Association of oestrogen-protein complexes with the membranes of ZR 75-1 human breast cancer cells

323 [HETEROGENEITY OF OESTROGEN RECEPTORS IN HUMAN BREASTTUMOURS. J.R.Puddefoot,E.Anderson, O.J.A.Gilmore*. Department of Biochemistry,Medical College of St.Bartholomews Hospital, Charterhouse Square,London.EClM 6BQ. *Breast Unit,St. Bartholomews Hospital,West Smithfield, London. EClA 7BE. up to 4ow, of ” oestrogen receptor positive ” (ER+ve) breast tumours do not respond to endocrine therapy.Data is presented here that could explain the relative hormone insensitivity of isome tumours within the ER*ve group. Biopsies of breast tumour from SO patients were obtained at a cellular area of tumour was aelected and placed in liquid nitrogen within 10 mins. transported to our laboratory. Tissue was pulverised to a powder and a homogenate was made in 1:lO dilution with buffer (30% glycerol, 50 mMNeH2P04, 1.5 mMEDTA, 10 mklMTG, pH7.4). 2OOOg supernetant was prepared: Cytosol (100~1 I 0.23 - 0.6 mg protein ) was incubated in a Of 300 ul wltttvarymg concentrations of H-oestradiol-17-B (20 nM - 0.156 nH ),. H ligand was separated from bound using dextran coated charcoal (0.5% charcoal, 0,059. and subjected to Scarchard analysis. This revealed that not only do diffefgnt contain different concentrations of the oestrogen receptor (O-300 fmols mg protein)bur importantly the dissociation constant fKd) of their receptors showed a ten-fold (Kd from 0.067nM - 0.718nM). A further observation revealed that lo/SO of the turnouts knalysed clearly contained two specific oestrogen binding componentq, one of very high affinity 0.075fQ.18nM, n- 10 [ERI rangingfrom 1 - 111 fmols mg protein ). The Ofher with a lower (Kd 2.24$X).53nM, n* 10 [ERJ ranging from 10 - 513 fmols mg protein ).We suggest that oestrogen receptor mey be present in different states of activation and that those tumours ontaining high concentrations of ER with high affinities are more sensitive co oestrogen than t:hose containing low ER with low affinities. _ 324 STEROID RECEPTORS ItN BRXAST CANCER TISSUES OF INDIAN WOMEN AS AMARKER OF ~O~OI?E ~~P~~~Y; UrInila Verma, Murtigesan, Kt, KaPUr,

BIM*L~, Laumas, K.R. and Farooq, A. Dept. of ReproductiveBiology and Surgery*,AZ.1 India Instituteof Medical Sciences,New Delhi - 110029, INDIA Determination of estrogen and progesteronereceptorsin breast carcinoma (EC) plays a ‘marker’ role in selectingpatients for therapeiatic strategies. In this seudy 181 breast cancer tumors from Indian women were screenedfor steroid receptor estimations. Cytoaol estrogenreceptor (ER > value of 3 ant 10 fmole/mg protein served as cut off value for pre and post&enopausalwomen 50 out of 69 (56.1%)premenopausaland 51 out of 92 (55.4%) respectively. > was estimatedin 35 BC postmenopausalBC tumours were ER+. Nuclear ER ( tissues, 61.9% of BC tumors in p&menopausal and % of postmenopausalstate with a cut off value of 70 fmoles/lOOlugDNA. ProgesteroneFeWJtOr r;r) onwith ER was measured in 53 BC (23 pre and 30 POFtmenOPaUSal) tiskeg. mly 23.9% and 19% of pre and postmenopausaltumors had both ERz and pR while 4% and 48.6% tWiOrs from pre and POStILVZnOPaUSal State Were patients are ER- PR'. Based on ER and PR receptor value of BC tiSSUeS, be&g %reated with tamo%ifen. The results suggested estimationof PR aonmitb ER has a better predictivevalue for response to the hormone therapy.

-325 SSOCIATIONOF ORSTR~EN~PROTEIN COMPLEXES WITHTHE CARS OF ZR 75-l HUMAN BREASTCANCER Medical College of ELLS. Anderson, E. and Gilmore, O.J.A.*. Department of Biochemistry, 1St. Bartholomew’s Hospital, Charterhouse Square, London. ECl. U.K. *Breast Unit, St. Bartholomew’s Hospital, West Smithfield, London. ECI. D.K.

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In the ZR 75-l human breast cancer cell line, oestrogen is sequestered into tightly-bound omplexes with protein which are disrupted by trypsin. Dye exclusion tests show no effect of zrypsinon cell integrity suggesting trypsin acts at the cell membrane. Cultured ZR 75-1 cells ere preincubated in hormone-free Dulbecco’s Modified Eagles Medium f24h), resuspended in rebs Ringer bicarbonate buffer with 0 .2% w/v glucose (KRBG) and incubated in a total of 5ml RBG, to which test compounds were added, for Ih at 37’C. Total oeetrogen, consisting of oestror ith smaller amounts of oestradiol and an unidentified, non-polar compound, were extracted with thy1 acerate and measured with a radioreceptor assay (RRA) based on the rat uterine oestrogen eceptor. Incubation of the cells with trypsin (BDH, Zmg/ml) followed by separate extraction of with the cells ~ he cells and medium showed that the trypsin-releasableoestrogen was associated (e.g. trypsin: 674237pg vs. cantrol: 552t21 pg oestradiol equiv./t06cells, meanfS.E, n*6, pCO.O! nd not the medium (376*90 and 386i34 pg respectively). Calcium ionophore CA231871 dibutyryl CM had no effect upon the extracted oesrrogen k nd phorbol ester (12-O-tetradecanoyl-l3-acetate) asurable by RRA. Treatment of the cells with the membrane disruptant, KSCN, (0.5M) caused a arge, highly significant increase in extractable oestrogen (659i38 to 3397zt355 pg, n=6, pCO.00 Fereater than caused by trypsin (1164f41 pg, n-6, p
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