A monoclonal antibody to mammalian angiotensin II AT1 receptor recognizes one of the angiotensin II receptor isoforms expressed by the eel (Anguilla anguilla).

Using labelled ligand-binding methods, previous studies have identified specific angiotensin II receptors (Ang II-Rs) in eel liver, kidney and intestine membranes. Isoelectric focusing on polyacrylamide gels also showed that there are two Ang II-R isoforms in eel liver, focusing at isoelectric points (pI) 6.5 and 6.7. These may have different functions. In contrast, eel enterocyte plasma membrane and renal brush border membranes contain only the pI 6.5 form. To characterize the eel receptors more fully, a newly developed monoclonal antibody (6313/G2) which selectively recognizes the AT1 subtype of mammalian Ang II-R was used. In ligand-binding experiments, the preincubation of eel liver membranes with 6313/G2 antibody eliminated the specific [3,5-3H]Tyr4-Ile5-Ang II binding. Moreover, Ang II-receptor complexes from solubilized liver membranes, which were immunoprecipitated by 6313/G2-coated beads, had a pI of 6.5. In immunoblotting experiments, the antibody recognized the isoform focusing at pI 6.5 in eel intestine and liver preparations, but not the liver pI 6.7 isoform. Immunoblotting of SDS gels showed that the antibody bound to a single protein of molecular mass of 75 kDa in eel liver, gill and kidney and to a doublet of molecular mass of about 74 and 75 kDa in intestinal membrane preparations. Immunocytochemistry of paraffin-embedded and cryostat sections of eel liver, kidney, intestine and gill showed that antibody 6313/G2 bound to uniformly distributed intracellular sites and cell surface membranes in proximal tubular cells, absorptive intestinal cells, hepatocytes and chloride cells. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine. The data suggest that the previously described Ang II-R from eel liver, kidney and intestine may be similar to the mammalian AT1 subtype.

Multiple isoforms of the oestrogen receptor in endometrial cancer.

We evaluated the presence and variability of oestrogen receptor (ER) isoforms in endometrial cancer by using [3H]oestradiol-labelled ERs and the H222 monoclonal antibody obtained from the Abbott enzyme immunoassay kit. Using isoelectric focusing (IEF), endometrial ER was shown to be composed of four different species, with pI values of 6.1, 6.3, 6.6 and 6.8, indistinguishable from the isoforms found in normal rat uterus, and human breast and larynx carcinomas. The isoforms at pI 6.3, 6.6 and 6.8, all sedimenting at 4S by sucrose gradient fractionation, showed, on two-dimensional SDS electrophoresis, relative masses of 50, 70 and 65 kDa respectively, equal to the masses previously found in breast cancer. These isoforms did not alter their pI values during IEF fractionation performed in a linear gradient of urea, while the pI 6.1, sedimenting at 8S, generated a new isoform at about 9 mol/l urea with pI 7.2 and a relative mass of 65 kDa. The urea-dissociated isoform (pI 7.2) was able approximately to double the antibody binding with respect to the nondissociated oligomer, which suggested that some epitopes are 'masked', i.e. not accessible to the antibodies when ER is present in its complexed form. The evidence thus suggested that the oligomer at pI 6.1 contained a single 65 kDa ER form which, as a monomer, focused at pI 7.2. The variability in the ER isoform profile found in endometrial cancer was similar to the variability previously reported in breast and larynx carcinomas. The balance between these isoforms could be a dynamic parameter involved in the functionality of this receptor and consequently in cell transformation.

Effect of tumor size on the association between ps2 and cathepsin-d in primary breast-cancer.

Previous studies have shown that pS2 and cathepsin D are linked in lymph node positive (N+) tumours, but not in tumours from lymph node negative (N-) patients. The purpose of this study was to understand whether or not size would effect the relationship between pS2 and cathepsin D. Findings were further extended to some subgroups of tumours obtained stratifying for T and N and particularly to the small (TI) but aggressive (N+) cancers (T1/N+) and to those of size greater than 2 cm (T2 and T3) but yet node negative (T2+T3/N-). Oestrogen (ER) and progesterone (PR) receptors, pS2 and cathepsin D concentrations were therefore assayed in 355 primary breast cancers. ER, PR, pS2 and cathepsin D did not correlate to nodal status and size of the tumours; no significant differences in the expression of these four biological factors between infiltrating ductal carcinomas without special features (NOS) and non-NOS carcinomas were found. Multivariate analysis performed among cathepsin D, ER, PR and pS2 indicated that, in T1 tumours, pS2 was the most important variable and the best predictor in cathepsin D determination, while such association was absent in T2 and T3 tumours. pS2 and cathepsin D significantly associated also in tumours obtained from N+ patients, and such correlation was highest in T1 tumours with positive axillary nodes (N+/T1). pS2 and cathepsin D did not associate in tumours taken from N- patients. Considering the NOS carcinomas, correlation between pS2 and cathepsin D in the N+, T1 and N+/T1 subgroups was higher in the poorly differentiated grade 3 with respect to grade 1 and grade 2 cancers. The data suggest that pS2 could have a role in cathepsin D expression and we hypothesise that such control could be an early biological event occurring in the development and progression of particularly aggressive (N+/grade 3), small (T1) breast cancers.

Enzyme-linked immunosorbent assay of HER-2/neu gene product (p185) in breast cancer: its correlation with sex steroid receptors, cathepsin D and histologic grades.

One hundred and nine primary breast cancers were analyzed to assess the presence of the HER-2/neu gene product (p185), the oestrogen (ER) and the progesterone (PR) receptors, and the total cathepsin D status. An enzyme-linked immunosorbent assay (ELISA kit, Oncogene Science Inc.) was used for the evaluation of p185 in pellets obtained after a 100,000 x g centrifugation, ER and PR were measured by enzyme immunoassay (EIA kit, Abbott Laboratories), and the total cathepsin D content was evaluated by immunoradiometric assay (IRMA kit, CIS Biointernational). We showed that the ELISA kit is feasible to quantify the p185 present in breast cancer cell membranes, and that the detector antibody recognises a protein of apprroximatly M(r) 185,000. The detected antigen was inversely related to both ER and PR, but it did not correlate to total cathepsin D. No significant differences were found in the expression of p185, ER, PR, cathepsin D between infiltrating ductal carcinomas without special features (NOS) and non-ductal (non-NOS) carcinomas. Nevertheless, in NOS carcinomas, a trend was observed in the p185 levels expressed by the tumours with different histological grades, in that p185 concentration was higher in the poorly differentiated grade 3 with respect to grade 2 and grade 1.