Atsuyuki Okuda

Genetic analysis of control of proliferation in fibroblastic cells in culture. I. Isolation and characterization of mutants temperature-sensitive for proliferation or survival of untransformed diploid rat cell line 3Y1

Mutants temperature sensitive for proliferation or survival were isolated from an untransformed diploid clone of fibroblastic rat cells (3Y1), according to an isolation protocol that selected for mutants defective at 38.5‡C (selection temperature) in undergoing the transition from quiescent to proliferating state while maintaining viability at 38.5‡ C. Of the 108 temperature-sensitive clones isolated, 32 were examined for survival in sparse cultures at 39.8‡ C (nonpermissive temperature) and classified into four classes. Results of temperature shift-up experiments suggest that functions defective in 11 of the 32 mutants are necessary not only for changing from the quiescent to proliferating state but also for maintenance of the proliferating state. Of the 32 mutants, 17 were assigned to eight complementation groups. Results of the physiological characterization of the representative mutants of each of the eight complementation groups are presented.

Abortive transformation of rat 3Y1 cells by simian virus 40: Viral function overcoming inhibition of cellular proliferation under various conditions of culture

Resting cultures of nonpermissive rat 3Y1 cells were infected with simian and T antigen expression and entry into S phase were examined under various conditions of culture. In the complete absence of serum from the medium or at an extremely high cell density, the cells delayed T antigen expression and entry into S phase, leaving the interval between the two events constant. Results using the viral mutants deleted in the coding region for the small t antigen ruled out the role of this antigen in induction of S phase. From these and other results presented, we conclude that the large T antigen induces S phase with the same efficiency under different conditions of cultures. We also present the evidence that the large T antigen function is required and is sufficient for entry into S phase in the second as well as in the first generation.

Control in previous and present generations of preparation for entry into S phase and the relationship to resting state in 3Y1 rat fibroblastic cells

In both the presence and absence of serum, 3Y1 rat fibroblastic cells synchronized at early S phase by aphidicolin entered M phase 6 h after removal of aphidicolin. However, in the second generation their entry into S phase in the presence of serum was delayed due to the deprivation of serum in the first generation. A similar delaying effect in the second generation was observed when the resting cells were stimulated by serum and then deprived of serum during a period of 8 h preceding mitosis. In both cases, the interval between mitosis and entry into S phase in the second generation was almost equal to that required for the resting cells to enter S phase when stimulated by serum. A similar delaying effect was also observed when the cells, synchronized at early S phase, were kept in suspension culture in the presence of serum for a period in the first generation. Results of a similar type of experiments using various combinations of growth factors showed that, when the G1 period in the second generation was shortened by exposure to growth factors in the first generation, and when the resting cells were stimulated to enter S phase, the same combination of growth factors was required. These and previous results suggest that the preparation for entry into S phase is controlled in both previous and present generations of 3Y1 cells.

The continuum model: An experimental and theoretical challenge to the G1 model of cell cycle regulation

The dominant and current view of cell cycle regulation is that the controls for cell proliferation reside primarily in the Gl phase of the division cycle. This current or classical model is deeply ingrained in cell biology, and has been described very well [ 1, 21; therefore we will only summarize below the basic support of the Gl model. Over the past decade, however, an alternative proposal, the continuum model, has emerged that suggests that there are no Gl-specific events or controls, and that all of the evidence for the importance of the Gl phase can be explained in terms of a continuous process that occurs in all phases of the division cycle [3-71. These early reports addressed previous experimental results and explained them in terms of the continuum model. In a sense, therefore, these analyses were theoretical rather than experimental. The basic ideas of the continuum model are presented in Fig. 1. Recently there has emerged a set of experiments that has been stimulated by the ideas of the continuum model, and these experiments support the basic ideas of the continuum model. This means that the continuum model has been a stimulus to experimental tests, and therefore has an experimental as well as a theoretical base. We will describe these experiments below, and explain how they support the continuum model.

Kinetic analysis of entry into S phase in resting rat 3Y1 cells stimulated by serum: Effects of serum concentration and temperature

The kinetics of entry into S phase after stimulation of resting 3Y1 cells by serum was examined in relation to serum concentration, temperature and the time at which the serum was withdrawn or at which the temperature was shifted. The kinetics of entry into S phase could be represented not only by a lag phase followed by a negative exponential curve (fit 1), but also by a normal distribution of the reciprocals of the time required for cells to enter S phase (velocities) (fit 2). As the temperature was lowered below 37 degrees C, the exponential slope decreased and the lag period increased (fit 1), and both the mean velocity and its standard deviation decreased (fit 2). As the serum concentration decreased below 10%, the exponential slope decreased without change in the lag period (fit 1), and the mean velocity decreased with increase in the standard deviation (fit 2). The cells which did not enter S phase within 8 h on removal of serum, stopped or delayed entry into S phase. In this case the lag phase was not changed (11 h). When serum was removed just before the end of the lag phase, no effect was seen on the kinetic curve. When the temperature was shifted at any time, including after the lag phase, the characteristics of the kinetic curve (lag phase, synchrony) changed. These facts indicate that there is a serum-non-requiring, but temperature-dependent period before S phase. Most of the asynchrony in entry into S phase under conditions of low serum seems to be generated during the serum-requiring period presumably by the random transition to the state in which cells are committed to enter S phase or by the variability of reaction rates at unpredictable times due to undeterministic effects.

Serum-dependent control of entry into S phase of next generation in rat 3Y1 fibroblasts: Effect of large T antigen of Simian virus 40☆

When rat 3Y1 fibroblasts are deprived of serum in S phase and/or G2 phase in the first generation, the cells delay entry into S phase in the second generation for the duration of the serum deprivation. We can now show that when resting 3Y1 cells are infected with Simian virus 40 (SV40), the removal of serum through S and G2 phases in the first generation does not markedly delay entry into S phase in the second generation. These observations suggest that the serum-dependent control of entry into S phase of the second generation continues from the first generation, and that the abolition of this control by infection with SV40 in the first generation involves the mechanism operative when the resting cells are stimulated to enter S phase (of the first generation) by infection with SV40.

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture.

SummaryA semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbeccos modified Eagles medium (DMEM) were insulin transferrin epidermal growth factor, poly-d-lysine, bovine albumin, oleic acid, and bovine α-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetat bovine serum (FBS), and colonies, albeit of smaller sizes, diddform. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium.When trypsinized 3Y1 cells were seeded with the medium lacking α-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding α-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.

Transient increase in the c-fos mRNA level after change of culture condition from serum absence to serum presence and after cycloheximide addition in rat 3Y1 fibroblasts

When 3Y1 cells resting at a saturation density were mitotically stimulated with serum, the c-fos mRNA level markedly increased in a short period of time and then decreased rapidly to an undetectable level. Subsequent serum deprivation followed by serum re-addition or subsequent cycloheximide addition caused a transient re-increase in the c-fos mRNA level. These results can be explained by assuming that the continuous expression of the c-fos gene at a minimum level is necessary for the eventual initiation of S phase, and that the over-expression of the c-fos gene occurs when the control of the gene expression is transiently disturbed by the change of the culture condition.

Increase in c-fos and c-myc mRNA levels in untransformed and SV40-transformed 3Y1 fibroblasts after addition of serum: Its relationship to the control of initiation of S phase

When rat 3Y1 fibroblasts were exposed to serum after 7.5 h of S, G2, and M phases in the absence of serum, the c-fos and c-myc mRNA levels markedly increased. This marked increase was also observed when density-arrested cells were stimulated with fresh serum to initiate proliferation. Increase in the c-fos and c-myc mRNA levels was not observed in cells that had traversed 7.5 h in these phases in the presence of serum. Cells passing through S, G2, and M phases in the absence of serum delayed entry into the next S phase approximately 8 h compared to control cells incubated in the presence of serum. Also, when density-arrested cells were stimulated with serum for 5 h, then deprived of serum for 8 h, and then incubated in serum again, the c-fos and c-myc mRNA levels increased. In this last case, the total excess time of serum exposure required to enter S phase was only 2 h, indicating that cells had not returned to the initial density-arrested state during the serum deprivation period. The increase in c-fos and c-myc mRNA levels following addition of serum after incubation in the absence of serum was also observed in SV40-transformed 3Y1 cells. The entry of SV40-transformed cells into S phase was not markedly affected by the absence of serum. These results can be explained by assuming that there is a process leading to the initiation of S phase that is operating or accumulating continuously in all cell cycle phases. In 3Y1 cells the expression of the c-fos and c-myc genes is required at any cell cycle phase, and the increase in c-fos and c-myc mRNA levels in response to changes in serum concentration simply reflects the possible overexpression due to the delay of a hypothesized negative feedback regulation. In SV40-transformed 3Y1 cells, the process leading to the initiation of S phase operates normally in response to growth factors, and the SV40 large T antigen supplements or enhances the process in the absence of the growth factors.

Cell-cycle independent increase in heat production in response to growth factors in cultured rat fibroblasts: Interpretation by continuum model

We measured the heat output from rat 3Y1 fibroblastic cells by stopped-flow method using a flow microcalorimeter. When the resting cells were stimulated to initiate DNA synthesis with growth factors, the heat output increased. Although cells normally progressed through S and G2 phases in the absence of any growth factor, cells increased the heat output in response to the growth factors during the progression through these phases. These results are consistent with the continuum model in which the preparation for the initiation of S phase occurs continuously and cumulatively between adjacent S phases not restricted in G1 phase.