The control of differentiation and proliferation in normal stem cells is complex and is mediated by both positive and negative signals. The expression of defects in the coupling or integrated control of cellular differentiation and proliferation are of etiological significance in neoplastic transformation. Carcinogenesis is a multistep process that involves the development of defects in a variety of regulatory mechanisms in the affected cell. In some cases, the autonomy of cancer cell growth is known to be caused, at least in part, by the aberrant production and response to growth factors, i.e., autocrine stimulation of proliferation. Recent studies suggest that one of the potential mechanisms by which differentiation is abrogated or inhibited in neoplastic cells is the autocrine secretion of differentiation-inhibiting factors. These studies have shown that transforming growth factor type-beta (TGF-beta) and cachectin/tumor necrosis factor (TNF), both of which are known to be anomalously secreted by some tumor cells, are potent inhibitors of distinct steps of differentiation in 3T3 T mesenchymal stem cells as well as other cell types. We hypothesize that the development of some cancers and their inability to differentiate properly involves the aberrant production and response to differentiation-inhibiting factors as well as growth factors. We propose to further define the mechanisms of carcinogenesis by determining how the activation of autoregulatory growth and differentiation control mechanisms contribute to aberrant proliferation and/or differentiation in cancer cells. First, we will determine if TGF-beta or cachectin/TNF exert their effect by inhibiting the expression of known differentiation-specific genes. Second, the aberrant expression of TGF-beta and cachectin/TNF will be characterized in nontransformed cells with single differentiation defects or fully transformed cells with cannot differentiate. Third, the potential existence of other differentiation-inhibiting factors will be determined by testing serum-free conditioned media from these same cells for the ability to inhibit differentiation of the parental 3T3 T cells. In addition, the autocrine production of growth factors will be characterized to provide a better understanding of autocrine differentiation and/or proliferation defects in cancer. Differentiation-specific gene expression, TGF-beta and cachectin/TNF expression, and growth factor expression will be studied by Northern blot analysis, Western blot analysis, or by the measurements of activity present in conditioned medium. Neutralizing antibodies are available for some of these factors and will be used to block their actions on the secreting cells and verify autocrine activity. We anticipate obtaining information which will provide new insight into the mechanisms regulating growth and differentiation control of normal and tranformed cells.
