Apoptosis induction by the photosensitizer verteporfin: identification of mitochondrial adenine nucleotide translocator as a critical target.

We report that the photosensitizer verteporfin kills lymphoma cells by an apoptotic process involving a dissipation of the mitochondrial inner transmembrane potential (deltapsim). Light-activated verteporfin-induced apoptosis was abolished by transfection with Bcl-2, a procedure reported to inhibit the mitochondrial permeability transition pore complex (PTPC). Verteporfin triggered the deltapsim loss in isolated mitochondria in vitro, and this effect was suppressed by bongrekic acid and cyclosporin A. Verteporfin plus light also permeabilized proteoliposomes containing the semipurified PTPC or the purified PTPC component adenine nucleotide translocator (ANT), yet had no effect on protein-free control liposomes. Verteporfin phototoxicity on ANT proteoliposomes was mediated by reactive oxygen species and was prevented by recombinant Bcl-2 or the adenine nucleotides ATP and ADP. In conclusion, verteporfin belongs to a class of clinically used chemotherapeutic agents acting on PTPC and ANT.

Analysis of a heterogeneous group of human breast carcinoma associated glycoproteins bearing the Tn determinant.

The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.

Purification and characterisation of a breast-cancer-associated glycoprotein not expressed in normal breast and identified by monoclonal antibody 83D4.

Monoclonal antibody (mAb) 83D4 was generated using formol-fixed paraffin-embedded human breast carcinoma tissue as the immunogen. Previous studies demonstrated that it was reactive with breast carcinoma tissues, but not with normal breast. The antigen identified by mAb 83D4 was detected, using ELISA, in MCF7 breast carcinoma cell line membrane extracts, in primary breast and colon carcinoma tissue extracts and in pleural effusion fluid from patients with metastatic breast cancer. No reactivity with 83D4 was found in either human milk fat globule membranes or skimmed milk. 83D4 reactive antigen was found to be a heterogeneous high molecular weight (MW) protein (apparent Mr:300-400 to over 1000 kDa) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The antigen was purified from MCF7 cells, breast and colon carcinomas and effusion fluid, by perchloric acid solubilisation followed by immunoaffinity chromatography with 83D4. The immunopurified antigen from MCF7 cells and pleural effusion fluid was further analysed by gel filtration and ion-exchange chromatography, which confirmed the high MW and indicated the charge heterogeneity of the reactive molecules. The 83D4 reactive antigen strongly bound to wheat-germ agglutinin and weakly to peanut lectin. No binding was found with lentil lectin or concanavalin A. Antigenic activity was strongly reduced by trypsin and subtilysin digestion and by treatment with sodium periodate, but it was not affected by neuraminidase. These results imply the glycoprotein nature of the 83D4-defined antigen and the involvement of carbohydrate, but probably not sialic acid, in the epitope. Purified 83D4 antigen did not display reactivity for mAb HMFG-1, directed against a polymorphic epithelial mucin, PEM, using ELISA, but bound mAb CC49 and weakly mAb B72.3, antibodies which define a tumour associated glycoprotein, TAG-72. Moreover CC49 and 83D4 showed similar reactivity pattern in immunoblotting assays. A double determinant radioimmunoassay confirmed that 83D4 antigen carries epitopes for mAb B72.3 and CC49. Competition radioimmunoassays clearly distinguished the 83D4 defined epitope from those recognised by B72.3 and CC49, demonstrating that antibody 83D4 identifies a unique epitope. It is suggested that the antigens identified by mAb 83D4 and by mAb B72.3 and CC49 may form part of the same family of carcinoma associated glycoproteins.

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method.

This report describes a method for the production of murine monoclonal antibodies (MAbs) against cellular antigens preserved during formol fixation and paraffin embedding of human tissues in an attempt to select markers that would be useful in immunopathology. Hybridomas were prepared using spleen cells from mice immunized with cell suspensions obtained from formalin-fixed paraffin block sections of a human breast carcinoma. A monoclonal antibody 83 D4 was selected, which was reactive with paraffin embedded breast carcinoma tissues, but not with normal breast. The reactive antigen has a high molecular weight (400-1000 kD) and was detected on the cell surface of live human breast cancer cell lines and on frozen tissues sections. These results demonstrate that the MAb 83 D4 identifies a native breast tumor associated epitope conserved during tissue fixation and embedding and could be used as an immunohistochemical marker.