Antonio Mastroianni

Characterization of two monoclonal antibodies directed against the 67 kDa high affinity laminin receptor and application for the study of breast carcinoma progression.

Two monoclonal antibodies (mAbs), designated MLuC5 and MLuC6, were produced against a human small cell lung carcinoma cell line. They were found to exhibit a superimposable reactivity on different cell lines and on platelets. Moreover, they both immunoprecipitated a 67 kDa molecule from the membrane of the reference target cells. Immunodepletion and cross-inhibition tests indicated that the two mAbs recognize two epitopes closely localized on the same molecule. The MLuC5 mAb was further characterized for its reactivity on platelets. Immunoprecipitation and ELISA assays demonstrate that this mAb recognizes the 67 kDa high afinity laminin receptor. MLuC5 reactivity was evaluated by immunohistochemistry on a variety of normal and tumor tissues, in particular breast specimens including normal epithelium, dysplastic lesions, in situ carcinomas, invasive primary carcinomas and distant metastases. The laminin receptor was found to be strongly expressed in 50% of the infiltrating carcinomas, whereas in situ carcinomas and benign lesions, as well as the normal mammary epithelium, were only weakly and focally positive. In metastatic lesions MLuC5 reactivity was only found in 11% of the samples tested, independently of the site of origin of the lesion.

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

SummaryIn this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 104 receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.

Alteration of Laminin Production in Small-Cell Lung Carcinoma: Possible Correlation with the Absence of the Basement Membrane

Basement membranes are frequently absent in small-cell lung cancer (SCLC). To study this phenomenon, the production of laminin by SCLC cells, both in vitro and in vivo, has been investigated and compared with the laminin production by lung carcinomas of other histotypes (non-SCLC, NSCLC). Immunoradiometric and immunoperoxidase tests, respectively, carried out on culture supernatants and cells using antilaminin rabbit antiserum, revealed that in 1 (H446) out of 8 SCLC cell lines tested and in the NSCLC line Calu3, laminin was detectable both in the culture medium and in the cytoplasm of the cells. After treatment of an SCLC-negative cell line (N592) with monensin, a molecule which inhibits protein secretion, laminin became detectable in the cytoplasm. Similar results were obtained by FACS analysis on cells permeabilized with saponin. Northern analysis indicated that the laminin B1 gene was transcribed. The level of mRNA for the B1 laminin subunit in the N592 cells was twice and 4 times higher than that found in the laminin-secreting Calu3 and H446 cell lines. The production of laminin in SCLC and NSCLC surgically resected samples using immunoperoxidase staining of cryostatic sections was also investigated. The results indicate that 85% of the NSCLC cases tested showed diffuse staining in the cytoplasm of the tumor cells and strong staining at the basement membrane level, whereas a similar staining was only found in 15% of the SCLC cases tested. The treatment of SCLC cells with differentiating agents in vitro has been shown to induce the adhesion of these cells. In fact, n-butyric acid induced the disaggregation of floating-clump cells and single-cell adhesion in the N592 line, whereas after treatment with retinoids the clumps of the N592 cells were still present, but 30% of these were found to adhere to the plate. However, no increase in the laminin production was found in the cytoplasmic or culture medium under these conditions.