In the past project period it was demonstrated that TGFbeta1 rapidly suppresses expression of the protooncogene c-myc at the level of transcriptional initiation in keratinocytes, and that expression of c-myc is necessary for proliferation of these cells. We have identified the TGFbeta control element (TCE) in the human c-myc promoter and further shown that the product of the retinoblastoma tumor susceptibility gene (pRB) is probably necessary for TGFbeta suppression of c-myc, at least in certain cell types. Preliminary data indicate that the tumor suppressor, p53, can also regulate c-myc transcription. Studies on carcinoma cell lines have shown that abnormalities in the rb gene correlate with the loss of the growth inhibitory response to TGFbeta1. Also in the past project period, studies with MMTV-TGFbeta1 transgenic mouse lines have shown a phenotype of inhibition of end-bud growth and branching morphogenesis. Additional studies have shown that focal administration or local overproduction of TGFbeta1 can cause increased connective tissue formation, including angiogenesis, and immunosuppression. Studies with MMTV-TGFalpha transgenic lines in another laboratory have demonstrated that misregulation of TGFalpha in breast epithelial cells leads to hyperplasia and an increased incidence of spontaneous and DMBA-induced tumorigenesis. Based on this and other data, it is hypothesized that in early steps of mammary carcinogenesis, over-expression of TGFalpha promotes, and over-expression of TGFbetas retards or suppresses, the carcinogenic process. With mutations in the carcinoma cells that result in loss of TGFbeta growth inhibition, endogenous TGFbetas then accelerate tumor progression through paracrine effects on stroma, including angiogenesis, and local immunosuppression. It is further hypothesized that the specific alterations that can cause loss of the growth inhibitory response to the TGFbetas are rb deletions, p53 mutations, and/or c-myc misregulation. These hypotheses will be tested through the following specific aims: (1) Continued studies on determining the role of pRB and p53 in the TGFbeta pathway for suppression of c-myc transcription and growth inhibition; characterizing a newly isolated cDNA clone encoding a protein that specifically binds the TCE; and characterizing the effect of p53 on c-myc transcription. (2) Transfection studies to explore the effect of c-myc, pRB and p53 on TGFbeta1 growth inhibition and the effect of TGFbeta1 over-expression in selected carcinoma cell lines. (3) Transgenic mouse studies on development and susceptibility to tumor formation resulting from misregulation of TGFbeta1S223,225, including crossbreeding of homozygous MMTV-TGFalpha and MMTV-TGFbeta1S223,225 lines to determine effect of transgene coexpression on mammary tumor formation; examining the effect of expression of TGFalpha and TGFbeta1S223,225 under the control of the whey acidic protein (WAP) promoter; crossbreeding MMTV-myc and MMTV- TGFbeta1S223,225 or WAP-myc and WAP-TGFbeta1S223,225 transgenic mice to determine effects on breast tumor formation; and experiments with cytokeratin and metallothionein promoters to determine the effects of misregulating TGFbeta1S223,255 on different tissues.
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