We present a model to account for several major observations on growth control of animal cells in culture. This model is tested by means of kinetic experiments which show that exponentially growing animal cells whose ability to synthesize total protein has been inhibited with cycloheximide (by up to 70%) grow at rates approximately proportional to their rates of protein synthesis. However, virtually the entire elongation of the cell cycle occurs in the part of the GC phase that depends on a high concentration of serum in the medium. This part of the cycle has earlier been suggested to lie prior to the restriction point-i.e., the point beyond the main regulatory processes of G1. The remainder of the cycle, from restriction point to mitosis, is markedly insensitive to these concentrations of cycloheximide as well as to growth regulation. We quantitatively account for the specific lengthening of that part of the cycle involved in growth regulation by assuming that cells must accumulate a specific protein in a critical amount before they can proceed beyond the restriction point. The lability of this protein (half-life about 2 hr) makes its accumulation unusually sensitive to inhibition of total protein synthesis by cycloheximide. Its production appears to depend on growth factors provided by serum. The model can also account for greater variations of G1 durations as the growth of cell populations is made slower. It also predicts two sorts of quiescence: one of cells slowly traversing GC, in slightly suboptimal conditions; the other of cells that enter Go under inadequate conditions. Transformation of different sorts could create cells with altered variables for initiation, synthesis, or inactivation of the regulatory protein or could altogether eliminate the need for the protein.The proliferation rate of animal cells is largely determined by the relative times that they spend in the cell cycle as opposed to quiescence. In each cycle an initiation event is required to start another round of the cell cycle; in its absence cells enter a quiescent state in which they remain until initiation occurs (1). Growth appears to be determined by the cyclic occurrence of this initiation event; completion of the cycle is relatively unaffected by conditions of growth (2). Similarly, for bacterial growth (3) and for synthesis of nucleic acids and proteins, control is by frequency of initiation; subsequent events proceed at constant rates.We have defined completion of the growth-controlling event in the cell cycle as the restriction point R (1). When a growing culture (of 3T3 mouse cells) is shifted to a condition (medium containing 0.5% serum) that does not support continued growth (4, 5), R can be calculated from the fraction of cells that cannot leave the G1 phase to lie about 2 hr before the beginning of DNA synthesis (6). Work with chicken cells earlier indicated that R lies somewhere in mid-GC (7).The role of R point control is accentuated by studies of cells transformed to tumorigenicity by DNA tumor viruses (8-10). Wh...
The synthesis of both cytoplasmic and nuclear proteins has been studied as quiescent, serum-deprived Swiss mouse 3T3 cells are stimulated to transit the cell cycle. In serum-arrested cells a 200,000 dalton cytoplasmic protein and a 51,000 dalton nuclear protein were found to be preferentially synthesized. In serum-stimulated cells the first major protein whose synthesis was seen to increase had a molecular mass of 42,000 daltons. This protein also showed the greatest change in synthesis during the transit from Go to S phase. Its synthesis rose to a maximum 4-6 hr after stimulation and then declined as cells entered S phase. The protein was present in both nuclear and cytoplasmic extracts. It was identified as actin on the basis of its mobility on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels. Other proteins synthesized preferentially by stimulated cells had molecular masses of 57,000 daltons (cytoplasmic), 33,000 daltons (cytoplasmic and nuclear), and 15,000 daltons (nuclear). The synthesis of the 57,000 and 33,000 dalton proteins increased gradually after stimulation and remainec high during S phase. The 15,000 dalton proteins began to be synthesized as cells entered S phase. The preferential synthesis of these proteins provides biochemical markers for the transition from quiescence to proliferation.Changes in biochemical properties during progression of the cell cycle have been widely described (1-3). However, the detection of marker proteins specific for different phases of the cell cycle is sorely lacking. Such markers would greatly facilitate more detailed analysis of biochemical events associated with the cell cycle.Crucial X 15 mm culture dishes in 25 ml of medium. On the following day the cultures were shifted into medium containing 0.5% serum and the cells were allowed to grow in this medium for [38][39][40][41][42][43][44][45][46][47][48] hr. Cells were stimulated to transit from Go to S by replacing the 0.5% serum medium with medium containing 20% serum. In order to monitor the entry of cells into S, cells were plated in 150-mm culture dishes containing coverslips and arrested in Go by serum deprivation as described above.[3H]-Thymidine at 0.5 ,uCi/ml (1 Ci = 3.7 X 1010 becquerels) was then included in the 20% serum medium used to stimulate the Go to S transit. Coverslips were processed for autoradiography (12) at the indicated times.Cultures were labeled for 2-hr periods with [3H]isoleucine (105 Ci/mmol; 20 or 40 ,uCi/ml) or ['4C]isoleucine (296 mCi/ mmol; 5 ,Ci/ml) in medium containing 1% (1 mg/liter) of the normal Dulbecco's medium isoleucine concentration. The cultures were then washed three times with cold phosphatebuffered saline and the cells were scraped off the plate with a rubber policeman. Cells were fractionated into cytoplasmic and nuclear fractions by using the method of Becker and Stanners (13). The nuclei were pelleted by a 2-min centrifugation at 600 X g. The supernatant was centrifuged at 250,000 X g for 30 min in a Beckman L5-65 ultracentrifuge (SW 50....
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