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Biomedical subjects

M Shearer

Publications and source records attributed to M Shearer.

At least 19 recordsLinked to original sources
PubMed1992

Studies of clonal cell lines developed from primary breast cancers indicate that the ability to undergo morphogenesis in vitro is lost early in malignancy.

In an attempt to obtain cell lines from the different components found in primary breast cancers, we used a low-calcium medium to culture epithelial cells of mixed phenotype and a recombinant T antigen containing retrovirus to immortalize cells found in these cultures. In each case, the histology of the sample used for culture was examined in detail and the best growth was obtained from samples associated with a substantial in situ or benign component and from lobular rather than ductal carcinomas. Clonal cell lines were developed from each of 4 tumours: 1 infiltrating ductal (tumour number 2), 2 infiltrating lobular (tumours 3 and 5) and 1 mucoid (tumour 6). To try to identify the phenotype and origin of the cell lines, immunohistochemical markers, histological analysis of tissue sections and behavioural markers were used. All the cell lines expressed mainly luminal epithelial cell markers, but the basal epithelial keratin, keratin 14, was also expressed homogeneously or heterogeneously. Growth in agar was seen with some but not all cell lines derived from only 1 tumour (tumour 5) and tumour development in nude mice was observed (with low efficiency) with cell lines from only 1 tumour (tumour 6). The data suggest that the cell lines obtained from the infiltrating ductal carcinoma (tumour 2) developed from cells cultured from the associated benign component, while the cell lines from tumours 3, 5 and 6 may each have developed from a cell in an early stage of malignancy. When tested for their ability to undergo morphogenesis on extracellular matrix components, cell lines from tumour 2 made well-developed ductal-alveolar-like structures, while those from the other tumours did not.

Animals↗
PubMed1992

Collagen-induced rapid morphogenesis of human mammary epithelial cells: the role of the alpha 2 beta 1 integrin.

The cell line MTSV1-7, originally derived by immortalizing mammary epithelial cells cultured from human milk was able to form three-dimensional structures in collagen gel. We have now found that these cells, cultured as a monolayer, are able to undergo rapid morphogenesis forming ridges and balls around collagen fibres, when soluble collagen type I is added to the medium. Monoclonal antibodies to the alpha 2 (P1E6) and beta 1- (mAB13) subunits of VLA-2, but not to the alpha 3-subunit (P1B5) of VLA-3, could block this collagen-induced rapid morphogenesis (CIRM). The effect of the antibodies on cell attachment, spreading, and migration on collagen gels was analyzed to identify alpha 2 beta 1 dependent steps which might be involved in CIRM. The results suggest that while other proteins, besides alpha 2 beta 1, are also involved in cell attachment and migration, cell spreading was specifically blocked by antibodies to the VLA-2, but not to the VLA-3 integrin. The results demonstrate that the alpha 2 beta 1 integrin plays a crucial role in the collagen-induced morphogenesis of human mammary epithelial cells and implicate the process of VLA-2-dependent cell spreading as an important step in this morphogenesis.

Antibodies, Monoclonal↗
PubMed1991

Expression of the gene coding for a human mucin in mouse mammary tumor cells can affect their tumorigenicity.

The human epithelial mucin which is the product of the MUC1 gene is expressed by many carcinomas, including those of breast, ovary, colon, and lung. The core protein is aberrantly glycosylated in the tumors resulting in the exposure or appearance of novel epitopes. To examine the possibility of using the MUC1 gene and its products in active immunization against breast and other carcinomas, we have developed a syngeneic mouse model, by transfecting the gene into the mouse mammary epithelial tumor cell 410.4. An 8.3-kilobase EcoRI fragment of the gene was transfected using the expression vector pEMSV scribe alpha 2. Transcripts of the correct size, initiating from the transcriptional start site seen in human cells, were observed in the transfectants. The mucin was expressed in the cytoplasm and in the membrane, and the glycosylation pattern appeared to be similar to that seen in human tumor cells, since the core protein epitopes recognized by antibodies HMFG-1, HMFG-2, and SM-3 were exposed. The 410.4 transfectants expressing the human mucin showed a reduction in tumor incidence at low inocula and a delay in tumor growth at higher inocula. Pretreatment with 10(4) transfected cells could inhibit the development of tumors from a subsequent inoculum of 10(6) transfectants, but had no effect on the tumor development of the untransfected 410.4 cells. Our results suggest that the human mucin expressed by the 410.4 cells may mobilize an immune response which inhibits tumor development. They also indicate that the mouse model will be useful for evaluation of efficacy of immunogens based on the MUC1 gene and its product.

Animals↗
PubMed1991

Efficient immortalization of luminal epithelial cells from human mammary gland by introduction of simian virus 40 large tumor antigen with a recombinant retrovirus.

When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell found in the terminal ductal lobular units. Luminal epithelial cells cultured from milk, which have limited proliferative potential, have now been immortalized by introducing the gene encoding simian virus 40 large tumor (T) antigen. Infection with a recombinant retrovirus proved to be 50-100 times more efficient than calcium phosphate transfection, and of the 17 cell lines isolated, only 5 passed through a crisis period as characterized by cessation of growth. When characterized by immunohistochemical staining with monoclonal antibodies, 14 lines showed features of luminal epithelial cells and of these, 7 resembled the common luminal epithelial cell type in the profile of keratins expressed. These cells express keratins 7, 8, 18, and 19 homogeneously and do not express keratin 14 or vimentin; a polymorphic epithelial mucin produced in vivo by luminal cells is expressed heterogeneously and the pattern of fibronectin staining is punctate. Although the cell lines have a reduced requirement for added growth factors, they do not grow in agar or produce tumors in the nude mouse. When the v-Ha-ras oncogene was introduced into two of the cell lines by using a recombinant retrovirus, most of the selected clones senesced, but one entered crisis and emerged after 3 months as a tumorigenic cell line.

Animals↗
PubMed1991

Relationship between outlet obstruction constipation and obstructed urinary flow.

Ten women with symptoms and radiological features of outlet obstruction constipation underwent urodynamic bladder studies. The results were compared with ten age- and sex-matched controls. The mean (s.e.m.) peak flow rate for patients was 19.4 (6.4) ml/s compared with 32.1 (7.2) ml/s for controls (P less than 0.05). The mean (s.e.m.) voiding time for patients was 62.9 (23.7) s against a corresponding value of 15.6 (6) for controls (P less than 0.05). The mean (s.e.m.) bladder volume in patients was 482 (80) ml compared with a control value of 254 (112) ml (P less than 0.03). The mean (s.e.m.) detrusor pressure during the voiding phase was 53.3 (12) cmH2O. These results demonstrate that patients with outlet obstruction constipation have a generalized pelvic floor disorder resulting in obstructed urinary flow.

Constipation↗
PubMed1989

Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway.

We have selected mutations in genes encoding components of the signaling pathway for alpha interferon (IFN-alpha) by using a specially constructed cell line. The upstream region of the IFN-regulated human gene 6-16 was fused to the Escherichia coli guanine phosphoribosyltransferase (gpt) gene and transfected into hypoxanthine-guanine phosphoribosyltransferase-negative human cells. These cells express gpt only in the presence of IFN-alpha. They grow in medium containing hypoxanthine, aminopterin, and thymidine plus IFN and are killed by 6-thioguanine plus IFN. Two different types of mutants were obtained after treating the cells with mutagens. A recessive mutant, selected in 6-thioguanine plus IFN, was completely resistant to IFN-alpha but responded normally to IFN-gamma and, unexpectedly, partially to IFN-beta. A constitutive mutant, selected in hypoxanthine-aminopterin-thymidine alone, was abnormal in expressing endogenous genes in the absence of IFN. Both types revert infrequently, allowing selection for complementation of the defects by transfection.

Blotting, Northern↗
PubMed1988

The development of hypoprothrombinaemia following antibiotic therapy in malnourished patients with low serum vitamin K1 levels.

A group of nine well-nourished patients, with normal serum vitamin K1 levels (mean 546, range 310-1350 pg/ml), maintained normal prothrombin times (PTs) and factor VII clotting activities throughout a 7 d course of i.v. cefotetan disodium, an N-methyl-thiotetrazole (NMTT) containing cephalosporin antibiotic. However, 11 of 20 patients, with acute intra-abdominal sepsis and initially normal PTs who underwent emergency surgery, developed prolonged PTs (INR 1.4-3.1) associated with reduction in factor VII activities (0.74-0.38 u/ml) after 3-7 d of antibiotic therapy. Nine of these 11 patients had clinical evidence of malnutrition and nine had subnormal serum vitamin K1 levels (mean 119, range 43-354 pg/ml) on admission. Seven received cefotetan but four were treated with a non-NMTT-containing cephalosporin or antibiotics belonging to other groups. The nine patients who maintained normal PTs all had normal nutritional status and normal serum vitamin K1 levels (mean 279, range 103-915 pg/ml) at presentation. The PT is a relatively insensitive indicator of vitamin K stores, and malnourished patients with low serum vitamin K1 levels are at risk of developing hypoprothrombinaemia following intravenous antibiotic therapy.

Adult↗
PubMed1987

Epitopes of human interferon-alpha defined by the reaction of monoclonal antibodies with alpha interferons and interferon analogues.

Five monoclonal antibodies reactive with human interferon (HuIFN)-alpha 2, but not with HuIFN-alpha 1, have been analyzed for their reaction with a series of IFN analogues and hybrid IFN molecules. Using analogues containing alpha 1 or gamma substitutions, it was shown that amino acids in the 107 to 113 region are implicated in the epitopes recognized by four of the five antibodies tested. Surprisingly, two of the antibodies that did not react with [alpha 1(113 to 114)112 to 113]alpha 2 also did not react with a truncated IFN-alpha 2(4 to 155). The presence of an epitope determined by amino acids at 112 and 113 and by the amino and carboxyl ends of the molecule supports a model for IFN where the carboxyl- and amino-terminals are adjacent to the proposed reverse turn around amino acid 110 to 115. The fifth alpha 2 specific antibody whose reaction with HuIFN-alpha 2 is not affected by the above substitutions and truncations recognizes IFN or IFN hybrids that, like alpha 2, have arginine at position 120, but does not react with IFN that, like alpha 1, have lysine at position 120. Amino acids 107 to 113 and 120 lie in regions of the molecule that have high hydrophilicity and are probably structurally involved in the epitopes recognized by the antibodies. Under conditions of antibody excess, the antibodies described here inhibit binding of HuIFN-alpha 2 to both human and bovine cells.

Animals↗
PubMed1987

Regulation of cell growth by interferon.

Interferons can regulate growth and differentiation in a wide range of cell types. These mechanisms are currently being examined. Interferons inhibit the growth of tumour cells and are thus potential anti-cancer agents. They can also inhibit normal cell growth in vitro, and stimulate tumour cell growth in vitro. They may also be involved in some autoimmune diseases. This review examines the effect of interferons on cell proliferation, function, and growth, focusing primarily on in vitro cell systems.

Animals↗
PubMed1987

The effects of cefotetan disodium on haemostasis.

A 7-day course of intravenous cefotetan disodium was given to nine patients. No significant changes were observed in haematological or biochemical parameters and serum vitamin K1 levels, prothrombin times, factor VII levels, thrombin times and activated partial thromboplastin times remained within the normal ranges throughout the treatment period in all patients. There was no evidence of clinical bleeding in any patient although in two the bleeding time was prolonged up to 13.0 min after 7 days' therapy. Notably, adenosine-5-diphosphate (ADP)-induced platelet aggregation responses were significantly increased (P less than 0.05) at the end of the treatment period. These data indicate that cefotetan disodium at a dose of up to 4 g daily can be used without risk of a bleeding diathesis. In situations associated with vitamin K1 deficiency, potential prolongation of the prothrombin time should be avoided by prophylactic vitamin K1 administration.

Adult↗