Targeting of HLA-DR molecules transduces agonistic functional signals in cutaneous melanoma.

The role of human leukocyte antigens (HLA) class II molecules in transducing intracellular signals in immune cells is well established. Solid tumors of different histotype can also express HLA class II antigens; however, their intracellular signaling ability is essentially unknown. Due to the frequent expression of HLA class II molecules in primary and metastatic lesions, cutaneous melanoma was utilized to investigate whether the engagement of HLA-DR molecules transduces functional intracellular signal(s).

Endothelin B receptor blockade inhibits dynamics of cell interactions and communications in melanoma cell progression.

Phenotypic and genotypic analyses of cutaneous melanoma have identified the endothelin B receptor (ET(B)R) as tumor progression marker, thus representing a potential therapeutic target. Here, we demonstrate that activation of ET(B)R by endothelin-1 (ET-1) and ET-3 leads to loss of expression of the cell adhesion molecule E-cadherin and associated catenin proteins and gain of N-cadherin expression. Exposure of melanoma cells to ET-1 leads to a 60% inhibition in intercellular communication by inducing phosphorylation of gap junctional protein connexin 43.

Application of an immunodiagnostic method for improving preoperative diagnosis of nodular thyroid lesions.

Background: Thyroid cancer is the most common endocrine malignant disease, but preoperative diagnosis remains a challenge. Fine-needle aspiration cytology has greatly improved the clinical management of thyroid nodules, but the preoperative characterisation of follicular lesions is very difficult. Many patients are thus referred to surgery more for diagnosis than for therapeutic necessity.

Role of endothelin-1 in neovascularization of ovarian carcinoma.

Endothelin-1 (ET-1) is overexpressed in ovarian carcinomas and acts, via ET(A) receptors (ET(A)R), as an autocrine growth factor. In this study we investigate the role of ET-1 in the neovascularization of ovarian carcinoma. Archival specimens of primary (n = 40) and metastatic (n = 8) ovarian tumors were examined by immunohistochemistry for angiogenic factor and receptor expression and for microvessel density using antibodies against CD31, ET-1, vascular endothelial growth factor (VEGF), and their receptors.

Transactivation of the epidermal growth factor receptor in endothelin-1-induced mitogenic signaling in human ovarian carcinoma cells.

Endothelin (ET)-1 is produced in ovarian carcinoma cells and is known to act through ET(A) receptors as an autocrine growth factor in vitro and in vivo. In OVCA 433 human ovarian carcinoma cells, ET-1 caused phosphorylation of the epidermal growth factor receptor (EGF-R) that was accompanied by phosphorylation of Shc and its recruitment complexed with Grb2. These findings suggested that an EGF-R/ras-dependent pathway may contribute to the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) 2 and mitogenic signaling induced by ET-1 in these cells.

Expression of endothelin 1 and endothelin A receptor in ovarian carcinoma: evidence for an autocrine role in tumor growth.

In the present study, we have investigated the expression of endothelin 1 (ET-1) and the ET(A) receptor (ET(A)R) and ET(B) receptor (ET(B)R) in primary (n = 30) and metastatic (n = 8) ovarian carcinomas and their involvement in tumor growth. By reverse transcription-PCR and Northern blot analysis, we detected ET-1 mRNA in 90% of primary and 100% of metastatic ovarian carcinomas. ET-1 mRNA expression was significantly higher in tumors than in normal ovarian tissues (n = 12; P < 0.

Activation of mitogenic signaling by endothelin 1 in ovarian carcinoma cells.

Endothelin 1 (ET-1) is produced in ovarian cancer cell lines and has been shown to act through ET(A) receptors as an autocrine growth factor to promote tumor cell proliferation in vitro. In OVCA 433 cells, the efficacy of ET-1 as a stimulus of [3H]thymidine incorporation was equivalent to that of epidermal growth factor. ET-1 also stimulated the rapid expression of c-fos, an action mediated by ET(A) receptors.

Autocrine actions of endothelin-1 as a growth factor in human ovarian carcinoma cells.

The production of endothelin 1 (ET-1) and its receptor-mediated actions on calcium signaling and growth responses were analyzed in human ovarian carcinoma cells. Immuno-reactive ET-1 was released from three of four ovarian tumor cell lines as a function of time in amounts ranging from 56 to 74 fmol/10(6) cells. Reverse-phase HPLC and radioimmuno-assay of conditioned media from tumor cells revealed a single peak coeluting with authentic ET-1.

Immunotoxins to the HER-2 oncogene product: functional and ultrastructural analysis of their cytotoxic activity.

Two immunotoxins were prepared using monoclonal antibodies (mAb) directed towards two distinct epitopes of the gp185HER-2 extracellular domain, and the type I ribosome inactivating protein (RIP) plant toxin saporin 6. Cell protein synthesis inhibition assay reveals that the immunotoxins display a potent and specific cytotoxicity that is characterized by a slow rate, since the time required to inhibit incorporation of radiolabeled leucine completely ranges from 36 h to 60 h depending on the target cell line and the immunotoxin. Because this feature may hamper the immunotherapeutic use of these conjugates we analysed this further by studying the early phases of internalization of immunotoxins by immunoelectron microscopy.

Characterization of cytotoxic activity of saporin anti-gp185/HER-2 immunotoxins.

The oncogene HER-2/neu encodes a trans-membrane receptor of 185 kDa with tyrosine-kinase activity. Over-expression of this molecule has been reported in a significant proportion of human breast and ovarian carcinomas, characterized by a poor clinical prognosis. Two monoclonal antibodies (MAbs), recognizing distinct epitopes of the gp 185 extracellular domain, have been utilized in the present study for the production of immunotoxins (ITs) by conjugation to the type-1 RIP (ribosome-inactivating protein) plant toxin saporin 6 (SAP).

Production and characterization of two immunotoxins specific for M5b ANLL leukaemia.

We describe the production and functional characterization of 2 monocytic-cell-lineage-specific immunotoxins constructed with saporin emitoxin (SAP) from Saponaria officinalis. Interest in the production of these immunotoxins, of possible clinical relevance, has been raised by the availability of 2 MAbs of high specificity for circulating monocytes and M5b ANLL, thus envisaging their potential use in bone-marrow purging. SAP emitoxin was selected on the basis of the low cytotoxicity in unconjugated form, as opposed to highly specific cytotoxicity and favourable pharmacokinetical properties in the conjugated form.

Saporin 6 conjugated to monoclonal antibody selectively kills human melanoma cells.

A novel human melanoma specific immunotoxin is described, which has been produced utilizing the murine monoclonal antibody (mAb) Ep2, IgG2a isotype, recognizing an epitope of the glycoprotein/proteoglycan high molecular weight-melanoma associated antigen. mAb Ep2 has been chemically conjugated by a disulphide bond, using the bifunctional reagent SPDP, to the plant toxin Saporin 6 (SAP) extracted from seeds of Saponaria officinalis. Cytotoxicity studies performed in vitro on melanoma cells have shown that Ep2/SAP immunotoxin efficiently kills antigen expressing cells and that its IC50 is approximately 1 x 10(-10) M, while not affecting the viability of antigen-negative melanoma cells at doses as high as 1 x 10(-7) M, therefore indicating a therapeutic index of Ep2/SAP immunotoxin higher than 1000.

Retrovirus-derived shuttle vectors in the generation of murine bispecific MoAbs to be used in ex vivo and in vivo immunotherapy.

Mouse monoclonal antibodies specific for CD3, FcR and a melanoma associated antigen have been produced. Drug resistence of such hibrydomas has been obtained tranfecting them with plasmids containing genes conferring specific resistence. Retrovirus derived shuttle vectors with high transfection efficiency have been used for transfection.

Effect of recombinant human leukocyte, fibroblast, and immune interferons on expression of class I and II major histocompatibility complex and invariant chain in early passage human melanoma cells.

Twenty-five early-passage (less than or equal to 8) melanoma cell lines, isolated from ten patients with metastatic melanoma, were analyzed by a combination of serological, immunochemical, and molecular methods for mRNA levels, synthesis, and surface expression of MHC class I and class II antigens prior to and following exposure to recombinant human leukocyte (IFN-alpha A), fibroblast (IFN-beta), and immune (IFN-gamma) interferon. All the cell lines expressed variable levels of HLA class I gene products that were up-regulated to different extents upon exposure to specific interferons (IFNs). HLA class II antigens were expressed in 22 of the 25 melanoma lines and IFN-gamma increased the levels of class II mRNA, protein synthesis, and surface expression in all cultures displaying baseline expression.

Modulation of the antigenic phenotype of early-passage human melanoma cells derived from multiple autologous metastases by recombinant human leukocyte, fibroblast and immune interferon.

We have investigated the relationship between in vitro cultivation of autologous melanoma metastases derived from different patients and their levels of expression of class-I and -II major histocompatibility complex (MHC) antigens and melanoma-associated antigens (MAAs). Cell cultures were established from 23 individual metastatic melanoma lesions from 10 patients and were tested early after isolation (between 3rd and 10th passages) for both constitutive expression and modulation by recombinant human leukocyte (IFN-alpha), fibroblast (IFN-beta) or immune (IFN-gamma) interferon of MHC antigens and MAA. All of the melanoma cell lines displayed altered antigen expression following IFN treatment.

Gene transfer by retrovirus-derived shuttle vectors in the generation of murine bispecific monoclonal antibodies.

The present study reports on the use of gene transfer by retrovirus-derived shuttle vectors in the generation of hybrid hybridomas secreting bispecific monoclonal antibodies. neo- and dhfr- genes were infected into distinct murine hybridomas, thus conferring a dominant resistance trait to geneticin (G418) and to methotrexate. The vectors employed were replication-deficient and dependent on complementation by a helper virus provided by the irradiated packaging lines.

Gene transfer by retrovirus-derived shuttle vectors in the generation of murine bispecific MAbs.

This report deals with the use of gene transfer by retrovirus-derived shuttle vectors in a novel model aimed at the generation of hybrid hybridomas secreting bispecific monoclonal antibodies (biMAbs). Following this approach, two genes conferring dominant resistance trait to the neomycine analogue geneticin (G418) and to methotrexate (MTX) respectively, were infected in two established hybridoma lines, each producing a well characterized MAb. The vectors used here were replication-deficient, being dependent on the complementation of helper virus provided by packaging lines.

mAb KUL/05 identifies a denaturation-resistant determinant shared by class II MHC products DR, DQ and DP.

mAb KUL/05, a novel murine monoclonal antibody, reacts with molecules displaying the typical tissue distribution and molecular profile of class II MHC antigens. An extensive scrutiny employing serological and immunochemical assays on DR homozygous and DR alpha- mutant cell lines has shown that this reagent displays some additional, interesting features, namely mAb KUL/05 (a) binds in a broadly monomorphic fashion to cells of DR1 through seven specificities, (b) recognizes a determinant shared by a large proportion of DR, DQ and DP beta chains from most haplotypes, in both their monomeric and alpha chain-associated forms, and (c) reacts with frozen, acetone-fixed, as well as conventional, formalin-fixed, paraffin embedded tissues. Thus, mAb KUL/05 is likely to represent a useful adjunct for the study of the expression of class II MHC products in normal and pathological tissue specimens.

Accessory cell function of human melanoma cells in mitogen-induced T cell proliferation.

Six out of eight human melanoma cell lines were found to be able to function as accessory cells in PHA-induced proliferation of autologous and allogeneic T cells. The accessory cell function of the melanoma cell lines appears to be similar to that of monocytes, requires the presence of viable cells, and does not correlate with the cell surface binding sites for PHA and with the level of expression of HMW-MAA and of HLA Class I antigens. HLA Class II antigens do not appear to play a major role in these phenomena, since there is no relationship between level of expression of HLA Class II antigens and accessory cell function of melanoma cells.

Recombinant human IFN-gamma, but not IFN-alpha or IFN-beta, enhances MHC- and non-MHC-encoded glycoproteins by a protein synthesis-dependent mechanism.

Despite quantitative as well as qualitative differences, all three types of IFN (IFN-alpha, IFN-beta, and IFN-gamma) modulate the synthesis as well as the expression of class I and class II histocompatibility Ag and a melanoma-associated Ag located in the plasma membrane as well as the cytoplasm of human melanoma cells. By employing inhibitors of RNA and protein synthesis it was demonstrated that IFN-alpha and -beta increase the expression of histocompatibility products and this tumor-associated Ag by a process not requiring new protein synthesis. In contrast, IFN-gamma does require de novo protein synthesis for its modulatory activity.

A murine monoclonal antibody strictly reactive with human peripheral blood monocytes.

We have produced and characterized a novel murine monoclonal antibody (LAM7) of IgG1 isotype which appears specific for peripheral blood monocytes (PBM) on the basis of histochemical and functional studies. By indirect immunofluorescence, including FACS analysis, the antibody reacts with 90 +/- 6% of PBM and with monocytic leukemias, while it is totally unreactive with B and T lymphocytes, platelets, granulocytes, peripheral macrophages, dendritic cells, large granular lymphocytes, and nonmonocytic leukemias. The antigen-presenting capacity of peripheral blood mononuclear cells is abolished by treatment with MoAb LAM7 in an antiglobulin-complement-mediated cytotoxicity test, and restored by addition of purified PBM.

A cytoplasmic human melanoma associated antigen as a marker of activation in lymphoid cells.

A cytoplasmic glycoprotein, originally identified by the monoclonal antibody 465.12S in melanoma tumors, is significantly increased in epithelial cells of different histotype following transformation. In the present study we show that the cytoplasmic melanoma associated antigen (cyt-MAA) is drastically enhanced in lymphoid cells by polyclonal and allogeneic stimulation, as well as by transformation.

Regulation of the expression of class II genes of the human major histocompatibility complex in tumor cells.

The control of expression of human class II MHC genes has been studied in lymphoid and melanoma cells. Specific unmethylation of all restriction sites nearby the promoter regions has been detected in all cell lines and tissues studied, irrespective of their ability to express class II MHC products. The main functional role of DNA methylation appears, on the contrary, to be the regulation of a fraction of the nucleotide polymorphism of class II MHC genes.

Regulation of the antigenic phenotype of human melanoma cells by recombinant interferons.

The ability of interferons to modulate the antigenic phenotype of tumor cells may involve alterations in the transcription, translation, membrane expression and shedding of Major Histocompatibility Complex (MHC) and Tumor Associated Antigens (TAAs). In the present study we have investigated possible mechanisms by which recombinant human interferons, IFN-alpha, -beta and -gamma, alter the antigenic profile of long- and short-term human melanoma cultures. IFN-alpha and -beta induced similar changes in the synthesis, expression and shedding of two TAAs, a HMW-MAA and a Cyt-MAA, in the established melanoma cell line Colo 38, whereas IFN-gamma exerted a differential effect on these melanoma associated antigens.

Gene products of the HLA-D region in normal and malignant tissues of nonlymphoid origin.

Indirect immunofluorescence staining with monoclonal antibodies has shown a differential distribution of HLA-DR, DQ, and DP antigens in normal tissues of nonlymphoid origin. The distribution of HLA-DP antigens is similar to that of HLA-DR antigens, while that of HLA-DQ antigens is more restricted. Malignant transformation of cells of nonlymphoid origin may be associated with the appearance of the gene products of the HLA-D region.