which malignancy was suppressed and a malignant segregant derived from this hybrid. A monoclonal antibody, designated Ca1, was found that discriminated absolutely between the hybrids in which malignancy was suppressed and the malignant segregants. This antibody detected an antigen present in the cell membranes of a wide variety of malignant human cells lines but not of diploid human cell strains. The antigen was found in very low concentrations, if at all, in homogenates of normal adult or fetal tissues. It could be immunoprecipitated by the Ca1 antibody from extracts of malignant cells but not from extracts of non-malignant cells. After reduction, the immunoprecipitated antigen separated in sodium dodecyl sulphate acrylamide gels as two bands with proximate molecular masses of 390 000 and 350 000. These two components had a properties of glycoproteins with a high carbohydrate content; both bound the Ca1 antibody. The antigenic determinant resisted boiling at 100 degrees C and extraction by range of organic solvents. The binding of the Ca1 antibody to the antigen was substantially reduced by treatment of the antigen with neuraminidase, and the antigenic determinant was largely destroyed by certain endoglycosidases and by extensive proteolysis. Pending its further characterisation, this antigen had been called the Ca antigen.

A new marker for human cancer cells, 2 immunohistochemical detection of the Ca antigen in human tissues with the Ca1 antibody.

The Ca1 antibody has been used in an immunohistochemical procedure to detect the Ca antigen in sections of tissues routinely embedded in paraffin wax. A representative sample of benign and malignant tumours from all the systems of the human body has been examined. The majority of malignant tumors express the Ca antigen. The exceptions are: prostatic carcinomas, testicular teratocarcinomas and seminomas, some sarcomas, some lymphomas, malignant brain tumours, neuroblastomas, and melanomas. The antigen is least readily detected in epithelial malignancies of the alimentary system, particularly of the colon. The Ca1 antibody does not react with any benign tumour. The only normal tissues that react specificity with this antibody are the epithelium of the fallopian tube and the transitional epithelium of the urinary tract. The Ca1 antibody also readily distinguishes malignant cells in smears of malignant effusions. These findings indicate that the Ca1 antibody may be useful in the diagnosis of malignancy in routine clinical practice where the morphological interpretation of the biopsy or cytological smear is in doubt.