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S. Marsigliante
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V. A. Baker
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J. Puddefoot
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S. Barker
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G. P. Vinson
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ABSTRACT

The variability in the profile of oestrogen receptor (ER) isoforms in breast tumours has been studied.

Using low-resolution isoelectric focussing (IEF), two major ER isoforms with isoelectric point (pI) values of 6.1 and 6.6 could be identified, with corresponding sedimentation coefficients in sucrose density gradients of 8 S and 4 S respectively.

Using high-resolution IEF or immunoblotting, the pI 6.6 form (4S) was shown to be composed of three different species, with pI values of 6.3, 6.6 and 6.8, while the oligomeric pI 6.1 protein (8 S) did not show charge heterogeneity. Data were obtained on the soluble receptors from supernatants of 42 ER-positive primary breast tumour homogenates using high-resolution IEF to obtain ER isoform profiles. It was found that 54.7% of tumours contained the isoforms at pI 6.6 and 6.1, while only 11.9% contained the full complement of isoforms (pI 6.1, 6.3, 6.6 and 6.8). Of the tumours studied, 11.9% contained isoforms of pI 6.1, 6.6 and 6.8, with 14.3% containing isoforms with pI 6.1, 6.6 and 6.3. Very few tumours contained only one isoform, with 4.8% of tumours containing a single isoform at pI 6.1 and 2.4% of tumours containing only the isoform at pI 6.6.

All four ER isoforms were also shown to be present in some tumours by immunoblotting using antibody H222 and, in addition, high-resolution IEF indicated that all isoforms bind oestradiol, diethylstilboestrol and tamoxifen.

The variability in the ER isoform profile may have a bearing on the known variability of tumour response to endocrine therapy and prognosis.

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J R Puddefoot
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V A Baker
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B Bakkers
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S Marsigliante
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S Barker
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C Panahy
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A W Goode
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R Carpenter
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G P Vinson
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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.

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