The Bag-1 inhibitor, Thio-2, reverses an atypical 3D morphology driven by Bag-1L overexpression in a MCF-10A model of ductal carcinoma in situ.

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.

Paramagnetic microspheres as immunological markers for light and electron microscopy.

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.

An improved fluorescence probe cytotoxicity assay.

The phenanthridine dye, ethidium bromide, which is actively excluded by viable cells, undergoes a significant fluorescence enhancement at 5900 A upon binding intracellular double-stranded polyribonucleotides. A rapid and sensitive assay of antibody mediated cytotoxicity to cells grown in vitro has been developed using this phenomenon. In this communication, we describe this fluorescence probe cytotoxicity assay and a sensitive electro-optical system designed to measure the fluorescence enhancement of ethidium bromide as it intercalates with intracellular polyribonucleotides. Basic characteristics of the fluorescence enhancement resulting from the interaction of ethidium bromide and non-viable cells are presented as well as examples of this assay as it has been used to study surface membrane neoantigens of cells tranformed by the oncogenic DNA virus, SV40.

Phenotypic modulation of T-lymphoblastic leukaemic cells with phorbol ester and leucocyte conditioned medium.

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.

A case of small-cell Sézary's syndrome with null-cell features.

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.

Surface marker and other characteristics of Gaucher's cells.

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.

Lymphocyte subpopulations in Hodgkin's disease and non-Hodgkin's lymphoma and the effects of radiotherapy.

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.

Characterization of the receptor for IgM present on human B lymphocytes.

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.

Evidence for the presence of a receptor for IgM on the pathological cells of Sézary's syndrome.

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.

Purification and quantification of T and B lymphocytes by an affinity method.

Affinity surfaces were produced by coupling human immunoglobulin (HGG) to the surface of tissue culture grade plastic-ware with a water-soluble carbodiimide followed by treatment with anti-HGG antisera. Surface immunoglobulin SIg) bearing human B lymphocytes attach to these surfaces when centrifuged on to them and unattached cells could be recovered by inverting the trays or dishes. Optimal cell attachment conditions could be rapidly evaluated by counting cells attached to representative areas of multi-well trays and percentage of SIg-bearing cells quantified. Evidence was obtained for cell attachment through Fc receptors as well as SIg using unrelated antigen--antibody-coated trays. This could be prevented by using the F (ab')2 fragments of the antisera. Under these conditions specific attachment through K and lambda light chains could be achieved with normal and chronic lymphocytic leukaemic lymphocytes. Using tissue culture plastic Petri dishes and relatively small quantities of antiserum, larger numbers of lymphocytes could be processed to produce T lymphocytes containing less than 1 per cent of contaminating SIg-positive cells.

A new monoclonal antibody (CH-F42) recognizes a CD7- subset of normal T lymphocytes and circulating malignant cells in adult T-cell lymphoma-leukemia and Sézary syndrome.

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.

Absence of a receptor for fixed C3 on the hairy cells of leukaemic reticuloendotheliosis.

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.

Differing surface marker characteristics in plasma cell dyscrasias with particular reference to IgM myeloma.

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.

Chronic myelomonocytic leukemia with paraproteinemia but no detectable plasmacytosis: a detailed cytological and immunological study.

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.

Typical hairy-cell leukaemia with IgGk paraproteinaemia.

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.

Morphological and immunological similarity of the monocytes from pure and mixed monocytic leukaemias.

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.