Epidermal growth factor stimulates proliferation and DNA synthesis in ciliate cells

We studied the effect of murine epidermal growth factor on cell proliferation and DNA synthesis in macronuclei of ciliate Tetrahymena pyriformis G1. Mitogenic effect of epidermal growth factor on proliferation-induced tetrahymena cells has been revealed. This effect is due to the induced progression of cells at G1 and, consequently, their earlier entering DNA synthesis phase of the first cell cycle. Epidermal growth factor had no mitogenic effect on the resting cells from stationary culture (G0 phase) whose development is independent of the growth factors in the medium.     

Reactivation of stationary Tetrahymena : A contribution to the question of G0 state

Stationary cells of Tetrahymena were reactivated to exponential growth phase by transfer to fresh medium. The sequence of resuming cell cycle events was analysed by scoring the division index, the labelling index for macro- and micronuclei and the increase in cell number. By long-term labelling it was found that all cells replicate in stationary phase cultures. They also divide eventually. Upon transfer to fresh medium a small fraction of cells (about 3%) divide immediately, whereas the rest divide 3 h later after having replicated their macronuclear DNA. The kinetics of entry into the S phase indicates that these cells have a lag period of about 2 h before they resume progress through the cell cycle. It takes more than 1 h until all cells have begun replication. These data show that in stationary cultures all cells proceed through the events of the cell cycle. The cell cycle phases are extended differentially, G1 taking the largest part. During G2 cells pass very slowly through a certain stage close to division. Under the present conditions there is no indication for cells being in a resting state that is not part of the cell cycle, from which they can be restimulated and which has been called the G0 state. The criteria to demonstrate a resting state of this nature are discussed.     

Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. Quiescent cells contained very low levels of replication initiation factors, and their entry into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase.     

Epidermal Growth Factor Induces Proliferation and DNA Synthesis in Ciliate Cells

We studied the effect of murine epidermal growth factor on cell proliferation and DNA synthesis in macronuclei of ciliate Tetrahymena pyriformis Gl. Mitogenic effect of epidermal growth factor on proliferation-induced tetrahymena cells has been revealed. This effect is due to the induced progression of cells at G ₁ and, consequently, their earlier entering DNA synthesis phase of the first cell cycle. Epidermal growth factor had no mitogenic effect on the resting cells in a stationary culture (G ₀ phase) whose development is independent of the growth factors in the medium.     

Selective effects of interferon on distinct sites of the T lymphocyte triggering process

Lectin- and antigen-induced proliferation of murine T cells consists of two major events, namely, a rapid induction of susceptibility to growth factors and a later-occurring, accessory cell-dependent production of T cell growth factors (TCGF). The mechanism by which interferon (IFN) inhibits T cell responses was studied accordingly. A decrease of Con A-induced proliferation was observed in the presence of increasing amounts of IFN. The reduced proliferative response in such cultures was found to be due to an accumulation of cells in the G0/G1 phase of the cell cycle. Furthermore, the results show that IFN did not inhibit the early events in T cell triggering, because the acquisition of responsiveness of resting T cells to TCGF was unaltered in the presence of IFN, nor did it interfere with production of TCGF. In contrast, IFN was found to interfere with the TCGF-dependent T cell blast growth. Cytofluorometric analysis of the proliferative phase revealed that IFN exerts its effect on T cells, which have entered the proliferative cycle, by a postmitotic accumulation in G0/G1, thus reducing the proliferating population. The results demonstrate that IFN primarily affects the later phase of proliferative activity after T cell triggering, leaving the helper cell functions untouched.     

Cyclic GMP metabolism in relation to the regulation of cell growth in BALB/c3T3 cells.

BALB/c3T3 cell homogenates have guanylate cyclase in both 105000 g paniculate and soluble fraction. The activity in particulate fraction was much higher than that in the soluble fraction. Both enzyme activities were 2- to 3-fold greater in the resting state (G0 phase) than in the logarithmically growing state. In addition, cGMP-phosphodiesterase activity was 2-fold greater in resting than in growing cells. When G0-arrested cells entered into the G1 phase by serum addition, cGMP levels rapidly increased, whereas guanylate cyclase activities did not change within 30 min after serum addition. Four hours after serum addition, these activities had, however, decreased to one third and remained at that low level throughout the G1 phase. The relationship between cell growth and guanylate cyclase activity is discussed.     

T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro.

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

A Highlights from MBoC Selection: Trehalose Is a Key Determinant of the Quiescent Metabolic State That Fuels Cell Cycle Progression upon Return to Growth

When conditions are unfavorable, virtually all living cells have the capability of entering a resting state termed quiescence or G0. Many aspects of the quiescence program as well as the mechanisms governing the entry and exit from quiescence remain poorly understood. Previous studies using the budding yeast Saccharomyces cerevisiae have shown that upon entry into stationary phase, a quiescent cell population emerges that is heavier in density than nonquiescent cells. Here, we show that total intracellular trehalose and glycogen content exhibits substantial correlation with the density of individual cells both in stationary phase batch cultures and during continuous growth. During prolonged quiescence, trehalose stores are often maintained in favor over glycogen, perhaps to fulfill its numerous stress-protectant functions. Immediately upon exit from quiescence, cells preferentially metabolize trehalose over other fuel sources. Moreover, cells lacking trehalose initiate growth more slowly and frequently exhibit poor survivability. Together, our results support the view that trehalose, which is more stable than other carbohydrates, provides an enduring source of energy that helps drive cell cycle progression upon return to growth.     

CELL CYCLE COMPARTMENT ANALYSIS OF CHINESE HAMSTER CELLS IN STATIONARY PHASE CULTURES

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G₁ and G₂ portions of the cycle; the relative proportion of these cells in G₁ increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G₂ than in G₁. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.     

Enzymatic epoxidation: synthesis of 7,8-epoxy-1-octene, 1,2-7,8-diepoxyoctane, and 1,2-Epoxyoctane by Pseudomonas oleovorans.

The kinetics of the enzymatic formation of 7,8-epoxy-1-octene, 1,2-7,8-diepoxyoctane, and 1,2-epoxyoctane by growing and resting cell suspensions of Pseudomonas oleovorans are described. Formation of 1,2-epoxyoctane occurs concurrently with exponential growth on 1-octene, providing that 1-octene is in excess. Conversion of 1,7-octadiene to 7,8-epoxy-1-octene by cells growing on octane lags behind exponential growth and continues into the stationary phase, terminating upon cell death. Formation of 1,2-7,8-diepoxyoctane does not begin until the cells are well into the stationary phase and also continues until cell death. Results with growing and resting cell suspensions suggest that the various substrates compete for the same enzyme system; that viable cells are essential for substrate transport and epoxidation by whole cells; and that whole cells may concentrate and sequester the epoxides, rendering them unrecoverable by our current methods.     

Formation of the population composition of a stationary cell culture and the participation of nonproliferating cells with a doubled DNA content in regulating its density

The conditions of the cultivation of chick embryo diploid cells were alternated (prolonged maintenance with or without medium replacement, with or without consequent cell replating in fresh medium). In different times of culture growth, the cell DNA content was assessed by cytophotometry; the percentage of non-labeled mitoses after incubating the cells with 3H-thymidine and colcemide, as well as the cell density were determined. The phenomenon, detected earlier, of the accumulation of cells containing 4c DNA during the transition of the culture from logarithmic into the stationary phase of growth, was confirmed. These cells were shown to differ in their ability to survive in conditions of stationary culture and by proliferative potential. The fraction of cells reversibly arrested in G2-period was described, by which fraction the change of the cell population size is occurring after the decrease of its proliferation rate. The transitional stage is distinguished at the beginning of the stationary phase of culture growth. During this stage the stabilization of structural and numerical composition of the population is taking place.     

Lymphocyte cell-cycle analysis by flow cytometry. Evidence for a specific postmitotic phase before return to G0

We studied the cell cycle of lectin-stimulated human lymphocytes, making use of a flow cytometer. The RNA and DNA content of large numbers of individual cells was determined by supravital staining with acridine orange. The present study confirmed previous observations by others of a progression from G0 through G1 and S phase to G2/mitosis during the first 3 d in culture. It was also found that on subsequent days stimulated cells, before their return to G0, remained stationary in a state in which they contained the G0 complement of DNA and approximately twice the G0 complement of RNA. Cell-cycle manipulation with vinblastine and 5-bromo-2-deoxyuridine (BUdR) revealed that previous passage through both S phase and mitosis was required for entry into this newly observed late phase. In addition, there was high correlation (r = 0.973, P less than 0.001) between the number of cells in the late phase and measured [3H]thymidine uptake. It therefore appears that, in this system, stimulated cells remain in a distinct cell-cycle phase for a number of hours before their return to the resting state.     

Vitamin D receptor expression is linked to cell cycle control in normal human keratinocytes

To improve our understanding of the cutaneous vitamin D system, we studied vitamin D receptor (VDR) gene regulation in cultured human keratinocytes. Because VDR and its ligand 1 alpha,25-dihydroxyvitamin D(3) have been implicated in epidermal growth control, we investigated VDR expression as related to cellular proliferation by using different cell cycle synchronization protocols. Keratinocytes, deprived of growth factors, were forced into quiescence and a concomitant loss of VDR expression was observed. Mitogenic stimulation of these G(0) cells however quickly upregulated VDR levels several hours ahead the G(1)-S transition point. Growth arrest at the G(1)-S border by mimosine treatment or at the metaphase by nocodazole also downregulated VDR levels but a restoration of VDR expression was again quickly achieved after reentering the cell cycle. These findings indicate that VDR expression in keratinocytes is restricted to actively cycling cells, but not limited to one particular phase of the cell cycle.     

Inhibition of endothelial cell proliferation by platelet factor-4 involves a unique action on S phase progression

Modulation of endothelial cell proliferation and cell cycle progression by the "chemokine" platelet factor-4 (PF-4) was investigated. PF-4 inhibited DNA synthesis, as well as proliferation of endothelial cells derived from large and small blood vessels. Inhibition by PF-4 was independent of the type and the concentration of stimuli used for the induction of endothelial cell proliferation. Inhibition of cell growth by PF-4 was reversible. The effects of PF-4 were antagonized by heparin. Cell cycle analysis using [3H]thymidine pulse labeling during traverse of synchronous cells from G0/G1 to S phase revealed that addition of PF-4 during G1 phase completely abolished the entry of cells into S phase. In addition, PF-4 also inhibited DNA synthesis in cells that were already in S phase. In exponentially growing cells, addition of PF-4 resulted in an accumulation of > 70% of the cells in early S phase, as determined by FACS (Becton-Dickinson Immunocytometry Systems, Mountain View, CA). In cells synchronized in S phase by hydroxyurea and then released, addition of PF-4 promptly blocked further progression of DNA synthesis. These results demonstrate that in G0/G1-arrested cells, PF-4 inhibited entry of endothelial cells into S phase. More strikingly, our studies have revealed a unique mode of endothelial cell growth inhibition whereby PF-4 effectively blocked cell cycle progression during S phase.