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1.
Oncogenesis ; 5: e215, 2016 Apr 04.
Article in English | MEDLINE | ID: mdl-27043661

ABSTRACT

Mammary MCF-10A cells seeded on reconstituted basement membrane form spherical structures with a hollow central lumen, termed acini, which are a physiologically relevant model of mammary morphogenesis. Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional protein overexpressed in breast cancer and ductal carcinoma in situ. When present in the nucleus Bag-1 is predictive of clinical outcome in breast cancer. Bag-1 exists as three main isoforms, which are produced by alternative translation initiation from a single mRNA. The long isoform of Bag-1, Bag-1L, contains a nuclear localisation sequence not present in the other isoforms. When present in the nucleus Bag-1L, but not the other Bag-1 isoforms, can interact with and modulate the activities of estrogen-, androgen- and vitamin D-receptors. Overexpression of Bag-1 mRNA in MCF-10A is known to produce acini with luminal filling reminiscent of ductal carcinoma in situ. As this mRNA predominantly overexpresses the short isoform of Bag-1, Bag-1S, we set out to examine whether the nuclear Bag-1L isoform is sufficient to drive premalignant change by developing a Bag-1L-overexpressing MCF-10A model. Two clones differentially overexpressing Bag-1L were grown in two-dimensional (2D) and three-dimensional (3D) cultures and compared with an established model of HER2-driven transformation. In 2D cultures, Bag-1L overexpression reduced proliferation but did not affect growth factor responsiveness or clonogenicity. Acini formed by Bag-1L-overexpressing cells exhibited reduced luminal clearing when compared with controls. An abnormal branching morphology was also observed which correlated with the level of Bag-1L overexpression, suggesting further malignant change. Treatment with Thio-2, a small-molecule inhibitor of Bag-1, reduced the level of branching. In summary, 3D cultures of MCF-10A mammary epithelial cells overexpressing Bag-1L demonstrate a premalignant phenotype with features of ductal carcinoma in situ. Using this model to test the small-molecule Bag-1 inhibitor, Thio-2, reveals its potential to reverse the atypical branched morphology of acini that characterizes this premalignant change.

2.
Blood ; 76(7): 1361-8, 1990 Oct 01.
Article in English | MEDLINE | ID: mdl-1698478

ABSTRACT

We describe a new rat immunoglobulin M monoclonal antibody (CH-F42) that recognizes a subset (1.5% to 8%) of normal peripheral blood T lymphocytes. The phenotype of these cells was determined, using dual-color immunofluorescence, to be CD2+, CD3+, CD4+, CD5+, CD7-, CD8-. They do not express T-cell activation markers, and are positive for UCHL1 (CD45RO), but negative for 2H4 (CD45RA). The antigen was expressed on circulating malignant cells in Sézary syndrome (four of four cases) and adult T-cell lymphoma-leukemia (ATLL) (four of six cases) and negative in a variety of other hematologic malignancies tested. These included chronic and acute lymphoid leukemias of B and T lineage, together with chronic and acute myeloid leukemias. However, normal CH-F42+ cells do not display any of the ultrastructural features associated with Sézary or ATLL cells. The marked similarities between these conditions together with the shared expression of an otherwise very restricted surface antigen (CH-F42) provide strong evidence for the existence of a common normal counterpart. Preliminary characterization studies of the antigen, which is also expressed by K562 and Jurkat cells, suggest the CH-F42 antigen is an O-linked, sialated glycan on a glycoprotein.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , Leukemia-Lymphoma, Adult T-Cell/pathology , Neoplastic Cells, Circulating/immunology , Sezary Syndrome/pathology , T-Lymphocyte Subsets/immunology , Antigens, CD7 , Biomarkers, Tumor/analysis , Cell Line , Cell Transformation, Neoplastic/immunology , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Humans , Leukemia-Lymphoma, Adult T-Cell/immunology , Microscopy, Electron , Phenotype , Sezary Syndrome/immunology , Stem Cells/immunology , Stem Cells/ultrastructure
3.
J Immunol Methods ; 115(2): 209-17, 1988 Dec 09.
Article in English | MEDLINE | ID: mdl-3058806

ABSTRACT

M-450 Dynabeads are magnetizable polystyrene microspheres 4.5 micron in diameter to which antibodies of IgM isotype can be physically adsorbed. Antibody-coated Dynabeads can be used to label cell surfaces and then to separate the rosetted cells by application of an external magnetic field. We demonstrate here, using cell lines K562 and U937 and the previously undescribed monoclonal antibodies CH-F42 and CH-E25, that Dynabeads can also be used to label cells at the ultrastructural level. Dynabeads can therefore provide a useful bridge between light and electron microscopy. The preservation of specific rosettes at the ultrastructural level without the formation of artifactual aggregates requires rapid but gentle fixation in dilute suspension. We have achieved this by fixing briefly in a large volume of buffered glutaraldehyde followed by neutralization of excess glutaraldehyde with ethanolamine.


Subject(s)
Magnetics , Microscopy, Electron , Microscopy, Phase-Contrast , Microspheres , Rosette Formation , Animals , Antibodies, Monoclonal , Cell Line , Immunoglobulin M , Leukemia, Erythroblastic, Acute/pathology , Microscopy, Electron/methods , Microscopy, Phase-Contrast/methods , Polystyrenes , Rats , Rosette Formation/methods , Tumor Cells, Cultured/ultrastructure
4.
Leuk Res ; 10(2): 211-20, 1986.
Article in English | MEDLINE | ID: mdl-3485224

ABSTRACT

Human T-lymphoblastic leukaemic cell line Karpas-45 (K-45) can be induced by phorbol 12-myristate 13-acetate (PMA) to become phenotypically more mature. Marker studies show that the percentages of E-rosette-positive (E+) and UCHT1+ cells are increased after exposure to PMA. Fluorescence of UCHT4+ and 2D1+ cells is increased although their percentages remain almost unchanged. OKT4+ and peanut agglutinin (PNA)-positive cells are greatly reduced. Terminal deoxynucleotidyl transferase (TdT) becomes negative. PMA treatment of K-45 cells after deletion of UCHT4+ cells suggest that marker changes detected by UCHT2, OKT4 and sheep red blood cells (SRBC) occur mainly in the UCHT4+ cells. The proliferation of PMA-treated K-45 cells is dramatically inhibited and the cell size becomes smaller. These results indicate that K-45 cells are induced to differentiate phenotypically towards a suppressor/cytotoxic T cell. However functional maturation can not be demonstrated. Phytohemagglutinin-stimulated leucocyte conditioned medium (LCM) is able to suppress DNA synthesis of K-45 cells and reduce the PNA+ proportion. It seems that LCM can induce a limited degree of differentiation of K-45 cells.


Subject(s)
Leukemia, Lymphoid/pathology , Lymphocytes/cytology , Phorbols/pharmacology , T-Lymphocytes/cytology , Tetradecanoylphorbol Acetate/pharmacology , Adult , Antibodies, Monoclonal , Cell Line , Female , Fetal Blood , Humans , Immunoglobulin G/analysis , Leukemia, Lymphoid/immunology , Phenotype , Pregnancy , Rosette Formation
5.
Clin Radiol ; 35(5): 353-7, 1984 Sep.
Article in English | MEDLINE | ID: mdl-6236011

ABSTRACT

Examination of peripheral blood lymphocyte subsets in untreated patients with Hodgkin's disease and non-Hodgkin's lymphoma revealed depressed ratios of helper/suppressor T-cells, assessed with monoclonal antibodies OKT4 and 8. Non-Hodgkin's lymphoma and Hodgkin's disease patients studied from 1 to 204 months following treatment by radiotherapy also demonstrated depressed ratios, although the patients were clinically free of disease. The failure of the helper T-cells to recover following apparently successful treatment suggests that either the initial disease or the radiotherapy produces a virtually irreversible effect on T-cell subpopulations. Treated Hodgkin's disease patients also exhibited a long term increase in B-cell numbers but this effect could be correlated with splenectomy performed during staging laparotomy.


Subject(s)
Hodgkin Disease/immunology , Lymphocytes/immunology , Lymphoma/immunology , B-Lymphocytes/immunology , B-Lymphocytes/radiation effects , Hodgkin Disease/radiotherapy , Humans , Leukocyte Count , Lymphocytes/classification , Lymphocytes/radiation effects , Lymphoma/radiotherapy , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/radiation effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/radiation effects
6.
Br J Haematol ; 43(2): 215-21, 1979 Oct.
Article in English | MEDLINE | ID: mdl-508631

ABSTRACT

A case of hairy-cell leukaemia (HCL) with IgG(k) paraprotein is described. The typical clinico-pathological features of the patient are stressed, and it is shown that the hairy cells (HCs) were producing and secreting IgGk. The case is placed in a more general context by the demonstration that HCs from other typical cases of HCL produce and secrete IgG. These findings conclusively demonstrate that HCL is a B-cell disease.


Subject(s)
Hypergammaglobulinemia/immunology , Immunoglobulin G , Leukemia, Hairy Cell/immunology , Aged , Cytoplasm/immunology , Female , Humans , Leukocytes/immunology , Receptors, Antigen, B-Cell/analysis
7.
Cancer ; 44(3): 927-36, 1979 Sep.
Article in English | MEDLINE | ID: mdl-113079

ABSTRACT

A patient with chronic myelomonocytic leukemia with IgG K paraproteinemia, but no detectable plasmacytosis, is described. The patient was entering a blastic phase at the time of the most detailed studies. Cytological, cytochemical, and ultrastructural studies revealed a mixed myeloid proliferation with granulocytic forms predominating over monocytic elements. A variety of ultrastructural abnormalities, including defective granulation, was observed but no cells with highly developed rough endoplasmic reticulum were observed. Immunological marker studies showed that the mature myeloid cells possessed receptors for the Fc of IgG and weakly expressed the Ia-like P29/34 antigen. The mature myeloid cells also expressed both surface and intracytoplasmic Ig restricted to IgG K, and this IgG K persisted after 4 weeks in culture. A reverse plaque assay showed that the myeloid cells were capable of releasing IgG K in vitro, but studies involving the incorporation of radio-labeled amino acids showed no detectable Ig production by the myeloid cells. The possible interpretations of these data are discussed in some detail in relation to previous reports of paraproteinemia in myeloid proliferative disorders.


Subject(s)
Immunoglobulin G , Immunoglobulin Light Chains , Immunoglobulin kappa-Chains , Leukemia, Myeloid/complications , Paraproteinemias/complications , Aged , Humans , Leukemia, Myeloid/immunology , Leukemia, Myeloid/pathology , Male , Microscopy, Electron , Paraproteinemias/immunology , Paraproteinemias/pathology , Receptors, Fc
8.
Immunology ; 36(3): 569-77, 1979 Mar.
Article in English | MEDLINE | ID: mdl-312263

ABSTRACT

The presence of a receptor for the Fc of IgM (muFcR) was demonstrated on the pathological B cells of all of sixteen patients with hairy-cell leukaemia and most, but not all, of twenty-four cases of chronic lymphocytic leukaemia, by a rosette method employing ox erythrocytes sensitized with purified IgM (EAm). This muFcR was also demonstrated on a small population of normal human mononuclear cells from peripheral blood. Pathological B cells with this receptor (Bm) simultaneously expressed a different and distinct receptor for the Fc of IgG, and were detectable without preincubation in medium containing foetal calf serum (FCS). The muFcR on B cells was blocked by Fc5mu and IgM, but not by F(ab')2mu fragments, or by IgG, whether monomeric or aggregated. Monomeric IgM and IgM bound to its antigen blocked much more effectively than pentameric IgM. B cells also possessed surface immunoglobulin and the Ia-like P29, 34 antigen, and an antiserum to this antigen blocked the muFcR. The muFcR on B cells differs in a number of ways from the muFcR reported on T cells, and these differential characteristics are discussed in some detail. The muFcR was rapidly shed and resynthesized when washed Bm cells were maintained in medium not containing FCS and the general importance of this phenomenon in any study of muFcR is considered. It is suggested that Bm cells are memory cells and that the muFcR plays a part in the immune response.


Subject(s)
B-Lymphocytes/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin M/immunology , Humans , Leukemia, Hairy Cell/immunology , Leukemia, Hairy Cell/ultrastructure , Leukemia, Lymphoid/immunology , Microscopy, Electron , Receptors, Antigen, B-Cell/analysis , Rosette Formation
12.
Scand J Haematol ; 21(3): 233-42, 1978 Sep.
Article in English | MEDLINE | ID: mdl-280939

ABSTRACT

Morphological and immunological marker data on a patient with 'pure' monocytic leukaemia are presented and compared with those of 6 cases of clearly mixed myelomonocytic leukaemia with a variable monocytic component. In all patients studied, the leukaemic monocytes expressed a receptor for the Fc of IgG, and IgG sensitization markedly enhanced phagocytosis of ox erythrocytes. A variable, but lower, percentage of the leukaemic monocytes had a receptor for mouse C3, but the cells uniformly lacked surface immunoglobulin and receptors for the Fc of IgM and for unsensitised mouse erythrocytes. Cytochemical and ultrastructural study also showed no clear difference between the monocytes of the 'pure' and mixed monocytic leukaemias. This report therefore lends no support to the concept of distinct types of monocytic leukaemia.


Subject(s)
Leukemia, Myeloid/blood , Monocytes , Binding Sites, Antibody , Complement C3 , Esterases/blood , Female , Humans , Immunoglobulin Fc Fragments , Immunoglobulin G , Immunoglobulin M , Leukemia, Myeloid/immunology , Leukemia, Myeloid/ultrastructure , Middle Aged , Monocytes/enzymology , Monocytes/immunology , Monocytes/ultrastructure , Phagocytosis , Receptors, Antigen, B-Cell , Rosette Formation
14.
Clin Exp Immunol ; 31(3): 391-6, 1978 Mar.
Article in English | MEDLINE | ID: mdl-148985

ABSTRACT

It has been demonstrated that in two patients with T-cell Sézary syndrome the pathological cells express a receptor for EA(IgM) when cultured in a medium with foetal calf or autologous serum. A study of the time-course of this rosette formation revealed that a large proportion, but not all, of E-rosetting cells progressively expressed this receptor. In agreement with previous reports by others on the normal T-cell population bearing the IgM receptor, incubation at 37 degrees C in the presence of serum was shown to be a necessary condition. The implications of this pathological subpopulation of T-cells are discussed.


Subject(s)
Dermatitis, Exfoliative/immunology , Immunoglobulin M , Lymphatic Diseases/immunology , Binding Sites , Cells, Cultured , Dermatitis, Exfoliative/pathology , Humans , Lymphatic Diseases/pathology , Rosette Formation , Syndrome , T-Lymphocytes/immunology , Time Factors
15.
Clin Exp Immunol ; 31(3): 414-8, 1978 Mar.
Article in English | MEDLINE | ID: mdl-350460

ABSTRACT

Immunological marker studies using direct immunofluorescence and rosette methods were performed on the bone marrow and peripheral blood of an IgM myeloma, and on the peripheral blood of two cases of IgA myeloma and one IgG myeloma. These studies confirmed the peripheral blood involvement in plasma cell dyscrasias. In the IgA myeloma, the membrane and cytoplasmic immunoglobulin was restricted to IgA, and the IgG myeloma did not express immunoglobulin at the surface of the cells shown to contain cytoplasmic IgG. The IgM myeloma cells, on the other hand, expressed membrane immunoglobulin, including IgD, of a single light chain type. The majority of plasma cells secreting IgA or IgG did not express the IgG Fc receptor, but most of the cells from the IgM myeloma, including plasma cells, did express IgG Fc receptors.


Subject(s)
Immunoglobulin M , Multiple Myeloma/immunology , Receptors, Antigen, B-Cell/analysis , Bone Marrow/immunology , Fluorescent Antibody Technique , Humans , Immunoglobulin A , Immunoglobulin G , Leukocyte Count , Lymphocytes/immunology , Rosette Formation
16.
J Clin Pathol ; 30(10): 981-8, 1977 Oct.
Article in English | MEDLINE | ID: mdl-304070

ABSTRACT

A full surface marker study of the splenic storage cells in a case of Gaucher's disease largely substantiates the monocyte/histiocyte nature of Gaucher's cells. In addition, an apparent T-lymphocyte deficiency is demonstrated in the spleen and peripheral blood, and the possible significance of this finding is discussed.


Subject(s)
Gaucher Disease/immunology , Spleen/immunology , Cell Extracts/immunology , Child , Female , Gaucher Disease/pathology , Humans , Leukocyte Count , Receptors, Antigen, B-Cell/analysis , Rosette Formation , Spleen/ultrastructure , T-Lymphocytes/immunology
17.
Clin Exp Immunol ; 29(3): 442-6, 1977 Sep.
Article in English | MEDLINE | ID: mdl-589863

ABSTRACT

By the use of a rosette test employing yeast particles coated with C3 activated by the alternative pathway, we have demonstrated that the hairy cells of leukaemic reticuloendotheliosis (LRE) do not possess a receptor for C3. It is also shown that hairy cells display a consistently lower percentage of rosette formation with ox EAC than with ox EA IgM and this is attributed to varying degrees of steric hindrance of IgM or incomplete coating by complement. Blocking with monomeric IgM confirmed this interpretation. These results are used to reconcile previously conflicting reports concerning the presence or absence of a receptor for C3 on the hairy cells of LRE, and to suggest that the disease is accompanied by a severe reduction of normal B lymphocytes.


Subject(s)
Binding Sites , Complement C3/metabolism , Leukemia, Hairy Cell/immunology , Cell Membrane/immunology , Humans , Immunoglobulin M , Rosette Formation
20.
J Clin Pathol ; 29(9): 848-51, 1976 Sep.
Article in English | MEDLINE | ID: mdl-135771

ABSTRACT

A case of the small-cell variant of Sézary's syndrome (SS) is reported in which the SS cells lacked the surface-marker characteristics of both T- and B-cells. In particular, the SS cells failed to form E-rosettes even with a sensitive technique using 2-amino-ethylisothiouronium bromide (AET)-treated sheep red blood cells. The significance of these findings is briefly considered in relation to the existing literature.


Subject(s)
Dermatitis, Exfoliative/immunology , Lymphatic Diseases/immunology , Skin Neoplasms/immunology , Aged , B-Lymphocytes/immunology , Humans , Immune Adherence Reaction , Lymphocytes/ultrastructure , Male , Syndrome , T-Lymphocytes/immunology , beta-Aminoethyl Isothiourea
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