Abstract
In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.
Highlights
In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (FaslodexTM, fulvestrant)
Characterization of T47D and T47D-r—T47D-r were generated from the antiestrogen-sensitive human breast cancer cell line T47D after continuous cultivation in presence of 10Ϫ10 M
The proliferative response of T47D and T47D-r to E2 and increasing concentrations of the antiestrogen ZM 182780 was measured with the MTT assay (Fig. 2)
Summary
In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (FaslodexTM, fulvestrant). We established an antiestrogen-resistant breast cancer cell line as an in vitro model system in order to analyze differentially expressed genes and proteins. To this end, the human ER␣-positive breast carcinoma cell line T47D, which is growth-stimulated by 17-estradiol (E2) and sensitive to antiestrogens, was exposed to the pure antiestrogen ZM 182780 at a concentration of 10Ϫ6 M over a period of several months. From the ‡Research Laboratories of Schering AG, 13342 Berlin, Germany; and ¶Protein Chemistry Group, Max-Delbruck-Center for Molecular Medicine, 13092 Berlin, Germany
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