Detection of casein messenger RNA in hormone-dependent mammary cancer by molecular hybridisation

Abstract

THE growth and development of the normal mammary gland are regulated by the complex interaction of both peptide and steroid hormones¹. Whereas some breast cancers retain the hormone-dependent characteristics of normal mammary tissue^2,3, other tumours loose this responsiveness and grow autonomously. Following the pioneering work of Huggins⁴ on the hormonal dependence of both experimental and human breast cancer, investigators seeking a prognostic test have studied the relationship of specific oestrogen receptors to mammary cancer^5,6 to the response of the tumour to endocrine ablation^7,8, and to the levels of other steroid⁹ and peptide hormone receptors^10,11. A superior marker for hormone responsiveness would, however, be a measurable product of hormone action rather than the initial binding interaction. In the mammary gland, casein synthesis has been used as a specific biochemical marker of differentiated function and hormone responsiveness. In the studies reported here, a specific complementary DNA copy (cDNA) of rat casein mRNA has been utilised to study the effects of hormones on the expression of differentiated function in chemical carcinogen-induced mammary aden-carcinomas. It is shown that casein mRNA as detected by cDNA may be a useful indicator of hormone dependence in experimental breast cancer. The cDNA probe was synthesised using as a template a 15S casein mRNA fraction purified 180-fold from a lactating RNA extract by chromatography on dT-cellulose and several precise sizing techniques^12,13. The template mRNA fraction represented greater than 90% casein mRNA, as estimated by specific immunoprecipitation of the total protein synthesised in the wheat germ assay, and by a careful analysis of its kinetics of hybridisation with the cDNA probe¹³. The specificity of the complementary DNA copy of the 15S casein mRNA has been demonstrated previously¹³. This cDNA probe selectively hybridised to RNA from lactating tissue but not to rat liver poly(A)-containinig RNA and the resulting hybrid displayed a high Tm characteristic of a well basepaired duplex. In addition, the rate of hybridisation of the casein-specific cDNA to various RNA preparations was directly related to the casein mRNA activity of these same preparations determined by a cell-free translation assay.