Retinoids (RA) are potent differentiating agents, with significant potential both as chemopreventive and antineoplastic agent of breast cancer. We have established and characterized a series of novel estrogen (E2)-independent breast cancer cell line variants in which to identify the critical factors associated with RA-induced growth inhibition of E2- responsive and -unresponsive breast cancer. This was essential, since E2 also induces genes/proteins unrelated to growth regulation, and our analyses would be complicated by the effect of RAs on irrelevant E2- regulated genes in E2-dependent cells. Using these variants, we have identified genes/proteins closely associated with RA-induced growth inhibition. We applied both a 2-dimensional gel electrophoretic analysis of cellular homogenates and the RT-PCR """"""""differential display"""""""" of RNA obtained from treated and untreated cells. These analyses identified RA- induced (i.e. TRI-1, TRI-2) and RA-repressed genes (i.e. TRR-1), and proteins that are induced/repressed/modified by RA treatment, including Hl-10/numatrin (E2-regulated, protein kinase C and cdc2 kinase substrate), TRA-1 (Mr 35 kDa/pI 4.6), TRA-2 (Mr 55 kDa/pI 5.1), TRA-3 (Mr 58 kDa/pI 5.3). We also detected autoantibodies to HI- l0/numatrin in the sera of breast cancer patients. Based on these preliminary data, we hypothesize that PAs regulate the expression of specific genes associated with the inhibition of cell proliferation of human breast cancer cells, some of which also may be F2-regulated. We further hypothesize that TRI-1,TRI-2, TRR-1, HI-10/numatrin, TRA-1, TRA- 2, TRA-3 are at least partly responsible for mediating these effects. We also hypothesize that these RA-regulated genes/proteins will be expressed/repressed/modified in human breast tumors, and that their patterns of expression will predict response/resistance to RA therapies, and provide critical prognostic information Specifically, we will: (i) Determine the dose and time dependent regulation of TRA-regulated genes/proteins by RAs (e.g. 9-cis-retinoic acid; 4-hydroxyfenretinide). (ii) Obtain the full length cDNAs for the TRA-regulated genes/proteins. Full length cDNAs from PCR products will be obtained by cDNA library screening and/or """"""""rapid amplification of cDNA ends"""""""". (iii) For proteins, micro-sequence data will be obtained, and the cDNAs isolated by cDNA library screening using degenerate oligonucleotide probes or specific antisera. (iv) We will generate reagents (synthetic peptides, antisera, oligonucleotide probes) for screening human breast cancer biopsies and cell lines for expression. Oligonucleotides and synthetic peptides will be synthesized in the LCRC Sequencing and Synthesis Core Facility, and polyclonal antisera will be generated by the LCRC Animal Core Facility. (v) We will screen human breast cancer biopsies using either specific antibodies (immunohistochemistry), riboprobes (Northern/RNase protection), or oligonucleotide primers (RT- PCR), and sera for HI-10/numatrin autoantibodies by ELISA and/or Western. (vi) We will determine the nature of the interactions among RAs and antiestrogens using isobologram analyses.
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