The primary objectives of this research are A) to characterize the protein corresponding to a newly discovered calmodulin-like gene transcript, and B) to learn why expression of the gene is downregulated after transformation of human mammary epithelial cells (HMEC). This gene, designated NB-1, was recently clones using a probe enriched by subtractive hybridization for sequences preferentially expressed in a normal HMEC strain in comparison to a closely matched tumorigenic HMEC derivative. Evidence that NB-1 expression is often depressed in primary breast tumor tissues and that NB-1 transcripts are undetectable in tumor derived cell lines supports the possibility that loss of NB-1 expression is an important step in tumorigenic transformation of breast epithelial cells. The 85% amino acid sequence similarity between the NB-1 protein and calmodulin suggests that NB-1 encodes a Ca+2 binding protein with signal transduction properties. However, calmodulin is expressed at relatively constant levels in most cells, whereas NB-1 exhibits tissue specificity, regulation by TGF-beta and extracellular matrix, and absence in transformed cells. Our working hypothesis is that NB-1 protein functions as a regulatory molecule analogous to calmodulin, but that the range of proteins to which it binds and regulates differs from that of calmodulin. In order to gain a better understanding of the function and regulation of NB-1, a number of biochemical and molecular biological studies will be performed: 1) newly developed NB-1 specific antibodies will be used to localize NB-1 protein in cultured cells and tissues, 2) recombinant NB-1 protein will be used to identify proteins to which NB-1 protein binds and regulates, 3) human breast cell lines expressing transfected NB-1 genes will be compared with control transfectants to examine effects of NB-1 expression on tumor associated properties, 4) thioated antisense oligonucleotides will be used to block specific translation of NB-1 mRNA in normal HMEC, 5) recombinant NB-1 protein will be assayed in vitro for its ability to perform known calmodulin functions, 6) in vivo gene complementation studies will be performed in fungi, 7) transcription assays and promoter assays will be performed in order to identify NB-1 regulatory elements which function differently in normal and transformed HMEC. The experiments proposed will begin to address the role of NB-1 in the coordination of growth and differentiation in normal breast epithelium. Calcium mobilization is known to be a critical determinant of both of these processes in epithelial cells. Disruption of Ca+2 mediated events is also one of the earliest detectable alterations in some carcinogen exposed epithelial cells. Downregulation of NB-1 expression observed after in vitro or in vivo transformation of HMEC may contribute to or be required for the pathology of breast carcinomas.
| Edman, C F; George, S E; Means, A R et al. (1994) Selective activation and inhibition of calmodulin-dependent enzymes by a calmodulin-like protein found in human epithelial cells. Eur J Biochem 226:725-30 |
