We are interested in understanding how cell adhesion, and integrins in particular, control the proliferation of cells. This adhesion requirement is a classic feature of most nontransformed cell types, and loss of adhesion control (termed """"""""anchorage-independent growth) correlates closely with tumorigenicity in animals. Using the anchorage-dependent NRK and NIH- 3T3 fibroblasts as model systems, we have previously shown that the adhesion requirement for proliferation can be explained in terms of a discrete cell cycle transition that maps to late G1 and results, at least in large part, from the adhesion-dependent expression of cyclin A. We also find that TGF-beta induces anchorage-independent growth of responsive fibroblast cell lines by overriding the adhesion requirement for G1/S transit specifically. These data, taken together with the well-established stimulatory effects of TGF-beta on matrix protein and integrin synthesis, suggest that the subcellular effects of cell adhesion and TGF-beta might overlap. Indeed, the preliminary results in this application show that NRK cells pretreated with a beta1-integrin antisense oligonucleotide lose their ability to undergo anchorage-independent growth in response to TGF-beta. This result, along with others described in this application, demonstrate that the stimulatory effect of TGF-beta on 1 integrin synthesis mediates the stimulatory effect of TGF-beta on anchorage-independent growth. They also strongly suggest that at least one member of the alphabeta1 integrin family is involved in the adhesion effect on cyclin A expression and G1/S transit. This application is focused on identifying the specific integrin(s) that mediate the effects of TGF-beta and adhesion on the cell cycle.
In aim 1, we will prepare antisense oligonucleotides to specific integrin subunits within the alphabeta1 integrin family.
In aim 2, we will use these antisense oligonucleotides to modulate the effect of TGF-beta on surface integrin expression in nonadherent NRK fibroblasts. The induction of G1/S transit will be monitored in parallel, and the combined results will allow us to identify the specific heterodimer(s) that mediate the stimulatory effect of TGF-beta on anchorage-independence.
In aim 3, a similar antisense approach will be used to identify the specific integrin(s) that regulate cyclin A expression and G1/S transit in adherent cells.
In aim 4, we will prepare stable antisense transfectants to confirm the results obtained with antisense oligonucleotides and extend the key observations to other cell lines. Together, the results from these studies will allow us to establish the roles of specific alphabeta1 integrins in cell cycle control of anchorage-dependent and -independent growth.
