When subconfluent, Swiss 3T3 cells made quiescent by serum deprivation are stimulated with low concentrations of serum (ca. 1%), only a proportion of them (roughly 50%) enter S phase despite daily replacement with fresh, low-serum medium. The cells that fail to enter S phase are not incapable of doing so, since most of them initiate DNA synthesis after transfer to 10% serum. It would appear that individual cells vary in their growth factor requirements. Using time-lapse cinemicroscopy a few of the cells that respond to low serum were seen to give rise to several generations of progeny, while the majority of cells failed to divide at all, or divided once at most. Despite this, differences between cells in growth factor requirements do not seem to be heritable in the long term, since attempts to enrich for responding cells by prolonged culture in 1% serum have been unsuccessful. Rather, it would appear that the capacity to respond to low serum is an unstable property lost after a few generations in low serum. The loss of responsiveness shows parallels with "cellular senescence" and could conceivably result from decay of the platelet-derived growth factor-induced state of "competence." But regardless of why some cells respond to low serum while others do not, it is clear that the kinetics of entry into S phase after serum stimulation of quiescent 3T3 cells are not strictly first-order, since the labelling index plateaus after roughly 3 days at values substantially below 100%. As such, the kinetics, though not contradicting the transition probability model, cannot be taken to support it as was previously thought.

Epidermal growth factor (EGF) as well as prostaglandin F2 alpha (PGF2 alpha), when added to quiescent, confluent Swiss 3T3 cells, stimulate the initiation of DNA synthesis, which occurs with apparent first-order kinetics after a lag phase of 14-15 hr. These two growth factors appear to stimulate similar events; insulin enhances and hydrocortisone can inhibit the stimulatory effect of either. Here we show that the addition of EGF and PGF2 alpha together, however, results in a synergistic effect seen at the end of the lag phase, but only when EGF and PGF2 alpha are added within 6 hr of each other. Addition of one growth factor 10 or 15 hr after the other delayed the synergy for 15 hr after the addition of the second growth factor. Insulin further increased the rate of entry into the s phage stimulated by EGF and PGF2 alpha together, whereas hydrocortisone inhibited the stimulatory effect observed with either EGF or PGF2 alpha alone. These results suggest that, in spite of the common events responsible for the interactions with the two hormones, EGF and PGF2 alpha must have differences in their sequences of events that initiate DNA synthesis.

Fibroblast Growth Factor (FGF) stimulates quiescent Swiss 3T3 cells to initiate DNA synthesis and divide. Cells begin to enter the S-phase after a lag of 13--15 hr, and the rate of initiation of DNA synthesis in the population can be quantified by a first order rate constant, k. A subsaturating concentration of FGF may establish the lag phase, while the value of k is dependent on the FGF concentration present during the second half of the lag phase. Insulin and hydrocortisone enhance the effect of FGF by increasing k without changing the lag phase, and they can act when added at any time after FGF. Prostaglandin E1 (PGE1) causes a decrease in k and a lengthening of the lag phase, and acts only when added during the first 8 hr. None of these agents stimulate DNA synthesis in the absence of FGF.

Pairs of normally incompatible derivatives of R100-1 (one Chls TetR, the other ChlRTets) were forced to coexist in a recA host by selection for ChlR TetR cells. After many generations stable derivatives were isolated. The analysis of nine independent stabilization experiments showed that in each case TetR was translocated from the plasmid to the chromosome of the host. No evidence for the joint integration of other plasmid genes (those controlling transfer, antibiotic resistance, incompatibility, or origin of transfer replication) was obtained. One of the chromosomal TetR determinants was mapped close to metE.

Confluent, quiescent Swiss 3T3 cells in culture can be stimulated to initiate DNA synthesis and divide by addition of growth factors to the culture medium. Here we show that hydrocortisone and other steroids which have glucocorticoid activity inhibit the stimulation of these cells by epidermal growth factor (EGF) in contrast to their reported enhancement of stimulation by fibroblast growth factor (FGF). Binding studies using [3H]-triamcinolone acetonide show that Swiss 3T3 cells contain a single class of glucocorticoid receptor of uniform affinity (KD = 2.0 nM), and about 34,000 receptor sites per cell. Those steroids which displace bound [3H]-triamcinolone acetonide are also effective in inhibiting the stimulation of DNA synthesis by EGF in the presence or absence of insulin, and the concentration of triamcinolone acetonide required for one-half maximal biological effect is in the same range as the KD. A similar concentration is required for one-half maximal enhancement of the effect of FGF. These results suggest that both the inhibitory and stimulatory effects of glucocorticoids may be mediated via these receptors, the different effects thus being due to differences in the intracellular events triggered by each growth factor.