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.

[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.

[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.

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.