In the previous report (Porter et al ., in this issue) morphological changes in Chinese hamster ovary (CHO) cells during the cell cycle were described . In this report we describe the role of intercellular contact on these changes . We find that intercellular contact is required for cells to exhibit the morphologies Porter et al . described for S and G2 . When cells are synchronized by mitotic selection and plated onto cover slips at very low density such that no intercellular contact occurs, the cells remain in a G I configuration (rounded and highly blebbed through G1, S, and G 2) . This GI morphology is also observed in nonsynchronized log phase cells plated at low densities and allowed to grow for several generations . The addition of conditioned medium from confluent cultures does not induce low density cells to change morphology during the cell cycle . These results indicate that extensive intercellular contact is required for the complete expression of the morphological changes associated with the cell cycle (as described by Porter et al .) . It is concluded that although classic contact inhibition of movement and of growth may be absent in this transformed cell line, some contact-dependent response persists.

Cultured Chinese hamster ovary cells showed no significant change in generation time or fraction in the S-phase in the presence of 1 mM N6,02'-dibutyryl adenosine 3': 5'-cyclic monophosphate. Growth continued for at least two generations after expression of the morphological transformation induced by this cyclic AMP analog. Despite identical growth rates, apparent rates of DNA and RNA synthesis (incorporation of [IHluridine) were reduced up to 15-fold in log phase by 1 mM cyclic nucleotide. PIHiDeoxycytidine incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [,;H]thymidine demonstrated an inhibition of transport rate dependent on the concentration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 10 was decreased 21-fold by 1 mM cyclic nucleotide; half-maximal inhibition occurred at 6 MM. At 370, the pool size of acid-soluble thymidylate was strongly reduced by 1 mM cyclic nucleotide, and synergistic reduction of the pool size was found with 0.5 mM aminophylline. Phosphorylation of the acidsoluble intracellular label was unaffected by dibutyryl cyclic AMP. Inhibition of thymidine uptake is attributed to an observed decrease in thymidine kinase activity caused by growth in 1 mM dibutyryl cyclic AMP, and possibly to a simultaneous alteration in membrane permeability. Kinase-facilitated uptake of other metabolites may be regulated in a similar fashion by cyclic AMP. Adenosine 3':5'-cyclic monophosphate has proven to be a very common regulatory substance in biological systems. Its diverse functions include regulation of transcription in bacteria (1), intercellular communication in slime molds (2), and mediation of hormone action in mammalian tissues (3). Recently, cyclic AMP has been implicated in the control of cell growth and differentiation. Hsie and Puck (4) were among the first to suggest that cyclic AMP might control cellular differentiation, and the concept has been expanded by studies on macrophage and granulocyte cells (5) and mouse-adrenal tumor cells (6). The dibutyryl cyclic AMP (Bu2cAMP)-induced morphological transformation of Chinese hamster ovary (CHO) cells (4, 7) is coincident with greatly increased collagen production (7). Sheppard (8) clearly showed that spontaneously-and virally-transformed mouse cell lines could be restored to contact-inhibited growth by addition of Bu2cAMP and theophylline to the medium. Otten et al. (9) found an inverse relationship between growth rate and endogenous levels of cyclic AMP in 12 mouse-fibroblast cell lines. The involvement of cyclic AMP and Bu2cAMP in growth control is interesting in light of the observed alterations that they induce in plasma membrane properties. Bu2cAMP causes a large decrease in the agglutinability of mouse fibroblasts by wheat-germ agglutinin (8), and increased adhesion to plastic surfaces (10). We studied the effect of Bu2cAMP on DNA synthesis in CHO cells; our attention soon focussed on metabolite transport, because it appeared that this process was most severely affec...

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