Brief exposures of resting fibroblasts to okadaic acid stimulate DNA synthesis.

To study further the factors providing for cellular quiescence, we used okadaic acid (OA) at concentrations (0.1, 1, 10 or 100 nM) inhibiting type 1 and/or type 2A protein phosphatases in mammalian cell cultures. Brief (2 h) exposure of resting (0.2% serum for 72 h) NIH 3T3 mouse fibroblasts to OA with subsequent incubation of cells in a medium with 0.2% serum, stimulated DNA synthesis at all concentrations studied. Maximal stimulation was observed following pre-incubation of resting cells with 10 nM OA. Treatment of cycling cells (10% serum) with OA (2 h pulses at 12 h intervals for 72 h) prevented their exit to the resting state on transfer to a medium with 0.2% serum. Brief exposures of resting cells to OA did not affect the rate of protein synthesis. OA pulses in the late pre-replicative period had no effect on the entry of serum-stimulated cells into the S phase. Cell fusion experiments with resting (serum-deprived) and proliferating (serum-stimulated) NIH 3T3 cells, using radioautography with a double-labelling technique, revealed that pre-incubation of resting cells with OA for 2 h before and after fusion abrogates their ability to suppress the onset of DNA synthesis in the nuclei of proliferating cells in heterodikaryons. The results indicate that protein phosphatases of type 1 and/or 2A may be involved in the growth-arrest machinery that provides for cellular quiescence.

Transient inhibition of protein synthesis induces expression of proto-oncogenes and stimulates resting cells to enter the cell cycle.

There is evidence that resting cells are able to produce molecules with antiproliferative activity, some of which behave as short-lived repressor proteins. We suggest that transient inhibition of protein synthesis in resting cells would lead to a decrease in the levels of these negative growth regulators and might, therefore, promote mitogenic responses. We report that treatment of resting (serum-deprived) NIH 3T3 cells with cyclocheximide (CH) or puromycin induces expression of c-fos, c-jun and c-myc proto-oncogenes in a manner similar to that of platelet-derived growth factor (PDGF). Actinomycin D (Act D) abrogates the induction of proto-oncogene expression. Transient inhibition of protein synthesis by CH or puromycin also induces the resting NIH 3T3 and C3H 1OT1/2 cells to enter the cell cycle. Inhibition of new RNA or protein synthesis abolishes the proliferative response. These findings show that control mechanisms at both transcriptional and translational levels are operative in the resting cells treated with protein synthesis inhibitors. Cell fusion experiments with resting and serum-stimulated NIH 3T3 cells revealed that brief pre-incubation of resting cells with either PDGF, CH or puromycin abrogates their ability to suppress the onset of DNA synthesis in the nuclei of stimulated cells in heterodikaryons. However, the abrogative effect of PDGF disappeared in the presence of Act D, whereas the effects of protein synthesis inhibitors did not, indicating their independence of the induction of transcription. The data suggest that the observed effects of protein synthesis inhibitors are connected with elimination of some short-lived negative growth regulators, since a brief translational arrest is sufficient for the resumption of DNA synthesis in the nuclei of stimulated cells blocked by resting cells in heterodikaryons.

Protein synthesis inhibitors, like growth factors, may render resting 3T3 cells competent for DNA synthesis: a radioautographic and cell fusion study.

Serum-deprived (0.1-0.2%) resting NIH 3T3 mouse fibroblasts pre-incubated with cycloheximide (7.5 micrograms/ml), or puromycin (10 micrograms/ml), were fused with stimulated cells taken 10 h after changing the medium to one containing 10% serum, and DNA synthesis was investigated in the nuclei of monokaryons, homodikaryons and heterodikaryons using radioautography with the double-labelling technique. Pre-incubation of resting cells with inhibitors of protein synthesis for 1-4 h abolished their ability to suppress DNA synthesis in stimulated nuclei in heterokaryons. Three hours after the removal of cycloheximide from the medium, the resting cells acquired once again the inhibitory capacity for entry of stimulated nuclei into the S period. This inhibitory influence disappeared also in the case of post-fusion cycloheximide application as well as following an 8-12 h pre-treatment of resting cells with actinomycin D (1 microgram/ml) prior to fusion. Pre-incubation of resting cells for 12 h with PDGF (1 u/ml-1) followed by an 8-48 h incubation in serum-free medium stimulated the onset of DNA synthesis. A brief exposure (45 min) of resting cells to cycloheximide (7.5 micrograms/ml), or puromycin (7.5 micrograms/ml), exerted a similar effect, inducing by itself the entry of cells into the S period. The results support the assumption that acquirement, by resting cells, of competence for DNA replication includes as a necessary step the down-regulation of intracellular growth inhibitors whose formation depends on protein synthesis.

Growth factors and endogenous control of cell proliferation.

Incubation of resting (serum-deprived) NIH 3T3 mouse fibroblasts for 12 hours with PDGF1) stimulates the onset of DNA synthesis. A brief exposure (45 minutes) of resting cells to inhibitors of protein synthesis (cycloheximide or puromycin) exerts similar effect inducing by itself the entry of cells into the S period. Incubation with EGF1) following pretreatment with either PDGF or protein synthesis inhibitors does not enhance the number of cells synthesizing DNA. The results support the assumption that the acquirement, by resting cells, of competence for DNA replication includes, as a necessary step, the down-regulation of intracellular growth inhibitors whose formation depends on protein synthesis.

[The surface topography of the NIH 3T3 line in proliferating and resting states]. / Topografiia poverkhnosti kletok linii NIH 3T3 v sostoianii profileratsii i pokoia.

The surface topography of resting (serum-deprived) and proliferating (serum-stimulated) NIH 3T3 monolayer cell cultures has been examined by scanning electron microscopy. During G1 and S periods of the cell cycle the cells exhibited well pronounced surface microvilli localized mainly in the perinuclear zone, whereas serum deprivation led to a relatively smooth surface with few microvilli. The observed differences are not likely to be associated with the degree of cell spreading over the substrate, rather reflecting metabolic peculiarities of proliferating and resting cells.

[DNA synthesis in heterokaryons obtained by the fusion of proliferating cells of the NIH 3T3 line with cells in transition to the resting state]. / Izuchenie sinteza DNK v geterokarionakh, poluchennykh pri sliianii proliferiruiushchikh kletok linii NIH 3T3 s kletkami, perekhodiashchimi v sostoianie pokoia.

NIH 3T3 mouse fibroblasts cultured in the medium containing 0.5% serum for 2, 4, 8, 24, 48 and 72 hours were fused to cells stimulated for proliferation by replacing the medium with a fresh one containing 10% serum; DNA synthesis was studied in monokaryons, homo- and heterodikaryons using radioautography and double-labelling technique. Cells that were cultured in the medium with a low serum content for 72 hours exerted their inhibitory effect on the entry of stimulated nuclei into the S period in heterodikaryons, whereas cells deprived of serum for shorter periods failed to exert this effect. It thus appears that in cell fusion studies with NIH 3T3 cells, the effects of endogenous growth inhibitor(s) produced by resting cells may be revealed not earlier than by the 3rd day of serum depletion.

[Viability and proliferative activity of a Chinese hamster cell culture exposed to nitrosoureas in exponential and stationary growth phases]. / Zhiznesposobnost' i proliferativnaia aktivnost' kultury kletok kitaiskogo khomiachka pri deistvii nitrozomochevin v éksponentsial'noi i statsionarnoi fazakh rosta.

The lethal effect of antitumor nitrosourea chloroethyl derivatives on proliferating (exponential phase of growth) and non-proliferating (stationary phase of growth) cells is observed at a concentration 5-fold less than that of methyl derivatives revealed by the colony-formation technique. 1,3-bis(2-chlororoethyl)-1-nitrosourea is equally effective towards proliferating and non-proliferating cells, but chlorozotocin exerts a primary cytotoxic effect on proliferating cells. 1-methyl-1-nitrosourea at low concentration causes death more readily of proliferating cells than non-proliferating ones. However, studies on proliferative activity during the first hours after treatment with 1-methyl-1-nitrosourea revealed drug sensitivity in cells being at the early stationary phase of growth.

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.

[Dependence of the entry of NIH 3T3 cells into a period of DNA synthesis on the duration of their preliminary resting state]. / Zavisimost' vstupleniia kletok linii NIH 3T3 v period sinteza DNK ot dlitel'nosti ikh predvaritel'nogo prebyvaniia v sostoianii pokoia.

Using radioautography and cell fusion technique, we studied cell kinetics and functional properties of NIH 3T3 mouse fibroblasts stimulated to proliferate after being quiescent for 3, 7 and 14 days. The resting state was achieved by cultivating cells in the medium with 0.5% of serum, the stimulation being achieved by replacement of the depleted medium for a fresh one containing 10% of serum. It was found that the longer cells had been kept resting, the longer their prereplicative period lasted after the stimulation, the lesser was the fraction of cells that entered the S-period. Cell-fusion experiments revealed that the ability of the resting nuclei to suppress the onset of DNA synthesis in the nuclei of stimulated cells in heterodikaryons increased as the cells stayed in the resting state before fusion, and that the period of suppression was prolonged. The data are consistent with the idea of cells going into deeper resting states. It may be concluded that the resting cells undergo a gradual development resulting in the changes of their functional properties.

[DNA synthesis in the nuclei of resting and proliferating cells of the NIH 3T3 line in monokaryons, homo- and heterodikaryons]. / Sintez DNK v iadrakh pokoiashchikhsia i proliferiruiushchikh kletok linii NIH 3T3 v monokarionakh, gomo- i geterodikarionakh.

Serum-deprived (0.5%) resting NIH 3T3 mouse fibroblasts were fused with stimulated cells taken at 2 hour intervals after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in monokaryons, homodikaryons, and heterodikaryous using radioautography with double-labeling technique. The presence of the resting nucleus in the common cytoplasm has an inhibitory effect on the entry of the stimulated nucleus into the S period in the medium containing either 0.5 or 10% serum, but DNA synthesis continues. After a 24 hour stay in the common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterodikaryons still persists for at least 2 hours following stimulation. Preincubation of resting cells with cycloheximide for 4 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei. The data suggest that the resting cells produce an endogenous inhibitor of cell proliferation whose formation depends upon the synthesis of protein(s). When stimulated, cell can proliferate only upon decreasing the level of this inhibitor. The obtained results are consistent with the idea of a negative control of cell proliferation.

Onset of DNA replication in nuclei of proliferating and resting NIH 3T3 fibroblasts following fusion.

NIH 3T3 mouse fibroblasts arrested in medium containing 0.5% serum were fused with stimulated cells taken at 2-h intervals after replacing the medium with one containing 10% serum, and DNA synthesis was studied in mono-, homo- and heterokaryons using radioautography with double-labelling technique. The presence of a resting nucleus in a common cytoplasm with a stimulated nucleus from the prereplicative period has an inhibitory effect on the entry of the stimulated nucleus into the S period in medium containing either 0.5 or 10% serum, but ongoing DNA synthesis continues. After a 24-h stay in a common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterokaryons still persists, at least for 2 h following stimulation. Preincubation of resting cells with cycloheximide for 4 h abolishes their ability to suppress DNA synthesis in stimulated nuclei. The data suggest that resting cells produce an endogenous inhibitor of cell proliferation, whose formation depends upon the synthesis of protein. When stimulated, the cells can proliferate only after decreasing the level of this inhibitor. The results obtained are consistent with the idea of a negative control of cell proliferation.

[Reaction of Chinese hamster cell culture to the cytotoxic action of imuran in the early and late stationary growth phases]. / Reaktsiia kul'tury kletok kitaiskogo khomiachka na tsitotoksicheskoe deistvie imurana v rannei i pozdnei statsionarnoi faze rosta.

It has been shown that when cells are incubated for a long time without medium change, imuran exerts a much greater effect on the viability of cells in the late stationary phase of growth than in the early stationary phase. When nutrient medium supplemented with 10% serum was changed for the medium with 0.5% serum, or when a partial change of medium (one third of the volume) was carried out daily from day 4, these differences were only slightly pronounced. Thus, the degree of cell response to imuran in the early and late stationary phases of growth depends upon the way of cell maintaining in the resting state.

[Active metabolic processes in resting cells]. / Aktivnye metabolicheskie protsessy v pokoiashchikhsia kletkakh.

A number of intracellular metabolic processes are activated when cells proceed from proliferation to rest. These processes may be classified into 3 main categories: processes regulating the transition of cells from proliferation to rest; metabolic reactions necessary for the suppression of cell proliferation; processes required by a resting cell to maintain viability. Among the last category 2 groups of processes seem to be particularly important: a high rate of macromolecular turnover and an inhancement in the activity of catabolic enzymes. The high rate of RNA and protein turnover prevents the unbalanced cell growth and their death while the enhanced activity of catabolic enzymes maintains partial autolysis of resting cell compensating for the insufficient supply of nutrients due to a decreased external membrane permeability for some low molecular compounds. Thereupon, both these processes provide for the resistance of resting cells against unfavourable ambient effects allowing them to preserve proliferative potentials. Consequently the resting cells are in a special physiological state securing their self-maintenance under conditions of long-lasting absence of proliferation. The ability of cells to pass into a resting state may be regarded as a common feature of living systems acquired in the course of evolution. A co-ordinated activation of certain complexes of metabolic processes appears to be a general principle of resting cells existence in the animate nature.

[Causes of the preferential action of imuran on the viability and proliferative activity of a Chinese hamster cell culture in the stationary growth phase]. / Izuchenie prichin preimushchestvennogo deistviia i murana na zhiznesposobnost' i proliferativnuiu aktivnost' kul'tury kletok kitaiskogo khomiachka v statsionarnoi faze rosta.

It has been shown that the active principle of imuran, 6-mercaptopurine, exerts a similar though less pronounced effect on viability and proliferative activity of Chinese hamster cells by affecting them preferentially in the stationary phase of growth. This effect cannot be attributed to the inhibition by imuran of xanthine oxidase, as proposed earlier, since another inhibitor of this enzyme, allopurinol, appears more effective, causing more cell deaths during exponential growth phase rather than during stationary growth. Other possible grounds for different cytotoxic effects of imuran depending upon the proliferative status of a cell have been discussed.

[Metabolism of proliferating and resting cells]. / Metabolizm proliferiruiushchikh i pokoiashchikhsia kletok.

This review concerns the modern trends and experimental approaches to the study of cell cycle (cell population kinetics, cell structure and functions at various steps of the cycle,, etc.) and their input into the current views of cell proliferation controls. The resting state of the cell is considered and metabolic features of proliferating and resting cells are compared. Evidence is presented that resting cells are metabolically active and less resistant to the damaging factors that it has been previously supposed. The importance of this finding for biology and medicine, especially for cancer chemotherapy, is discussed.

Responses of proliferating and non-proliferating Chinese hamster cells to cytotoxic agents.

The effects of various cytotoxic chemicals, as measured by viable cell counts, colony-forming ability and proliferative capacity, have been studied using Chinese hamster cells in exponential and plateau (stationary) phases of growth. The proliferating cells were altogether more sensitive to the action of the drugs than non-proliferating cells. However, imuran (azathioprine) a purine antimetabolite, was more effective against the plateau-phase cells. The observed response of cells to imuran could be detected at a wide range of concentrations (1-100 microgram/ml). These findings are discussed in view of the possible ability of imuran to interfere with active metabolic processes in non-proliferating cells.

A comparative study of the effects of lucanthone (miracil D) and actinomycin D on the Chinese hamster cells grown in cultures.

Chinese hamster cells of an established clone line grown in monolayers were incubated for up to two hours with either lucanthone (0.3-30 mug/ml) or actinomycin D (0.06-0.10 MUG/ML) AND SUbjected to radioautographic investigations with 3H-uridine during the period of treatment. At concentration of 9 mug/ml lucanthone selectively inhibited the synthesis of nucleolar (ribosomal) RNA while the extranucleolar RNA synthesis proceeded at a high level. Similar results were obtained with 0.08 mug/ml actinomycin D. Protein synthesis and mitotic activity were also affected by lucanthone but the drug did not markedly interfere with DNA synthesis. Lucanthone appeared to be much less effective in cell killing than actinomycin D and its inhibitory effects on the nucleolar RNA synthesis and other cellular processes proved readily reversible. The results allow to conclude that lucanthone may be useful as a tool for studying RNA synthesis in animal cells.