Inhibition of DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes.

Abstract

3T3 plasma membranes were added to subconfluent cultures of SV3T3 cells in the presence of fusogens. If this protocol results in the introduction into the SV3T3 cell membrane of 3T3 plasma membrane components responsible for density-dependent inhibition of growth, then the SV3T3 cell cultures would be expected to show decreased rates of DNA synthesis as they approach confluence. Results of these experiments indicate that rates of DNA synthesis in SV3T3 cultures so treated were as much as 63% less than in untreated controls. This effect could not be attributed to the fusogens or to the 3T3 plasma membranes alone. This growth-inhibitory effect is specific for 3T3 membranes and is not observed when SV3T3 plasma membranes are fused with SV3T3 cell cultures. These data support the hypothesis that one aspect of the loss of densitydependent inhibition of growth in SV3T3 cells is a deletion or alteration in plasma membrane components and, further, that density-dependent inhibition of growth can be in part restored to SV3T3 cell cultures by fusing the cells with 3T3 plasma membranes.Inhibition of DNA synthesis has been observed in many cultured cell lines as those cultures attain monolayer confluence (1-3). Although the mechanism of this density-dependent inhibition of growth has never been fully elucidated, experiments with wounding have indicated that cell-cell contact is one requirement (4, 5). More recently, Whittenberger and Glaser (6) have shown that DNA synthesis can be inhibited in actively growing 3T3 cell cultures by the addition of isolated 3T3 plasma membranes. The effect was specific for 3T3 cells and was not seen when membranes were added to SV3T3 cell cultures. Those authors also showed that a similar inhibition could not be achieved by the addition of plasma membranes isolated from transformed SV3T3 cells.Subsequent research has shown that the growth-inhibiting effect of 3T3 membranes is probably due to one or more integral membrane proteins (7). Whittenberger et al. (8) and Raben et al. (9) have also reported the partial purification and characterization of a similar 3T3 plasma membrane protein capable of inhibiting growth in subconfluent cultures.The loss of density-dependent inhibitior~ of growth in SV3T3 cell cultures may be due to a deletion or alteration of the plasma membrane growth-inhibiting protein(s). Using a modification of a fusion technique developed by Poste and Nicholson (10), we have tested this possibility by fusing 3T3 plasma membranes to actively growing subconfluent SV3T3 cell cultures. If this fusion procedure results in the introduction of the normal complement of membrane proteins into the SV3T3 cell membrane, then a restoration of densitydependent inhibition of growth in the transformed cell culture might be expected to occur. We report here the results of such experiments. Up to 63% inhibition of DNA synthesis occurs as the fused SV3T3 cells reach confluence.These findings suggest that an alteration in membrane proteins contributes to the loss of density-dependent inhib...

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