Biochemical Society Transactions ( 1 992) 20 345s Expression of an oestrogen rebulated mRNA in breast tumour tissue. PATRICIA A. BURKE and CHRIS D. GREEN* School of Engineering and Science, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, U.K. *Department of Biochemistry, University of Liverpool, P.O.Box 147, Liverpool L69 3BX, U.K. It is estimated that 15,000 women die each year in the United Kingdom from breast cancer [1,2]. However, patient survival rates can be increased if the cancer is diagnosed and treated during the early stages of development. Traditionally, surgery is the preferred course of breast cancer treatment, however, breast tumours do have a pronounced tendency to metastasize forming secondary tumours at distinct sites within the body[3]. It is these metastases which can often prove fatal, since in general, their growth cannot be controlled by surgical methods and systemic treatment is required (Tamoxifen, an anti-oestrogen treatment is used in the management of breast metastases). Since the growth of approximately one third of breast tumours is hormonally regulated, a large number of breast cancers fail to respond to Endocrine therapy [4,5]. Attempts therefore, to isolate a reliable breast tumour marker for use in screening is still of paramount importance. In recent years a number of hormonally regulated mRNA species (pS2[6], oestrogen receptor(ER), progesterone receptor(PR)) have been isolated and have proven to be of some use in breast cancer screening [7,8]. In our laboratory a previously unidentified mRNA species was isolated from the breast cancer cell line ZR-75-1 [9]. This 367bp fragment (pLIVI) hybridizes to three mRNA species (4.4kb, 3.7kb and 2.3kb) in ZR-75-1 and MCF-7 breast cancer cell lines [lo]. In this study we used hybridization techniques to investigate the tissue specificity of pLlVl induction and to assess its suitablity as a breast tumour marker. Northern and dot blot analysis were performed on RNA samples (10-2Ougs) extracted from human breast tumour tissue, Rat liver, human placenta and a number of cell lines [ll]. Filters were hybridized sequentially, to a spectrum of radiolabelled probes (pS2, pLlV1, ER, 8-Actin) for 8-12 hrs at 65°C. Standard washing conditions were used, which included several low stringency washes (6-3 x SSPC/ 0.1% SDS) at room temperature and 42"C, followed by several washes at high stringency (2-0.1 x SSPC/ 0.1% SDS, 65OC). Each wash was followed by autoradiography at -70°C.

-4kb

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2 3 4

5 6 7 8 9 1011121314 Tumour Samples 3 -14

Fig. 1. Hybridisation of the pLIVI probe to a 4.4kb mKNA species in breast tumour tissue. Samples 1 and 2 are the Rat Liver and Human Placental RNA Controls respectively. Hybridisation conditions are described in the text.

The characteristic 3 band pattern observed when pLIVI hybridizes to RNA from oestrogen regulated breast cancer cell lines [ l o ] , was observed in 60% of the breast tumour samples screened. Surprisingly, a complete loss of pLIVI signal was experienced by the two lower molecular weight species (3.7 and 2.3kb) following a high stringency wash. (Fig.1) It is this latter observation which is difficult to explain, since in our results pLIVI would appear to share >95% sequence homology with each RNA species expressed in breast cancer cell lines, and a similar degree of homology with the major 4.4kb species found in some breast tumour tissue. However, the two minor RNA species detected in some breast tumour tissue would appear to have

Expression of an oestrogen regulated mRNA in breast tumour tissue.

Biochemical Society Transactions ( 1 992) 20 345s Expression of an oestrogen rebulated mRNA in breast tumour tissue. PATRICIA A. BURKE and CHRIS D. GR...
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