This proposal addresses the role of the human ST5 gene in signal transduction pathways and malignant transformation. First identified as a differentially regulated gene in a system of tumorigenic and non- tumorigenic HeLa/fibroblast somatic cell hybrids, further characterization suggests a specific interaction of ST5 gene products with signal transduction proteins and a marked down regulation of one of the three ST5 transcripts in a subset of human cancers. The three RNA species, 4.6, 3.1, and 2.8 kb in length, encode proteins of 126, 82, and 70 kDa, respectively. Alternative splicing accounts for the 4.6 and 3.1 kb mRNAs, which differ by 1260 nt present only in the larger message. The 2.8 kb species initiates at a distinct promoter located within an intron of the ST5 gene. In the HeLa/fibroblast system, only the non- tumorigenic hybrids express the 2.8 kb species. Analysis of several benign and malignant human epithelial cell lines suggests that down regulation of this message occurs frequently in human cancers.
Specific Aim #1 addresses the mechanism of this regulation. This analysis will employ a transient expression assay in which the regulatory elements of the internal promoter, cloned into a chloramphenicol acetyltransferase expression vector, are transfected into tumorigenic and non-tumorigenic cell lines. A mutagenic analysis of these regulatory elements will be performed to define the specific sequence elements responsible for specific regulation in nontumorigenic cells. Gel retardation assays and protein purification methods will be used to define the specific factors involved in regulation and how they are altered in the tumorigenic cells.
Specific Aim #2 involves an analysis of the role of the ST5 gene products in signal transduction and cancer. The 420 amino acids unique to the 126 kDa protein contain two proline rich domains with a total of seven potential SH3 binding motifs. A preliminary experiment demonstrates binding of ST5 to an isolated SH3 domain. These observations suggest the hypothesis that interaction with SH3, a common structural feature of signal transduction proteins, constitutes an important part of the in vivo function of this protein. Experiments designed to address this hypothesis will employ a filter binding assay to characterize the specific cellular proteins capable of interacting with the ST5 gene products. This information will then be applied to a series of transfection experiments which will analyze the biological consequences of the ST5-SH3 interaction. An observation derived from an antiserum generated against a portion of the ST5 protein forms the basis of Specific Aim #3. Western blot analysis with the affinity purified antiserum revealed aberrant protein bands in three cervical carcinoma cell lines. The expression of ST5 in cancers will be pursued further with a panel of antisera generated against several additional segments of the ST5 protein. This analysis will involve the use of RNA and protein blots to analyze expression in cancer cell lines and immunohistochemistry to study expression in sections of paraffin embedded tumor tissue.
|Majidi, M; Gutkind, J S; Lichy, J H (2000) Deletion of the COOH terminus converts the ST5 p70 protein from an inhibitor of RAS signaling to an activator with transforming activity in NIH-3T3 cells. J Biol Chem 275:6560-5|
|Hubbs, A E; Majidi, M; Lichy, J H (1999) Expression of an isoform of the novel signal transduction protein ST5 is linked to cell morphology. Oncogene 18:2519-25|
|Majidi, M; Hubbs, A E; Lichy, J H (1998) Activation of extracellular signal-regulated kinase 2 by a novel Abl-binding protein, ST5. J Biol Chem 273:16608-14|
|Lichy, J H; Majidi, M; Elbaum, J et al. (1996) Differential expression of the human ST5 gene in HeLa-fibroblast hybrid cell lines mediated by YY1: evidence that YY1 plays a part in tumor suppression. Nucleic Acids Res 24:4700-8|