Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

Inhibition of hepatocyte proliferation in vivo by a glycopeptide from rat serum

Abstract. The activity of a glycopeptide prepared from rat serum by treatment with trypsin and ultrafiltration was investigated in several in vivo proliferation systems. In baby rat hepatocytes synchronized by a subcutaneous injection of casein solution it caused a G₁-S block, stopping cells at the end of the G₁ phase and sending them back to the G₀ phase. The glycopeptide also caused a G₁-S block in young adult rats during the first semi-synchronized wave of proliferation that followed partial hepatectomy. Inhibition of hepatocyte proliferation by the glycopeptide was suppressed by blood proteins from normal rats but not from acute phase rats. α₁-acid glycoprotein, an acute phase protein, increased this inhibition and reversed the antagonistic effect of normal blood proteins. In normal baby rats a G₁-S block of non-synchronously proliferating hepatocytes was produced in two situations in which the antagonistic effect of normal blood proteins was eliminated: after treatment of the glycopeptide with leucine-aminopeptidase, and after mixing it with α₁-acid glycoprotein. The glycopeptide did not inhibit cell proliferation in kidney, submaxillary gland, or tongue epithelium. It seems to be the active component of a system that inhibits the proliferation of hepatocytes, probably by reducing their sensitivity to various mitogenic stimuli.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Abstract. Cellular uptake of [³H]thymidine ([³H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10^-18 mol/cell. The pool activity of G₁ cells was unmeasurable but rose to maximum values at the border of the G₁-S phase. It decreased again during G₂. The [³H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min^-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Normal G1/S transition and prolonged S phase within one cell cycle after seeding cells in the presence of an ornithine decarboxylase inhibitor

Abstract. We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G₁/S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine-flow cytometric method to more specifically study the G₁/S transition, the S phase length, and the progression of cells from S phase through G₂+ M and into G₁, after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G₁/S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G₁ phase occurred later than the effect on S phase progression. These results imply that the G₂+ M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO.     

Cell cycle progression in human cells following re-oxygenation after extreme hypoxia: consequences concerning initiation of DNA synthesis

Abstract. The initiation of DNA synthesis and further cell cycle progression in cells during and following exposure to extremely hypoxic conditions in either G₁ or G₂+M has been studied in human NHIK 3025 cells. Populations of cells, synchronized by mitotic selection, were rendered extremely hypoxic (< 4 p.p.m. O₂) for up to 24n h. Cell cycle progression was studied from flow cytometric DNA recordings. No accumulation of DNA was found to take place during extreme hypoxia. Cells initially in G₁ at the onset of treatment did not enter S during up to 24 h exposure to extreme hypoxia, but started DNA synthesis in a highly synchronous manner within 1.5 to 2.25 h after reoxygenation. The duration of S phase was only slightly affected (increased by ≅10%) by the hypoxic treatment. This suggests that the DNA synthesizing machinery either remains intact during hypoxia or is rapidly restored after reoxygenation. Cells initially in G₂ at the onset of hypoxia were able to complete mitosis, but further cell cycle progression was blocked in the subsequent G^ Following reoxygenation, these cells progressed into S phase, but the initiation of DNA synthesis was delayed for a period corresponding to at least the duration of normal G₁ and did not appear in a synchronous manner. In fact, cell cycle variability was found to be increased rather than decreased as a result of exposure to hypoxia starting in G₂. We interpret these findings as an indication that important steps in the preparation for initiation of DNA synthesis take place before mitosis. Furthermore, the change in cell cycle duration induced by hypoxia commencing in G₁ is of a nature other than that induced by hypoxia commencing in other parts of the cell cycle.     

THE ALTERATION OF THE REGENERATIVE ACTIVITY AND THE CELL CYCLE OF PARTIALLY HEPATECTOMIZED RAT LIVER FOLLOWING ADMINISTRATION OF d-GALACTOSAMINE

A single injection of d-galactosamine given to rats at different times after partial hepatectomy (PH) changes the pattern of regenerative proliferation. When administered during the pre-replicative phase of regeneration, the onset of DNA synthesis and the increase in labelling index after injection of ³H-thymidine are delayed by about 12 hr. The injection of d-galactosamine at 24 hr after PH inhibits the drop in DNA synthesis occurring normally during the following 12 hr period. This was detected by a high labelling index and by an increased specific activity of DNA. The findings indicate a lengthening of the S phase, while G₂ and M remain normal. Two modes of action of d-galactosamine on the cell cycle are discussed.     

CELL CYCLE PROPERTIES OF DIFFERENTIATING SPERMATOGONIA IN ADULT SPRAGUE-DAWLEY RATS

The duration of the mitotic cycle and of its components was analysed for each of the six successive generations of differentiating spermatogonia (A₁, A₂, A₃, A4, intermediate and B), using radioautographed whole mounts of seminiferous tubules from testes of adult Sprague-Dawley rats. Cell cycles were determined from two successive waves of per cent labeled metaphases obtained during the period of 81 hr after a single dose of ³H-thymidine. Except for the A₁ spermatogonia, all spermatogonial types (A₂ to B) had similar cell cycle durations of 41-42.5 hr and comparable pre-DNA synthesis phases (G₁) of 11-13 hr. Although the combined duration of DNA synthesis (S) and the post-synthesis phase (G₂) remained identical for all the cell types including A₁, there was a progressive lengthening of the S period at the expense of G₂ during the process of spermatogonial maturation. This change was most marked during the transition from A₁ to A₃ spermatogonia when the S period increased from 14 hr to 21 hr, and the G₂ phase shortened from 13 hr to 7.5 hr. This feature seems to be unique to germ cells and may be associated with an increasing amount of heterochromatin in the nucleus. Excluding the development of type A₁ cells, the entire process of spermatogonial maturation lasted for 208 hr. Combined data on cell cycle times indicated that every 313 hr or 13 days, a new sequence of spermatogonial differentiation was initiated by the A₁ cells. This was equivalent to the duration of one 'cycle' of the seminiferous epithelium as measured by other techniques.     

ON THE RESTING STAGES OF THE JB-1 ASCITES TUMOUR

Cytophotometric determination of single-cell DNA after repeated ³H-thymidine labelling of the JB-1 ascites tumour in the plateau phase of growth showed a massive accumulation of unlabelled cells with both G₁ and G₂ content. Autoradiography combined with cytophotometry or colcemid block demonstrated that some of these unlabelled cells were rapidly triggered into the cell cycle when plateau tumours were transferred to new hosts. This indicated that tumour cells may be held up in non-cycling stages corresponding to both the G₁ and the G₂ phase of the cell cycle.     

Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10⁻⁵M, 10⁻⁶M or 10⁻⁷M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10⁻⁵ M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G₀/G₁ phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P 0.05), 13.9% at 24 h (P 0.001), and 76.5% at 48 h (P 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P 0.005) and 12 h (P 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G₁/S transition by prolonging G₂/M and through decreasing the DNA synthesis rate     

Cell-cycle dependence of a ganglioside glycosyltransferase activity and its inhibition by enkephalin in a neurotumor cell line

Abstract: Rat glioma mouse neuroblastoma hybrid neurotumor cells (NG108-15), synchronized by amino acid deprivation, showed a cell-cycle-dependent peak of activity of a ganglioside N-acetylgalactosaminyl transferase 14-24 h following release from the cell cycle block (S/G₂ phase). Maximal expression of two typical lysosomal hydrolases, N-acetyl-β-hexosaminidase and β-galactosidase, occurred between 18 and 21 h following release (S phase), declining to G₁ phase levels during the peak of N-acetylgalactosamine (GalNAc) transferase activity. In addition, glycosyltransferase activity in G₂ phase cells showed an increase in apparent V_max (suggesting the presence of more enzyme/mg of cell protein) and apparent binding affinity for uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) (32 versus 14 M) when compared to transferase activity in the G₁ phase. However, the opioid peptide enkephalin [D-Ala², o-Leu⁵], which inhibits ganglioside GalNAc transferase activity in unsynchronized NG108-15 cultures, was much more inhibitory in whole cells 8 h after release from the cell cycle block (G₁ phase) than in cells 20 h after release (G, phase), with 50% inhibition occurring at 2 ± 10^-9M and 2 ± 10^-7M, respectively. These results suggest that the GalNAc transferase activity is regulated in more than one way during the cell cycle, since both V_max and K_m changes are observed, and that the cyclic AMP-dependent mechanism by which opiates reduce transferase activity is receptor mediated and cell cycle dependent.     

Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle

Abstract. Multivariate analysis of the expression of cyclin proteins and DNA content has opened new possibilities for the study of the cell cycle. By virtue of their cell cycle phase specificity, the expression of cyclins may serve, in addition to DNA content, as another marker of a cell's position in the cycle, and provide information about the proliferative potential of cell populations. Several applications of the methodology based on bivariate analysis of DNA content v . expression of B, E and D type cyclins are reviewed: 1 expression of cyclins by individual cells during their progression through the cycle can be studied, using exponentially growing cells without the necessity of cell synchronization or other perturbations of the cycle; 2 cells having the same DNA content but residing in different phases of the cycle (e.g. G₂ diploid v. G₁ tetraploid) can be distinguished; 3 cell transition from G₀ to G₁ and progression through G₁ (e.g. mitogen stimulated lymphocytes) can be assayed; 4 the population of proliferating cells can be distinguished from noncycling cells based on dual cell labelling with a G₁ and G₂ cyclin antibody; 5 cyclin restriction points can serve as additional cell cycle landmarks to map the point of action of antitumour drugs; 6 unscheduled expression of cyclins (e.g. the presence of cyclin B1 during G₁ and S) can be detected in several tumour transformed cell lines, possibly indicating disregulation of the machmery of cell cycle progression. The last finding 6 is of special importance, because such disregulation may be of prognostic consequence in human tumours.     

cAMP IN THE CELL CYCLE OF THE DINOFLAGELLATE CRYPTHECODINIUM COHNII (DINOPHYTA)

The second messenger cAMP is a key regulator of growth in many cells. Previous studies showed that cAMP could reverse the growth inhibition of indoleamines in the dinoflagellate Crypthecodinium cohnii Biecheler. In the present study, we measured the level of intracellular cAMP during the cell cycle of C. cohnii . cAMP peaked during the G₁ phase and decreased to a minimum during S phase. Similarly, cAMP-dependent protein kinase activities peaked at both G₁ and G₂+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O₂'-dibutyryl (Bt₂)-cAMP directly stimulated the growth of C. cohnii . Flow cytometric analysis of synchronized C. cohnii cells suggested that 1 mM cAMP shortened the cell cycle, probably at the exit from mitosis. The size of Bt₂-cAMP treated cells at G₁ was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates.     

EFFECTS OF PHYTOCHROME AND BLUE-NEAR ULTRAVIOLET LIGHT-ABSORBING PIGMENT ON DURATION OF COMPONENT PHASES OF THE CELL CYCLE IN ADIANTUM GAMETOPHYTES

Single-celled protonemata of the fern Adiantum capillus-veneris, kept under continuous red light, grew with a very low rate of cell division, and the cell cycle was arrested in the early G₁ phase. Cell division was induced by transferring the protonemata to the dark after various light treatments, and the duration of component phases in the cell cycle was determined by a continuous-labelling technique with 3H-thymidine. Blue light irradiation greatly reduced the duration of the G₁ phase but did not affect that of other phases. The greater the fluence of blue light, the shorter was the duration of G₁ phase was observed. In contrast, a brief exposure of red-light-grown protonemata to far-red light given immediately before the dark incubation showed no effect on the duration of G₁ S and M phases but significantly extended that of the G₂ phase. The effect of far-red light on the G₂ phase was reversed by red light, and the effects of red and far-red light were repeatedly reversible. The progression in the M phase was shown by means of a time-lapse video system to be not at all influenced by any pre-irradiation described above.     

La Synthèse de l'Acide Désoxyribonucléique au Cours du Cycle de Division de Trypanosoma mega

SYNOPSIS. Tritiated thymidine and autoradiographic methods were used to investigate the cyclic DNA synthesis in the culture form of Trypanosoma mega. It was found that the mean generation time of 18.9 hours comprises four successive periods: G₁, S, G₂ and D. The interphase lasts through the first three. S is the phase of DNA synthesis of both the nucleus and the kinetonucleus (kinetoplast). The cell divides during D, beginning with the division of the kinetonucleus. The respective durations of G₁, S, G₂ and D are 8.5, 7, 2 and 1.4 hours. The close time relationship between the two DNA synthesizing bodies is considered as bringing support to the old theory of the Binucleates and the possible genetic function of the kinetonucleus is suggested.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE CELL PROGRESS FROM M TO G1 AND S STAGES IN CULTURED MOUSE LEUKEMIA L5178Y CELLS

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G₁ stage. Actinomycin D in this concentration also significantly depressed uridine-³H uptake into G₁ stage cells, but did not suppress leucine-³H uptake by M and G₁ cells. This suggests that some proteins may be synthesized in M and G₁ stage cells by messenger RNA left over from the previous cell cycle. However, entry of G₁ cells into S stage would require synthesis of new messenger RNA near the beginning of G₁ stage. Puromycin (10 μg/ml) did not prevent M cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The site of blockage was about 0.7 hr before S stage or in the first two-third of G₁ stage. This might be the site where the cells synthesize new G₁ proteins necessary for entry to S stage.
Comparison of sensitivities of G₁ and G₂ stages to the two antibiotics reveals that the puromycin sensitivity of G₁ cells was similar to that of G₂ cells, but the actinomycin D sensitivity of G₁ was greater than that of G₂ cells.     

Cell Cycle-Dependent Expression of Specific Opiate Binding with Variable Coupling to Adenylate Cyclase in a Neurotumor Hybrid Cell Line NG108–15

Abstract: Monolayer cultures of neuroblastome × glioma hybrid (clonal) cell line NG108-15, synchronized by the isoleucine/glutamine deprivation method, showed maximal expression of opiate binding sities at the same point in the cell cycle at which prostaglandin E₁(PGE₁) had a maximum stimulatory effect of cyclinc AMP synthesis. However, the capacity of enkephalin (D-Ala²D-Leu⁵] to block the stimulation of cyclic AMP synthesis by PGE₁ was not related to the number of opiate receptors expressed. The K₁ for the inhibition of cyclic AMP synthesis by opioid peptides increased substanitilly during the period of the cell cycle at which maximal expression of opiate binding sites occurred, making the effectivel level of inhibition of adenylate cyclase activity by 0.1μM enkephalin [D-Al²D-Leu³] the same throght the cell cycle. Data are presented to suggest the enkephalin receptor coupling to adenylate cyclase, via a GTP-binding protein, is maximal during G₁ phase (which may approximate the state of the differentiated neuron) and minimal during S + G₂ phase, just prior to cell division, when many receptors are uncoupled.     

Induction of replicative senescence by 5-azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH-3T3 cells

Abstract. To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96 h exposures to 4 μM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G₁ compartments of the second and third cell cycles of serum stimulated cells. The exit from the G₀/G₁ compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2–3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G₂ phase arrests and delayed G₀/G₁ phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G₁ phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.     

Cell Cycle Phase Sensitivity to Cis-Dichloro-Bis (Isopropylamine) Trans-Dihydroxy Platinum (Iv) (Chip)

Abstract. Cis-dichloro-bis (isopropylamine) trans-dihydroxy platinum (IV) (CHIP) is a second generation platinum coordination complex now in Phase II clinical trials. In vitro studies with Chinese Hamster Ovary cell cultures show that CHIP is a phase-sensitive drug, being most cytotoxic to cells in early G₁ phase and least toxic to late S and G₁ phase cells. the dose-modifying factor between the drug sensitivity of cells treated in G₁ and in late S phase is 1.6. These findings and their clinical significance are discussed with respect to the phase sensitivity of other cytotoxic agents.