Muc-1 core protein is expressed on multiple myeloma cells and is induced by dexamethasone.

Monoclonal antibodies (MoAbs) that selectively identify Muc-1 core protein (MoAbs DF3-P, VU-4H5) determinants were used to identify the Muc-1 glycoform present on 7 multiple myeloma (MM) cell lines, 5 MM patient plasma cells, 12 MM patient B cells, as well as 32 non-MM cell lines and normal hematopoietic cells. Flow cytometry studies demonstrated that all MM cell lines, MM patient plasma cells, and MM patient B cells expressed Muc-1 core protein epitopes. Circulating B cells from 4 normal donors also expressed Muc-1 core protein. In contrast, Muc-1 core protein was absent on 28 of 32 non-MM neoplastic cell lines, 17 of which expressed Muc-1. Splenic and tonsillar B cells, CD34(+) stem cells, resting T cells, and bone marrow plasma cells obtained from normal donors both lacked Muc-1 glycoforms. We next studied the effects of estrogen, progesterone, and glucocorticoid receptor agonists and antagonists on Muc-1 expression, because consensus sequences for the response elements of these steroids are present on the Muc-1 gene promoter. These studies showed that dexamethasone (Dex) induced Muc-1 expression on MM cell lines, as determined by both flow cytometry and Western blot analyses. Dex also induced upregulation of Muc-1 on prostate and ovarian cancer cell lines. Time and dose-response studies demonstrated that Dex induced maximal cell surface Muc-1 expression by 24 hours at concentrations of 10(-8) mol/L. Dex induced Muc-1 upregulation could be blocked with a 10-fold excess of the glucocorticoid receptor antagonist RU486, confirming that Dex was acting via the glucocorticoid receptor. No changes in Muc-1 expression were observed on MM cells treated with estrogen and progesterone receptor agonists and antagonists or with RU486. These studies provide the framework for targeting Muc-1 core protein in vaccination and serotherapy trials in MM. In addition, the finding that Muc-1 expression on MM cells can be augmented by Dex at pharmacologically achievable levels suggests their potential utility in enhancing treatments targeting Muc-1 in MM.

MUC1 synthetic peptide inhibition of intercellular adhesion molecule-1 and MUC1 binding requires six tandem repeats.

We reported recently that breast cancer-associated MUC1 is a ligand for intercellular adhesion molecule-1 (ICAM-1; L. H. Regimbald et al., Cancer Res., 56: 4244-4249, 1996). We report here the results of a competitive indirect binding assay to detect the molecular requirements for binding between ICAM-1 and MUC1. The assay involved inhibition of the binding of recombinant human ICAM-1 to a murine breast adenocarcinoma cell line transfected with human MUC1. The addition of a library of human MUC1 synthetic peptides ranging from 9 to 24 amino acids (aa) showed minimal or no inhibition. However, a 120-aa peptide that corresponds to six tandem repeats of the human mucin MUC1 was as effective an inhibitor as purified tumor MUC1 and MUC1 epitope (PDTRPAP)-specific antibody (B27.29). We conclude that the number of MUC1 tandem repeats necessary for an ordered tertiary structure (D. Fontenot et al., Cancer Res., 53: 5386-5394, 1993) is also important for ICAM-1 recognition. These findings are similar to those described recently for MUC1 induction of T-cell anergy (B. Agrawal et al., Nat. Med., 4: 43-49, 1998). This suggests that the anergy induction by MUC1 may be due to ICAM-1 binding by MUC1.

Human single-chain Fv antibodies to MUC1 core peptide selected from phage display libraries recognize unique epitopes and predominantly bind adenocarcinoma.

The tumor-associated antigen MUC1 is overexpressed and underglycosylated in human adenocarcinomas of diverse origins, such as breast, ovary, and colon. We isolated and describe five human single-chain (sc) Fv antibodies specific for the MUC1 variable number of tandem repeats region isolated by in vitro selection from a large naive phage antibody library containing over 6 x 10(9) different scFv antibodies. A synthetic biotinylated 100-mer peptide corresponding to five tandem repeats of the MUC1 peptide core was used for selection. Two of the antibodies were highly specific for MUC1 as judged by ELISA and flow cytometry. In immunohistochemistry, antibody clone 10A stained MUC1 in the cytoplasm and membrane of adenocarcinoma cells of breast and ovary, whereas in normal epithelium, only cytoplasmic or no staining was observed. With antibody clone 10B, staining was less pronounced and was not always membrane associated in adenocarcinoma. Determination of the fine specificity of 10A and 10B using a novel "indirect epitope fingerprinting" ELISA showed that both antibodies recognize unique epitopes that have not been described for hybridoma-derived anti-mucin antibodies of mouse origin. The selected human antibodies, like many of the murine MUC1 antibodies, recognize epitopes on the protein core of MUC1 that are abundantly present in the underglycosylated form of cell surface mucin on adenocarcinoma. The best human scFv, clone 10A, appears to distinguish normal cells from adenocarcinoma cells, which makes it an attractive candidate for use in antibody-based tumor targeting.

Establishment of immunological probes to study human gonadotropin-releasing hormone receptors.

Monoclonal antibodies were raised to a synthetic peptide corresponding to amino acids 1-29 of the human gonadotropin-releasing hormone (GnRH) receptor. One of the two antibodies was found to recognise GnRH receptors on human pituitary gonadotrophs as determined by immunohistochemistry and supported by Western blotting. The antibody also bound to T47D human breast carcinoma cell line as determined by flow cytometric analysis.

Clinical applications of monoclonal antibodies against ovarian cancer-associated antigens.

Monoclonal antibodies (McAbs), reactive with tumour-associated antigens (TAAs) present on tumour cells, appear to offer new possibilities in the diagnosis and treatment of cancer (Table I). In addition to these prospects for clinical application, monoclonal antibodies also serve as useful instruments in basic cancer research. The hybridoma technology initiated by Köhler and Milstein in 1975, underwent a very rapid development and has now shown its potential in the field of oncology. This technique made it possible to produce very large quantities of homogeneous antibodies of a stable quality. These McAbs often recognize only one antigenic determinant, or epitope, of cell surface and other molecules. This high specificity is essential for in vivo applications, especially in therapeutic immunotargeting. A central question is whether the antibodies can reach and identify those antigens on ovarian tumour cells that are not shared with normal tissues. Various antibodies have been described in the field of gynaecological oncology, which are assumed to be capable of recognizing such ovarian tumour-related antigens. These McAbs, single or in combination, are capable of showing, unambiguously, the presence of various tumour-associated antigens on ovarian carcinoma cells either in tissue or, when antigen shedding occurs, in blood. However, these McAbs may also react with tumour-associated antigens present on endometrial, cervical, colorectal, breast or other carcinoma cells. The original immunogens used to generate these McAbs differ as to their origin: ovarian cancer cells, breast cancer cells, human milk-fat preparations, trophoblastic cells, endometrial cancer cells have been used as well as osteogenic sarcoma cells, epidermoid carcinoma cells and small-cell lung cancer, colorectal, pancreatic and laryngeal carcinoma cells. The histological distribution patterns of the antigens recognized by these McAbs vary widely: cross-reactions with normal tissue and with carcinomas different from those used as immunogen are frequently seen.

Glaucoma surgery in Caribbean Negroes on Curacao.

Fifty-four eyes of Negro patients of the Caribbean island of Curacao were operated upon for medically uncontrolled open-angle glaucoma. Two different types of surgical techniques were used: procedures with cautery under a scleral flap and trabeculectomy with cautery. A third group, a combined intracapsular lens extraction and trabeculectomy was also included. Trabeculectomy with cautery gave the most successful results: in about half of the eyes without medication and an additional third of the eyes with medication. Failures occurred in about 15%.

A new iris retractor.

I successfully used the iris-lasso, a new iris retractor in the form of a stainlless steel wire. The iris-lasso is simple to maneuver, suitable for microsurgery, and particularly useful in patients with small pupils.

Rapid separation of cells at unit gravity.

An apparatus is described for the rapid separation of cells at unit gravity. The apparatus is filled with a discontinuous density gradient, followed by the sample and then an overlay. The sedimentation chamber is turned from a vertical to a horizontal position before sedimentation takes place in order, (a) to form a continuous gradient out of the discontinuous gradient and, (b) to reduce the time required for the separation. After the sedimentation is completed the chamber is returned to a vertical position and the gradient fractionated by hydrostatic pressure using a newly developed floating device. The usefulness of the method is illustrated by the separation of leukemia cells in different phases of the life cycle.

Murine leukaemia virus group-specific antigen in tumor-resistant tetraparental AKR reversible CBA/H-T6 chimaeras.

Various facts are now known about the relative lymphoma resistance of a group of tetraparental AKR reversible CBA/H-T6 chimaeras derived by early embryo aggregation. Firstly, their tumour resistance is not due to the lack of the lymphomaprone AKR cells. Secondly, results showing titres of MuLV-gs antigen comparable with, and occasionally in excess of, those in the AKR suggest that the tumour resistance of the chimaeras is unlikely to be due to a lack of oncogenic leukaemia virus. However, in marked contrast to the AKR, antibody-viral antigen renal complexes in the chimaeras were minimal. Lack of viral antigens could not explain the relative lack of renal complexes. Absence of the corresponding anti-viral antibody is the most likely explanation and this has to be attributed to the CBA component of the tetraparental AKR reversible CBA/H-T6 chimaeras. We suggest that with tolerance to the leukaemia virus being maintained and in the absence of anti-viral antigenic complexes, tumour-specific sites can be recognized and thus tumours are eliminated. This hypothesis remains to be proven.