ON THE RESTING PERIODS IN THE CELL LIFE CYCLE*

The review is concerned with the transition of cells into a specific physiological state—the resting period—in which they may stay for an indefinite time interval without undergoing division or differentiation but retaining both of these potentials. When stimulated such cells may enter into mitotic cycle, divide and differentiate. No direct correlation between the onset of the differentiated state and the transition of cells through the mitotic cycle has been established. It cannot be excluded that sometimes cells may differentiate directly from the resting period. However, there is a large body of evidence that the entry of cells into mitotic cycle is a necessary prerequisite for subsequent differentiation. The susceptibility of cells to differentiative stimuli is retained during the mitotic cycle. The completion of mitosis itself does not imply that a cell will undergo differentiation; in the absence of adequate stimulus it may pass again into a resting period. According to what is known at present it is suggested that cells may pass into a true resting stage not only after completing mitosis but also after doubling their DNA content. It is also conceivable that a cell may pass into a resting period at different stages of its life cycle. The essential feature of the cell life cycle is the alternation of resting periods and periods of active proliferation. This general principle of organization provides conditions necessary for population-size control, cell differentiation, interaction of a given population with other systems, and the reactions of cells to a changing environment.     

Mathematical description and analysis of cell cycle kinetics and the application to Ehrlich ascites tumor

The linear and nonlinear aspects of the dynamics of the cell cycle kinetics of cell populations are studied. The dynamics are represented by difference equations. The characteristics of cell population systems are analyzed by applying the model to Ehrlich ascites tumor. The model applied for the simulations of the growth of Ehrlich ascites tumor cells incorporates processes of cell division, cell death, transition of cells to resting states and clearance of dead cells. Comparison of the results obtained with the model and the experimental data suggests that the duration of the mean generation time of the proliferating EAT cells increases with aging of the tumor. An attempt is made to relate the prolongation of cell mean generation time with processes of cell death and dead cell clearance. Studying the transition of cells to the resting states, it becomes apparent that in fact transition of proliferating cells to the resting states occurs somewhere close to the end of the cell cycle and with a rate that varies with the age of the tumor. Time course behavior of the cell age, cell size, and cell DNA distribution with aging of the tumor are obtained. Variations in average size and average DNA contents are determined.     

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.     

Supernatant from a cloned helper T cell stimulates most small resting B cells to undergo increased I-A expression, blastogenesis, and progression through cell cycle

Helper T cell clone 52.3 supernatant (52.3 SN) was previously shown to be able to stimulate gradient-purified murine resting B cells in the absence of any additional stimulus. However, the proportion of cells that were accounting for the thymidine uptake and the Ig production was unknown. In this paper, we have studied induced changes that can be measured at the single cell level, and have thus determined the frequency of resting B cells that respond to 52.3 SN. Results indicate that 52.3 SN induces an increased I-A expression and a cell size enlargement on virtually all resting B cells. A significant proportion (30%) of these cells later becomes large blasts. Acridine orange staining revealed that in the presence of 52.3 SN a large fraction of the resting B cells undergoes the G0 to G1 transition. Furthermore, 52.3 SN is able to induce at least 20% of the cells to continue through the cell cycle into S phase as indicated by propidium iodide staining of DNA. Finally, a fraction of the 52.3 SN-stimulated cells differentiate to Ig-producing cells. Our present results suggest that resting B cells express functional receptors for some lymphokines and that these lymphokines can act in the absence of membrane Ig occupancy. Our findings further support the existence of a B cell-activating factor acting in a MHC-unrestricted manner and responsible for the entry of resting B cells into cell cycle. The relationship between this factor and other lymphokines is discussed.     

Successive relaxation cycles during long-time cell aggregate rounding after uni-axial compression

The mean features of cell surface rearrangement during cell aggregate rounding after uni-axial compression between parallel plates are considered. This is based on long-time rheological modeling approaches in order to shed further light on collective cell migration. Many aspects of cell migration at the supra-cellular level, such as the coordination between surrounding migrating cell groups that leads to uncorrelated motility, have remained unclear. Aggregate shape changes during rounding are considered depending on the size and homogeneity of 2-D and 3-D cell aggregates. Cell aggregate shape changes that are taking place during successive relaxation cycles have various relaxation rates per cycle. Every relaxation rate is related to the corresponding cell migrating state. If most of the cells migrate per cycle, the relaxation rate is maximal. If most of the cells are in a resting state per cycle, the relaxation rate is nearing zero. If some cell groups migrate while the others, at the same time, stay in a resting state, the relaxation rate is lower than that obtained for the migrating cells. The relaxation rates per cycles are not random, but they have a tendency to gather around two or three values indicating an organized cell migrating pattern. Such behavior suggests that uncorrelated motility during collective cell migration in one cycle induces a decrease of the relaxation rate in the next cycle caused by an accumulation of cells in the resting state. However, cells have the ability to overcome these perturbations and re-establish an ordered migrating trend in the next cycle. These perturbations of the cell migrating state are more pronounced for: (1) more mobile cells, (2) a heterogeneous cell population, and (3) a larger cell population under the same experimental conditions.     

Cell cycle controls in higher eukaryotic cells: Resting state or a prolonged G1 period?

We express the viewpoint that control over cell growth in higher eukaryotes is achieved predominantly by regular transition of cells from proliferation to rest and vice versa as a result of coordinated interrelationship between intracellular growth inhibitors and extracellular growth factors. The resting state is considered as a special physiological state of a cell where the prereplicative reactions necessary for the onset of DNA synthesis are inhibited. Cells pass into a resting state at each successive cell cycle, with regard to the next cycle, once the threshold level of growth inhibitors has been attained. Cellular rest may thus initiate and proceed in parallel with conventional periods of the cell cycle but in a hidden way. Its termination strictly depends on the appropriate concentration of extracellular growth factors. In the absence of growth factors cells, after completing mitosis, pass into an overt state of rest metabolically different from any period of the cell cycle including G1.     

Molecular biology of the cell cycle

Genes and cDNA clones have been identified in animal cells that are cell cycle-regulated, i.e. they are preferentially expressed in a phase of the cell cycle. Some of these genes, including four oncogenes, are induced when G0 cells are stimulated to proliferate. Four approaches are described to identify the genes that regulate the transition of cells from a resting to a growing stage. The interrelationship among cell cycle-regulated genes, oncogenes, growth factors and receptors for growth factors points the way to a genetic dissection of cell cycle progression.     

The proto-oncogene c-myc is a direct target gene of Epstein-Barr virus nuclear antigen 2

Epstein-Barr virus (EBV) infects and transforms primary B lymphocytes in vitro. Viral infection initiates the cell cycle entry of the resting B lymphocytes. The maintenance of proliferation in the infected cells is strictly dependent on functional EBNA2. We have recently developed a conditional immortalization system for EBV by rendering the function of EBNA2, and thus proliferation of the immortalized cells, dependent on estrogen. This cellular system was used to identify early events preceding induction of proliferation. We show that LMP1 and c-myc are directly activated by EBNA2, indicating that all cellular factors essential for induction of these genes by EBNA2 are present in the resting cells. In contrast, induction of the cell cycle regulators cyclin D2 and cdk4 are secondary events, which require de novo protein synthesis.     

Cognate interactions between helper T cells and B cells. IV. Requirements for the expression of effector phase activity by helper T cells.

After activation with anti-CD3, activated Th (THCD3), but not resting Th, fixed with paraformaldehyde induce B cell RNA synthesis when co-cultured with resting B cells. This activity is expressed by Th of both Th1 and Th2 subtypes, as well as a third Th clone that is not classified into either subtype. It is proposed that anti-CD3 activation of Th results in the expression of Th membrane proteins that trigger B cell cycle entry. Kinetic studies reveal that 4 to 8 h of activation with anti-CD3 is sufficient for ThCD3 to express B cell-activating function. However, activation of Th with anti-CD3 for extended periods of time results in reduced Th effector activity. Inhibition of Th RNA synthesis during the anti-CD3 activation period ablates the ability of ThCD3 to induce B cell cycle entry. This indicates that de novo synthesis of proteins is required for ThCD3 to express effector function. The ability of fixed ThCD3 to induce entry of B cell into cycle is not due to an increase in expression of CD3, CD4, LFA-1, ICAM-1, class I MHC or Thy-1. Other forms of Th activation (PMA and A23187, Con A) also induced Th effector function. Furthermore, purified plasma membranes from anti-CD3 activated, but not resting Th, induced resting B cells to enter cycle. The addition of IL-4, but not IL-2, IL-5, or IFN-gamma amplified the DNA synthetic response of B cells stimulated with PM from activated Th. Taken together these data indicate that de novo expression of Th surface proteins on activated Th is required for Th to induce B cell cycle entry into G1 and the addition of IL-4 is required for the heightened progression into S phase.     

Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. 3. Electrophoretic patters of newly synthesized proteins in synchronized proliferating cells and resting cells

Electrophoretic patterns of newly synthesized proteins have been compared for hamster embryo fibroblasts in asynchronous cultures, mitotically synchronized cultures, and stationary phase cultures. Only proteins with molecular weight between 30,000 and 150,000, comprising 60–70% of the total cell proteins and excluding histones and collagen were included in the comparison. Although no significant differences could be detected between such patterns for cells at different stages of the cell cycle, significant differences were detected between patterns for cells in stationary phase and for proliferating asynchronous or synchronous cells in any stage of the cell cycle. These differences amounted to at least 5% of the newly synthesized cellular proteins. Much larger differences were detected between patterns from a nuclear fraction of proliferating and resting cells. These results indicate that normal cells in stationary phase are arrested in a state distinct from any phase of the normal cell cycle and may provide a biochemical marker for resting cells.     

Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli

Resting CD4(+) T cells are the best-defined reservoir of latent human immunodeficiency virus type 1 (HIV-1) infection, but how the reservoir is formed is unclear. Understanding how the reservoir of latently infected cells forms is critical because it is a major barrier to curing HIV infection. The system described here may provide an in vitro model of latent HIV-1 infection in resting CD4(+) T cells. We demonstrated that HIV-1 integrates into the genomes of in vitro-inoculated resting CD4(+) T cells that have not received activating stimuli and have not entered cell cycle stage G(1b). A percentage of the resting CD4(+) T cells that contain integrated DNA produce virus upon stimulation, i.e., are latently infected. Our results show that latent HIV-1 infection occurs in unstimulated resting CD4(+) T cells and suggest a new route for HIV-1 reservoir formation.     

Resonance in periodic chemotherapy: a case study of acute myelogenous leukemia

The effects of periodic chemotherapy administration are evaluated within the context of a G(0)model of the cell cycle. Parameters are estimated for normal bone marrow cells and malignant cells in acute myelogenous leukemia (AML). This model explicitly includes the resting G(0)phase and the feedback mechanism that recruits the cells back into the cell cycle. Periodic chemotherapy administration can induce resonance within our model under high cell kill rate where the average cell cycle times may change during the course of treatment, and therapeutic benefits from these resonances cannot be solely based on cell cycle times in untreated tissue. The depletion rate under chemotherapy and the regrowth rate may differ between the cell populations, and our analysis suggests that this favors the tumour cells. We were able to distinguish between the effects of cycle-non-specific, S -phase-specific and M -phase-specific drugs, and found that these can show differences in sharpness and location of the resonance phenomenon. We conclude that resonance chemotherapy (chronotherapy) is unlikely to be efficacious in the treatment of AML.     

Induction of proliferation in vitro of resting human natural killer cells: IL 2 induces into cell cycle most peripheral blood NK cells, but only a minor subset of low density T cells

We report that resting human peripheral blood natural killer (NK) cells proliferate in response to recombinant interleukin 2 (rIL 2), and addition of irradiated lymphoblastoid B cells significantly increase their proliferative response. Interaction of IL 2 with the Tac IL 2 receptor expressed on activated NK cells is necessary to maintain continued growth of these cells. Experiments in which NK cell mitosis is prevented by colchicine show that the majority of peripheral blood NK cells are induced into the first cell cycle over a 6-day culture period in the presence of rIL 2. The addition of the irradiated lymphoblastoid B cell line, Daudi, to colchicine blocked cultures does not increase the proportion of cells entering cell cycle in response to rIL 2 alone. In limiting dilution analysis, only 1/1700 B73.1+ cells grow clonally in response to rIL 2. The frequency of clonal growth of NK cells in response to irradiated Daudi cells alone is minimal, whereas the addition of irradiated Daudi cells to rIL 2 stimulated cultures resulted in a 10-fold increase in clonal frequency compared with the cultures in rIL 2 alone. Therefore, Daudi cells may act by maintaining continuous proliferation of the NK cells originally responsive to IL 2. Unlike NK cells, only a minimal proportion of peripheral blood T cells proliferate in response to IL 2. These IL 2 responsive T cells are characterized by a lower bouyant density than the majority of peripheral blood T cells. These results indicate physiologic differences between peripheral blood resting NK and T cells in their ability to be induced to cycle. IL 2 is a growth factor for both cell types, but although the presence of the growth factor is sufficient for quiescent NK cells to be induced into cycle, T cells require antigenic or other mitogenic stimuli to respond to IL 2. The small proportion of light density IL 2 responsive T cells might represent in vivo activated T cells.     

Burnet oration. T-cell survival and the role of cytokines

Typical T cells are long-lived resting cells. Despite their quiescent appearance, there is increasing evidence that T cells are subjected to continuous stimulation through contact with various stimuli, notably by self peptide/MHC complexes and cytokines. These stimuli keep T cells alive and also cause intermittent entry into cell cycle.     

Relationship of Friend murine leukemia virus production to growth and hemoglobin synthesis in cultured erythroleukemia cells.

The factors that control oncornavirus formation were analyzed in Friend leukemia cells that undergo hematopoiesis when treated with dimethyl sulfoxide. Suspension cultures of Ostertag FSD-1 cell line were found to enter a G or resting state at the end of their proliferative phase and to simultaneously cease producing helper and dependent components of Friend virus. Whereas the decline in virus production is at least 100-fold, rates of cellular RNA and protein synthesis are only slightly lower in resting than in growing cells. Both resting and growing cells contain similarly large concentrations of the viral proteins P(30) and P(12). Dimethyl sulfoxide induces hemoglobin synthesis in growing cells, but its effects on virus production appear to be indirect results of its action to inhibit cell growth and thus to delay entry of cells into the G resting state. Furthermore, variant cell lines were obtained with differing abilities to synthesize virus or hemoglobin. Some lines no longer produce infectious virus, although they all harbor murine leukemia virus genes which are expressed to varying extents. The major internal protein of these oncornaviruses, P(30), is synthesized in large amounts by all of the cell lines. These results suggest that Friend virus production is not coinduced with erythroid differentiation, as had been proposed, but rather is controlled by a cellular growth cycle.     

Controls on the cell cycle

The control of cell proliferation, in physiological terms, depends not so much on our understanding of the sequence of biochemical events unfolding as a cell progresses through its proliferation cycle, as upon the recognition by a tissue of the demands for functional cells of a particular type. After considering the modes of control possible, i.e. by recruitment of resting G0-state cells into cycle or by modifying the proliferative behaviour of already proliferating cells, haemopoietic tissue is used as a model to illustrate how the principles of proliferation control in specific cell lineages can be effected. Although the mode of stem cell control is different from that in the maturing populations, all depend on a co-ordination of negative feedback loops for inhibitor and stimulator which are specific to that cell population. The concept of a 'quantal' cell cycle is considered but its application to control in an adult steady-state tissue must be modified to take account of microenvironmental influences which are shown, by their cellular organization, to be an important feature in haemopoietic and probably all other tissues.