This is an application for continuation of a grant entitled """"""""Transcriptional Mechanisms of Carcinogenesis"""""""" which studies how alterations in transcription factor oncogenes contribute to transformation and tumorigenesis in mouse and human osteosarcomas. Although human cancers involve multiple steps and develop over considerable time, acute transforming viruses accelerate carcinogenesis and rapidly produce multiple tumors. Our work has focused on the transforming gene of the acute transforming viruses FBR, which produces multifocal osteosarcomas with an abundant collagen matrix. FBR v-fos was transduced from the c-fos proto-oncogene but has markedly different transcriptional and cellular functions than does c-fos. Our molecular dissection of transcriptional differences between v-fos and c-fos demonstrated a key role for myristylation of FBR v-fos protein. FBR v- fos is a gag-fos fusion and the myristylation occurs at the N-terminal glycine which is derived from the viral gag protein. Myristylation of FBR v-fos produced a loss of the usual c-fos transcriptional functions (AP-1 transactivation and SRE transrepression) but resulted in induction of new genes such as Type III collagen. This findings links a mutation in a transforming oncogene with transcriptional regulation of a gene which alters the tumor phenotype, producing a collagen matrix. This FBR- mediated induction of Type III collagen involves a differentiation- dependent mechanism which may be regulated by C/EBP. FBR v-fos also immortalizes primary embryo fibroblasts which may be mediated through interactions with Rb protein, since our studies demonstrate that FBR v- fos protein binds Rb protein in vitro. Studies of bone tumor induction in mice have shown that myristylated FBR v-fos produced bone tumors with abundant collagen matrix, but that mutant non-myristylated FBR v-fos does not produce tumors (despite equivalent expression and in vitro transformation efficiency). We hypothesize that FBR v-fos accelerates osteosarcoma induction by multiple, pleiotropic functions which simultaneously result in multiple events or """"""""hits"""""""" including: transformation, immortalization, and induction of a """"""""protective"""""""" collagen matrix. We further hypothesize that these events mimic the mutations found in human osteosarcomas by FBR v-fos binding to key tumors suppressor gene products. To test these hypotheses we propose the following specific aims: 1. Determine the mechanisms which induce collagen III transactivation by FBR v-fos; 2. Determine the mechanisms which normally repress collagen III transcription during adipocyte differentiation; 3. Analyze the potential functional role of collagen matrix induction by FBR v-fos during mouse osteosarcoma induction; 4. Determine the mechanisms which control immortalization by FBR v-fos. The ultimate goal of these studies determining specific molecular mechanisms of FBR v-fos induced osteosarcoma is to identify and study transcriptional events such as collagen III induction and immortalization which modulate the phenotype, contributing to tumorigenesis.

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National Cancer Institute (NCI)
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Chemical Pathology Study Section (CPA)
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Vanderbilt University Medical Center
Anatomy/Cell Biology
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United States
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Robinson-Benion, C; Jensen, R A; Holt, J T (2000) Analysis of cancer gene functions through gene inhibition with antisense oligonucleotides. Methods Enzymol 314:499-506
Abbott, D W; Thompson, M E; Robinson-Benion, C et al. (1999) BRCA1 expression restores radiation resistance in BRCA1-defective cancer cells through enhancement of transcription-coupled DNA repair. J Biol Chem 274:18808-12
Abbott, D W; Holt, J T (1999) Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation. J Biol Chem 274:2732-42
Abbott, D W; Freeman, M L; Holt, J T (1998) Double-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cells. J Natl Cancer Inst 90:978-85
Abbott, D W; Holt, J T (1997) Finkel-Biskis-Reilly osteosarcoma virus v-Fos inhibits adipogenesis and both the activity and expression of CCAAT/enhancer binding protein alpha, a key regulator of adipocyte differentiation. J Biol Chem 272:32454-62
Abbott, D W; Holt, J T (1997) Finkel-Biskis-Reilly mouse osteosarcoma virus v-fos inhibits the cellular response to ionizing radiation in a myristoylation-dependent manner. J Biol Chem 272:14005-8
Jotte, R M; Holt, J T (1996) Myristylation of FBR v-fos dictates the differentiation pathways in malignant osteosarcoma. J Cell Biol 135:457-67
Robinson-Benion, C; Holt, J T (1995) Antisense techniques. Methods Enzymol 254:363-75
Jotte, R M; Kamata, N; Holt, J T (1994) Myristylation-dependent transactivation by FBR v-fos is regulated by C/EBP. J Biol Chem 269:16383-96
Robinson-Benion, C; Li, Y X; Holt, J T (1994) Gene transplantation: combined antisense inhibition and gene replacement strategies. Leukemia 8 Suppl 1:S152-5

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