Activated ras oncogenes are involved in the formation of many types of malignant tumors in animals, and have also been linked to human carcinogenesis. The ras gene products are 21 kD proteins located at the inner surface of the cell plasma membrane. These proteins bind to guanine nucleotides and hydrolyze GTP to GDP. Thus, ras is believed to be involved in signal transduction mechanisms within cells. One approach to identifying such signal transduction pathways is to identify a physiological endpoint of the pathway, and use the end product to biochemically and/or genetically define other parts of the pathway. We have chosen the transcriptional activation of genes by ras in transformed cells as one physiological consequence of oncogene action. A seven base pair oligonucleotide, TGACTCT has been identified in both mouse and human genes as a ras-responsive enhancer element. A nuclear factor that specifically binds to the consensus sequences, termed RRF1, is found in both mouse and human transformed cells. RRF1 has an apparent molecular weight of 120,000 daltons. Several lines of evidence demonstrate that RRF1 is not the product by the c-fos and c-jun proto-oncogenes. The data indicate that ras activation of transcription is distinct from gene activation by phorbol esters, in that c-fos and c-jun protein products are not required for activation. In this proposal, we present plans for the continuation of this work. The ras-responsive nuclear factor will be further characterized, and the gene encoding this factor will be cloned. Experiments are proposed to define how, at the molecular level, the transcription factor is activated in ras-transformed cells. Finally, experiments are proposed to allow the identification and analysis of the component of the signalling pathway immediately upstream of RRF1.
The Specific Aims of this proposal are: 1. To characterize the ras-responsive nuclear factor (RRF1) from transformed cells. DNA-affinity chromatography is one technique that will be used to this end. 2. To understand how this nuclear factor is activated in ras-transformed cells. Biochemical assays for determining how RRF1 is activated will be set up with crude extracts from transformed cells. RRF1 purified by DNA affinity chromatography (or recombinant RRF1, see below) can be used to fine tune such assays. 3. To identify and purify the component of the signalling pathway immediately upstream of RRF1. Understanding how RRF1 is activated will provide an in vitro assay for purification of the upstream component. Standard techniques will be used to purify the signalling pathway component. 4. To clone the gene encoding RRF1. The correct gene will encode a protein that recognizes the sequence TGACTCT or AGACTCT with higher affinity than other AP1-related sequences (e.g., TGAGTAA) in both in vitro binding reactions and in transfection assays. The RRF1 gene product can be overexpressed in prokaryotic cells once the gene is cloned. 5. To test the role of RRF1 in transformation of cells by ras. The cloned RRF1 gene will be mutated so that constitutive trans-activators and dominant-negative effectors are obtained. These can be used to determine the role of the gene in ras transformation.
| Mathsyaraja, H; Thies, K; Taffany, D A et al. (2015) CSF1-ETS2-induced microRNA in myeloid cells promote metastatic tumor growth. Oncogene 34:3651-61 |
| Wallace, Julie A; Li, Fu; Balakrishnan, Subhasree et al. (2013) Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer. PLoS One 8:e71533 |
| Bronisz, A; Godlewski, J; Wallace, J A et al. (2012) Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320. Nat Cell Biol 14:159-67 |
| Wallace, Julie A; Li, Fu; Leone, Gustavo et al. (2011) Pten in the breast tumor microenvironment: modeling tumor-stroma coevolution. Cancer Res 71:1203-7 |
| Baran, Christopher P; Fischer, Sara N; Nuovo, Gerard J et al. (2011) Transcription factor ets-2 plays an important role in the pathogenesis of pulmonary fibrosis. Am J Respir Cell Mol Biol 45:999-1006 |
| Trikha, Prashant; Sharma, Nidhi; Opavsky, Rene et al. (2011) E2f1-3 are critical for myeloid development. J Biol Chem 286:4783-95 |
| Zabuawala, Tahera; Taffany, David A; Sharma, Sudarshana M et al. (2010) An ets2-driven transcriptional program in tumor-associated macrophages promotes tumor metastasis. Cancer Res 70:1323-33 |
| Wang, Hui; Karikomi, Matt; Naidu, Shan et al. (2010) Allele-specific tumor spectrum in pten knockin mice. Proc Natl Acad Sci U S A 107:5142-7 |
| Acharyya, Swarnali; Sharma, Sudarshana M; Cheng, Alfred S et al. (2010) TNF inhibits Notch-1 in skeletal muscle cells by Ezh2 and DNA methylation mediated repression: implications in duchenne muscular dystrophy. PLoS One 5:e12479 |
| Godlewski, Jakub; Nowicki, Michal O; Bronisz, Agnieszka et al. (2010) MicroRNA-451 regulates LKB1/AMPK signaling and allows adaptation to metabolic stress in glioma cells. Mol Cell 37:620-32 |
Showing the most recent 10 out of 36 publications
