Cell anchorage is required for the proliferation of almost all cell types and loss of this requirement (induction of anchorage-independence) is a hallmark of cell transformation. Among the growth factors isolated from normal tissue, TGF-beta is outstanding in its ability to induce anchorage-independent growth. However, this transforming effect of TGF-beta is restricted to a few fibroblastic cell lines (e.g. NRK cells become anchorage-independent in response to TGF-beta whereas NIH- 3T3 and normal human fibroblasts do not). Perhaps for this reason, the relationship between TGF-beta biology and anchorage-independence has never been resolved. Our data now indicate that induction of anchorage-independent growth represents an important aspect of TGF-beta pathology. Specifically, we find that (I) TGF-beta fails to induce anchorage-independent growth in cells that retain their normal adhesion requirement for expression of cyclin D1 and (ii) loss of this control--although non-transforming in itself-- renders cells susceptible to transformation by TGF-beta. Thus, loss of adhesion-dependent cyclin D1 expression is a prerequisite for transformation by TGF-beta. We now propose four specific aims to identify the molecular effect(s) induced by TGF-beta and determine how TGF-beta action complements the effect of constitutive cyclin D1 expression to induce anchorage-independence.
In aim 1, we will examine cell cycle progression from G0 to S phase to identify the subset of adhesion-dependent G1-cdk events that are stimulated by TGF-beta during induction of anchorage-independent growth: parallel studies will determine if TGF-beta has the same subcellular effect(s) on control cells that fail to undergo anchorage-independent growth.
In aim 2, we will ectopically express TGF-beta mediated cell cycle events to determine which of its effects are causal for TGF-beta mediated anchorage-independence. Finally, in aims 3 and 4, we will characterize the E2F-independent mechanism that regulates the adhesion-dependent expression of cyclin A and determine the effects of TGF-beta on E2F-dependent and E2F-independent cyclin A gene expression during induction of anchorage-independent growth.
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