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J Burchell

Publications and source records attributed to J Burchell.

At least 37 records · Page 2Linked to original sources
PubMed1993

Human models of breast cancer.

Experimental systems using human mammary tissue, secretions and tumours may be based on in vitro culture or on growth of tissue or tumour fragments in the nude mouse. In the development of in vitro culture systems, a detailed characterization of the cultured cells within the framework of the epithelial cell lineages found in vivo is crucial. Monoclonal antibodies are useful tools for defining the profile of antigens expressed by the basal and luminal cells in the normal gland and in distinguishing subclasses between these two major groups. When these same reagents are used to characterize breast cancers, the majority are found to show the phenotype of luminal cells, with a small subset showing some evidence of basal markers. Luminal epithelial cells cultured from milk or reduction mammoplasty tissue have a short life span in vitro but can be immortalized using SV40TAg. Demonstrably malignant cells are difficult to culture from primary breast cancer, but ER+ and ER- cell lines showing the luminal phenotypes have been readily developed from metastases: some ER- breast cancer cell lines show a more undifferentiated phenotype, and these may have developed from tumours expressing basal markers. As with in vitro culture, it is difficult to obtain tumour growth in the nude mouse from primary breast cancer specimens, and established cell lines are also difficult to grow in this animal. We have focused our studies on cell lines with the luminal phenotype developed from milk. These non-tumorigenic cell lines differ from breast cancer cell lines (a) in being able to form organized three dimensional structures in the presence of an extracellular matrix and (b) in the correct glycosylation of the polymorphic epithelial mucin, which is expressed and aberrantly glycosylated in cancers. These cell lines are therefore being used to study the mechanisms underlying morphogenesis and the processing of PEM, and also as recipients for oncogenes and proto-oncogenes.

Animals↗
PubMed1992

The product of the human MUC1 gene when secreted by mouse cells transfected with the full-length cDNA lacks the cytoplasmic tail.

The polymorphic epithelial mucin (PEM) is found as a cell associated transmembrane protein with an extracellular domain made up largely of tandem repeats and also as a soluble form in some body fluids and culture supernatants. To determine whether the soluble form can arise without the mechanism of alternative splicing mouse cells have been transfected with an expression construct containing the full-length cDNA, and the supernatants of the transfectants analyzed for the presence of the mucin. The presence of mucin in the supernatants could indeed be detected in a radioimmunoassay and by immunoprecipitation using monoclonal antibodies to the tandem repeat region of the core protein, indicating that release of the soluble form can occur without alternative splicing. The soluble form was not however precipitated with a polyclonal antiserum to the cytoplasmic tail, suggesting that it was released from the membrane by the action of a protease.

Animals↗
PubMed1991

Structure and biology of a carcinoma-associated mucin, MUC1.

Although mucins have been studied at the biochemical and biophysical level for some time, attempts to define their structures in detail were only partially successful because of their size and complexity. The advent of monoclonal antibodies reactive with these molecules introduced a new approach to structural studies by defining antigenic epitopes, by allowing purification of the mucin molecules by affinity chromatography, and by providing a means to clone genes coding for the core proteins. By their profile of reactivity with the normal and cancer-associated mucin in a particular tissue, the antibodies also defined a difference in the mucin derived from the two sources. It is now clear that this difference lies in the carbohydrate side chains, as the core proteins are identical. Because the mucins are tumor-associated antigens and the cancer mucins can express epitopes that are relatively tumor specific, this family of molecules is now being intensively studied. There is also considerable interest in elucidating the normal function of the mucin and in determining whether, through an altered structure, this function is subverted in malignancy. In the next few years we should expect that the structure of other mucins will be defined in the same detail as the product of the MUC1 gene. We should also expect to see the continued application of mucin-reactive antibodies in the clinic and the investigation of mucins as agents for immunotherapy of some cancers. As to the function(s) of these molecules, perhaps we will learn enough in the future to make a critical reappraisal of the name.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin.

Human mammary cells present on the cell surface a polymorphic epithelial mucin (PEM) which is developmentally regulated and aberrantly expressed in tumors. PEM carries tumor-associated epitopes recognized by the monoclonal antibodies HMFG-1, HMFG-2, and SM-3. Previously isolated partial cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 20-amino acid repeat units. We now report the full sequence for PEM, as deduced from cDNA sequences. The encoded protein consists of three distinct regions: the amino terminus consisting of a putative signal peptide and degenerate repeats; the major portion of the protein which is the tandem repeat region; the carboxyl terminus consisting of degenerate tandem repeats and a unique sequence containing a transmembrane sequence and a cytoplasmic tail. Potential O-glycosylation sites (serines or threonines) make up more than one-fourth of the amino acids. Length variations in the tandem repeat result in PEM being an expressed variable number tandem repeat locus. Tandem repeats appear to be a general characteristic of mucin core proteins.

Amino Acid Sequence↗
PubMed1990

Radioimmunoscintigraphy of ovarian tumors using a new monoclonal antibody, SM-3.

Radioimmunoscintigraphy with SM-3 monoclonal antibody produces results similar to those obtained with 123I-labeled HMFG-2. The tumor specificity of SM-3 in vitro is not as marked as that in vivo. SM-3 is, however, much easier to produce in tissue culture and, with the availability of 99mTc labelling, has allowed radioimmunoscintigraphy to be done as an outpatient procedure. This technique has important implications for the continuing management of patients with ovarian cancer.

Adult↗
PubMed1989

A short sequence, within the amino acid tandem repeat of a cancer-associated mucin, contains immunodominant epitopes.

The polymorphic epithelial mucin (PEM) appears to be the target molecule for many monoclonal antibodies (MAbs) which react with tumour-associated and epithelium-specific antigens. PEM contains a large domain made up of 20 amino-acid tandem repeats which are highly immunodominant as many of the antibodies reactive with this molecule recognize epitopes within this area. Using overlapping peptide octamers, we have precisely mapped the epitopes of 4 MAbs reactive with the tandem repeats including one, SM-3, which shows enhanced tumour specificity. We report that the core of the SM-3 epitope corresponds to the continuous amino acid sequence Pro-Asp-Thr-Arg-Pro. We also show that the epitopes recognized by 3 other antibodies, which show reactivity with normal and malignant tissues, map to within this area of the tandem repeat. However, none of these epitopes contain the proline found at the amino end of the SM-3 determinant. These results are consistent with the idea that, in the cancer-associated mucin, premature termination of the carbohydrate side-chains results in the exposure of the SM-3 epitope.

Amino Acid Sequence↗
PubMed1989

HMFG1 antigen: a new marker for carcinomatous meningitis.

Carcinomatous meningitis is a devastating metastatic complication of systemic carcinoma, which may occur insidiously, accompanied by a confusing spectrum of clinical symptoms and signs. In the absence of reliable diagnostic tumour markers, the diagnosis is established by the demonstration of malignant cells within the cerebrospinal fluid (CSF). Cytological techniques requiring skillful interpretation are occasionally negative in the presence of established disease, and when positive may indicate leptomeningeal malignancy of such advanced nature that effective palliation is difficult. Biochemical tumour marker technology offers the potential of reliable diagnosis in early disease states, prior to the appearance of exfoliated malignant cells. In a series of 100 patients, we assayed for an epithelial associated glycoprotein (HMFGI antigen) in CSF obtained at lumbar puncture. In 18 of 20 patients with carcinomatous meningitis, this high-molecular-weight glycoprotein was detectable in the CSF. The antigen was also present in 2 patients with neoplastic meningitis complicating lymphoma and medulloblastoma, but was not detected in the CSF of the remaining 78 patients.

Antigens, Neoplasm↗
PubMed1989

A core protein epitope of the polymorphic epithelial mucin detected by the monoclonal antibody SM-3 is selectively exposed in a range of primary carcinomas.

The monoclonal antibody (MAb) SM-3, which was raised to chemically deglycosylated milk mucin, reacts with an epitope present on the core protein of this mucin which we have referred to as PEM (polymorphic epithelial mucin). Although this mucin is abundantly expressed by both the lactating breast and breast carcinomas, the antibody SM-3 shows very little or no reactivity on the former but does react with 92% of breast carcinomas. Furthermore, SM-3 stains primary carcinomas of the lung, colon and ovary, but on the corresponding normal tissue the epitope is expressed at a much reduced level or not at all. These results indicate that an epitope masked in the normal mucin is exposed in the mucin produced by tumour cells, perhaps due to aberrant glycosylation. An extensive immunohistochemical study of other normal tissues reveals that the majority show only weak focal staining with SM-3 or none at all, the distal tubules and collecting ducts of the kidney, and sebaceous glands being the only normal tissues studied to show homogeneously positive staining.

Antibodies, Monoclonal↗
PubMed1988

A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats.

The nucleotide sequences of partial cDNA clones coding for the core protein of a human polymorphic epithelial mucin were determined, and a large domain was found to consist of a 60-base pair tandem repeat sequence. The cDNA clones were originally selected (Gendler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, M., and Taylor-Papadimitriou, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6060-6064) using three monoclonal antibodies which show differential reactivity with the mucin produced by normal and malignant breast. Two of the epitopes are exposed in the normally processed and cancer-associated mucin, while one epitope is unmasked only in the cancer-associated mucin (Burchell, J. M., Durbin, H., and Taylor-Papadimitriou, J. (1983) J. Immunol. 131, 508-513; Burchell, J., Gendler, S., Taylor-Papadimitriou, J., Girling, A., Lewis, A., Millis, R., and Lamport, D. (1987) Cancer Res. 47, 5476-5482). We show here that all three antibodies react with a synthetic peptide with an amino acid sequence corresponding to that predicted by the tandem repeat. Identification of the epitopes preferentially expressed on the cancer-associated mucin should allow a directed approach to the development of tumor-specific antibodies using synthetic peptides as immunogens.

Alleles↗
PubMed1988

[Immunolymphoscintigraphy. A new procedure for noninvasive lymph node staging illustrated by the example of breast cancer].

In the course of a prospective study 43 women underwent axillary immunolymphoscintigraphy (ILS) shortly before a planned operation for suspected carcinoma of the breast. The aim was to test the feasibility of this procedure for the noninvasive staging of lymph-nodes. Three antibodies were used: HMFG-1 (3 patients), HMFG-2 (13) and 3C6F9 (27). The diagnosis was confirmed in 35 patients. They underwent radical mastectomy with removal of the lymph-nodes. There were marked differences in the usefulness of HMFG-2 and 3C6F9 for ILS in these circumstances. There were too few patients to evaluate HMFG-1. Sensitivity and specificity of the test were 0.57 and 0.83, respectively, for HMFG-1 and 0.83 and 0.93 for 3C6F9.

Antibodies, Monoclonal↗
PubMed1987

Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin.

A mucin molecule, which has a molecular weight of greater than 400,000 and which carries tumor associated epitopes recognized by monoclonal antibodies HMFG-1 and HMFG-2, has been purified from human skimmed milk by affinity chromatography followed by passage through a size exclusion column. While treatment of the mucin with hydrogen fluoride for 1 h at 4 degrees C removed the peripheral oligosaccharides, treatment with HF for 3 h at room temperature removed all of its lectin binding ability and revealed a dominant polypeptide of about 68,000. This appears to be the size of the mucin core protein. Monoclonal antibodies have been developed that react with the stripped and partially stripped molecule but not with the intact mucin. From the initial screening on histological sections one of these antibodies, SM-3, reacts with 91% of breast carcinomas but shows little or no reactivity on benign mammary tumors, normal resting, pregnant, or lactating breast. It appears that this monoclonal antibody is reacting with an epitope that is usually masked by oligosaccharide moieties in normal cells but which is exposed, perhaps due to aberrant glycosylation, in malignant cells.

Amino Acids↗
PubMed1987

Immunological analysis of mucin molecules expressed by normal and malignant mammary epithelial cells.

Many existing MAbs raised against the human milk fat globule or against carcinoma cells recognise epitopes on high-molecular-weight glycoproteins. In a comparative ELISA assay a number of these antibodies have been shown to react with an extract of the human milk fat globule. In comparative immunoblots of cultured normal milk cells and breast cell lines, all were found to bind to large molecules which show some variation in molecular weight depending on the cell source. The HMFG-2 antibody, which is widely used in cancer diagnosis, also recognises epitopes on lower-molecular-weight components. In T47D cells these may be as small as 80,000 Mr, and with electron microscopy this cell line can be shown to accumulate HMFG-2-reactive components in the Golgi apparatus. Using an HMFG-2 affinity column we have immunopurified HMFG-2-reactive material from the 2 breast cancer cell lines MCF-7 and T47D and shown that all of the above antibodies react in a solid-phase ELISA with the purified material. In addition to high-molecular-weight components, the immunopurified material was found to contain lower-molecular-weight components including a glycoprotein of 68,000 Mr that was not, however, recognised by the HMFG-2 antibody on a Western blot. We have used this immunopurified material to generate new MAbs. All of these recognise the high-molecular-weight bands seen with the other antibodies, but 2 of them also recognise a band at 68,000 Mr in blots of MCF-7 and T47D. The second-generation antibodies show a spectrum of reactivity on tissues similar to HMFG-2 and one reacts at least as strongly as HMFG-2 with methanol-acetone-fixed sections of breast cancers.

Antibodies, Monoclonal↗
PubMed1987

Lack of beta-casein production by human breast tumours revealed by monoclonal antibodies.

An immunohistochemical study with four monoclonal antibodies to human beta-casein was carried out to examine the expression of this milk protein in a wide range of normal tissues, in 127 breast tumours and in a heterogeneous panel of 42 malignancies of other histogenesis. The only normal tissue stained positively by the antibodies was the mammary gland in late pregnancy, during lactation and in the post-lactational regression period. None of the tumours of non-mammary origin showed any staining. Furthermore, only two of 40 benign breast lesions and one anaplastic primary carcinoma with its metastasis (among 87 breast carcinomas) showed any reactivity. The immunohistochemical results were supported by immunoblotting data and suggested beta-casein expression has no role to play as a marker in the diagnosis or monitoring of human breast cancer.

Antibodies, Monoclonal↗
PubMed1987

Strategies for the development of monoclonal antibodies for in vivo imaging: their use in the imaging of ovarian carcinoma.

There are a number of strategies that have been used for the development of monoclonal antibodies which recognise tumour associated antigens. These include the use of whole tumour cells or membrane components as the immunogen, and the use of differentiation antigens, for example the human milk fat globule. The monoclonal antibody HMFG-2 was developed using the latter strategy and has been shown to react with a large molecular weight mucin-like molecule which appears to be highly immunogenic in the mouse. The HMFG-2 antibody is proving to be extremely useful in the localisation of ovarian tumours and is being used in a number of clinics. This antibody and its antigen have a number of characteristics which have contributed to its success in imaging ovarian carcinomas, including the repetitive nature of the antigenic epitope and the antibody's affinity.

Antibodies, Monoclonal↗
PubMed1987

Radioimmunoscintigraphy using monoclonal antibodies before second-look surgery in patients suffering from ovarian cancer.

19 patients with a known history of ovarian cancer were investigated by radioimmunoscintigraphy (RIS) to look for recurrent disease a few days before second-look surgery. The tumor-associated monoclonal antibody HMFG-2 (400 micrograms/patient) was injected intravenously after labeling with radioactive 123I (0.5-2.2 mCi/patient). Scans were reviewed for activity accumulations due to uptake of the tumor-associated antibody by tumor sites. In 15 out of the 19 cases the scan results correlated with the intraoperative findings. There were 2 false-positive and 2 false-negative scans, the latter in patients with subclinical disease. The smallest lesion detected by radioimmunoscintigraphy had a diameter of 1.5 cm. In 3 patients, tumor sites were identified that had been missed by all other routinely performed methods of investigation including transmission computed tomography. These data indicate that RIS is of considerable clinical value in the early detection and localization of recurrent ovarian cancer and may, therefore, improve the management of these patients.

Adult↗
PubMed1986

[Initial results of immunolymphoscintigraphy in the diagnosis of axillary lymph node involvement in breast cancer patients].

Sixteen patients suffering from breast cancer were investigated by the method of immunolymphoscintigraphy (ILS) to evaluate the axillary lymph node status. Two monoclonal tumour associated antibodies (HMFG-1 and HMFG-2) that are known to react strongly with a wide range of epithelial malignomas were used as tumour seeking agents in our recent study. All patients were operated a few days after the scanning procedure including a radical axillary lymph node dissection. Therefore the scan reports could be correlated in each case with the histology of the surgically removed probes. While HMFG-1 failed to show correct results, HMFG-2 gave accurate results in 9 of 13 cases of questionable lymph node involvement. Among the 4 false results, one turned out to be false positive and three false negative. If better antibodies or their fragments, respectively, are used, we are convinced this noninvasive approach will greatly improve preoperative diagnosis of lymphatic involvement in breast cancer patients.

Antibodies, Monoclonal↗
PubMed1986

Patterns of reaction of monoclonal antibodies HMFG-1 and -2 with benign breast tissues and breast carcinomas.

The monoclonal antibodies HMFG-1 and HMFG-2 are directed against different epitopes on a large molecule in the human milk fat globule and are also expressed on breast tumors. Here we examine the relative intensity of staining of the two antibodies in formalin fixed paraffin embedded tissue sections from malignant and benign mammary lesions. Most ductal carcinomas and their lymph node metastases stained more strongly with HMFG-2 whereas primary lobular carcinomas are more variable but the majority of their lymph node metastases stained more strongly with HMFG-1. Western blots of gel separated extracts of formalin fixed, paraffin embedded tumor tissue, stained with the antibodies in an ELISA assay, show that the molecular weight of components expressing the antigenic determinants varies. Smaller molecular weight components recognised by HMFG-2 and present in metastatic tumors may be lost during tissue processing. To assess the importance of the molecular weight parameter in tumor classification and prognosis it will be necessary to use frozen tissue.

Antibodies, Monoclonal↗