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ABSTRACT

We have generated hybridomas which secrete monoclonal antibodies to the AT₁ subtype of the angiotensin II receptor (AT₁ receptor). These were obtained after immunization of Balb C/c mice with synthetic peptides representing sequences from either the extracellular domain (residues 8-17) or the intracellular domain (residues 229-237) of the AT₁ receptor.

Hybridoma populations were first screened for the production of antibodies which bound to rat liver cells. Further selection, and cloning by limiting dilution, was carried out for antibodies which bound specifically to rat adrenal glomerulosa cells. Confirmation that the antibody designated 6313/G2 interacted with the angiotensin II receptor was obtained using COS-7 cells transfected with AT_1A receptor cDNA. In particular, the initial characterization of 6313/G2 showed specific immunofluorescence of vascular endothelium.

ABSTRACT

Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6·1, 6·3, 6·6 and 6·8. Of these the first (pI 6·1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6·3 and pI 6·8 were significantly correlated with the presence of progesterone receptors.

To characterize the isoforms more fully, ER isoforms labelled by [³H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-³H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6·3, 6·8 and 6·6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6·3, 6·8 and 6·6 isoforms, could form dimers.

We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.

ABSTRACT

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 non-dissociated 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.