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Oestrogen and insulin interact at the level of transcription in breast cancer cells.

‘the growth of the human breast cancer cell line, MCF-7, is stimulated by oestrogen and inhibited by antioestrogens both in vitro and in vivo [1,2]. MCF-7 cells may therefore serve as a model of those human breast tumours that are responsive to endocrine therapy. In addition to oestrogen receptors, MCF-7 cells possess receptors for, and respond to, a wide range of other hormones and growth factors [3]. Insulin is a mitogen for MCF-7 cells and synergises with oestradiol in stimulating cell growth [4]. We have investigated the interaction between insulin and oestradiol in regulating the expression of two oestrogeninducible genes, LIV-1 [5] and pS2 [6], in MCF-7 cells. MCF-7 cells were withdrawn from oestradiol by culturing them in phenol red-free medium, supplemented with dextrad charcoal treated serum, for 6 days. Withdrawn cells were then cultured in medium containing oestradiol(10-8111) with or without insulin (lpg/ml). Cells were harvested after 3 ,6 ,24 or 48 hours exposure to the hormondagents. LIV-1 and pS2 mRNA levels were determined by Northern blot [7]. The results are expressed as fold increase relative to the mRNA levels detected in untreated (control) cells. Nuclei were isolated from cells and the rates of LIV-1 and pS2 transcription were assayed using the run-on, in vitro transcription technique [7]. The resultant autoradiographs were quantified by densitometry and the LIV-1 and pS2 results were normalised relative to the actin (uninduced control) result. Results are expressed as fold increase with respect to the rate of transcription in untreated (control) cells. No hybridisation to the negative controls (rhodopsin, pBR322, pBluescript) was detected. Figure 1 presents the results from three such experiments. LIV1 and pS2 mRNA levels show a maximum increase in response to oestradiol and to oestradiol+insulin at 6hrs after hormone addition (Fig. l(a)). LIV1 mRNA was induced 1 1.3 fold by oestradiol alone and 26.1 fold by oestradiol+insulin. pS2 mRNA was induced 14.1 fold by oestradiol and 29.4 fold by oestradiol+insuhn. The mRNA levels declined only slowly from their 6hr maximum and remained at >70% of maximum by 48hrs. At all time points the oestradiol+insulin mRNA levels were 1.5 2.0 times that seen with oestradiol alone. It should be noted that the concentration of oestradiol used (10-8M) induces an optimal increase in LIV-1 and pS2, in the absence of insulin [7]. Evidently, induction by insulin occurs by a different but complementary mechanism to that of oestradiol. When the rate of LIV-1 and pS2 transcription was determined it was Seen that a maximal increase was reached by 3hrs after hormone addition (Fig.l(b)). LIV-1 transcription exhibited a 6.1 ( 9 . 5 ) fold increase in response to oestradiol and a 13.7 (9.4) fold increase in response to oestradiol+insulin. pS2 transcription was stimulated 8.6 (9.3) fold by oestradiol and 19.9 h0.3) fold by oestradiol+insulin. Transcription rate declined more rapidly from its 3 hr rate and by 24 hrs was <15% of maximum, in all cases. The more prolonged elevation of the mRNA levels must therefore reflect the stability of the messenger RNAs rather than their continued synthesis. Once again, insulin is able to produce a significantly (p< 0.001) greater increase in transcription than that induced by the optimal concentration of oestradiol alone. Our results show that the relatively long-lasting induction of LIV1 and pS2 mRNA by oestradiol and by oestradiol+insulin, results from a much briefer alteration in the rate of gene transcription. The reson for the rapid decline in transcription rate after 3 hrs is unclear but presumably results from a decrease in the amount or activity of the oestrogen receptor [9]. The different but complementary mechanisms of transcriptional stimulation used by oestradiol and insulin both require the oestrogen receptor. This example of cross-talk between two very different intracellular signalling systems is of interest in its own 30