Hirokazu Zaitsu

Serum-dependent regulation of proliferation of cultured rat fibroblasts in G1 and G2 phases

We reported that: (i) 3Y1tsF121 cells, a temperature-sensitive (ts) mutant of rat 3Y1 fibroblasts, are reversibly arrested either in the G1 or in the G2 phase, at the nonpermissive temperature. (ii) Cells retain the ability to resume proliferation at the permissive temperature after prolonged arrest in the G1 phase (for 5 days), whereas they lose it after prolonged arrest in the G2 phase (over 24 h). (iii) The G1 arrest is overcome at the nonpermissive temperature by the addition of fresh serum (H. Zaitsu and G. Kimura (1984) J. Cell. Physiol. 119, 82; (1985) J. Cell. Physiol. 124, 177). In the present study, the G2 arrest was overcome by exposing the cells to fresh serum, at the nonpermissive temperature. The G2 arrest occurred only at a higher cell density than that of the G1 arrest. The efficiency of the overcome was higher in the case of the G2 arrest than in case of the G1 arrest. When cells synchronized at the G1/S border by aphidicolin at the permissive temperature were released from the block, they divided in the absence of serum, at the permissive temperature. Even if they had passed through the previous G2 phase in a very high concentration of fresh serum at the permissive temperature, mitotic cells did not enter the S phase in the absence of serum, even at the permissive temperature. When the cells arrested in the G1 phase (not in G0) due to the ts defect were incubated in the absence of serum at the permissive temperature, only 34% entered the S phase and only 15% divided. These results suggest that (i) the ts defect in 3Y1tsF121 limiting cellular proliferation in both the G1 and the G2 phases is probably due to a single mutational event, and is a serum-requiring event. (ii) Preparation of the serum-requiring event which is required for the G2 traverse is completed in the G1 phase, under ordinary conditions. (iii) However, cells are able to fulfill the serum-requiring event in the G2 phase as well as in the G1 phase when the preparation is below the required level. (iv) The commitment to DNA synthesis is not necessarily a commitment to cell division. (v) Cells are arrested in the G1 phase more safely and more effectively than in the G2 phase, by the serum-related mechanism.

Elongation and shortening of time required for entry into S phase after release from G 1 and G 0 arrests in temperature-sensitive mutants of rat 3Y1 Cells

Temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts representing four separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly in the G1 phase when cells of randomly proliferating population at 33.8 degrees C are shifted to 39.8 degrees C (temperature arrest). We examined the time lag of the cellular entry into the S phase after release at 33.8 degrees C, both from the temperature arrest and from the arrest at 33.8 degrees C at a confluent cell density (density arrest). In the temperature-arrested cells, as the duration of temperature arrest increased, the time lag of entry into S phase after shift down to 33.8 degrees C was prolonged, in all four mutants. These observations suggest that the four different functional lesions, each causing arrest in the G1 phase, are also responsible for prolongation of the time lag of entry into the S phase in cells arrested in the G1 phase. The prolongation of the time lag in the temperature-arrested cultures was accelerated at a higher cell density, in medium supplemented with a lower concentration of serum, and at a higher restrictive temperature. In the density-arrested cells, as the duration of pre-exposure to 39.8 degrees C was increased, the time lag of entry into S phase at 33.8 degrees C after release from the arrest was drastically prolonged, in all four mutants. In 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203, when the density-arrested cells were prestimulated by serum at 39.8 degrees C for various periods of time, the time lag of entry into S phase after release from the density arrest at 33.8 degrees C was initially shortened, and then, prolonged progressively as the period of prestimulation increased. These findings, taken together with other data, show that all four ts defects affect cells in states ranging from the deeper resting to mid- or late-G1 phase. It is suggested that events represented by these four mutants are required for entry into the S phase and normally operate in parallel but not in sequence in cells in states ranging from the deeper resting to the mid- or late-G1 phases, though they may affect each other.

Simian virus 40 compensates a cellular mutational defect of a serum-dependent function controlling cell cycle progression in the G2 phase

Rat 3Y1tsF121 fibroblasts are arrested in the G2 phase at the nonpermissive temperature due to a temperature-sensitive (ts) defect, and the G2 arrest is overcome at the nonpermissive temperature by the addition of a large dose of fresh serum. When the G2-arrested cells which had been exposed to the nonpermissive temperature for 12 hr were shifted down to the permissive temperature, most divided within 12 hr. When the cultures prepared in parallel were infected with simian virus 40 (SV40) at the nonpermissive temperature, the G2-arrested cells divided as early as 6 hr after the expression of T antigen. The G2-arrested cells, which had been exposed to the nonpermissive temperature for 36 hr, lost both the ability to restore the G2 and M traverse at the nonpermissive temperature after the addition of fresh serum and the reversibility of the arrest upon shift down to the permissive temperature. However, SV40 induced these cells to divide at the nonpermissive temperature, as in the case of the reversibly arrested cells. A small t-antigen-deletion mutant (dl-884) also induced both types of the G2-arrested cells to divide at the nonpermissive temperature. These results suggest that (1) SV40 compensates or activates, in the G2 phase, the function regulating G2 and M transition by serum; (2) SV40 induces restoration of the irreversible G2 arrest; and (3) small t antigen is not responsible for these activities of SV40.

Deeper ‘GO’ Phase in Rat Fibroblasts Affects the Lag Time Required for T Antigen Induced DNA Synthesis, but not the Lag Time Required for SV40 T Antigen Expression

In density-arrested rat 3YltsG125 cells, the time required for entry into S phase after serum stimulation is prolonged with increase in duration of the arrest. When these cells were infected with simian virus 40, the time required for expression of T antigen did not change, but the time required for entry into S phase was prolonged with increase in duration of the arrest. The extent of prolongation of the interval between T antigen expression and entry into S phase correlated well with the extent of the prolongation detected with serum stimulation. These observations suggest that: (1) expression of T antigen was not affected by the level of cellular preparedness for entry into S phase, i.e., it did not depend on cellular functions directly involved in the preparedness, and (2) T antigen induced cellular DNA synthesis, at least in part, by a mechanism similar to that of serum-induced DNA synthesis.