Cell cycle specificity and reversibility of the inhibitory effect of epidermal G1-chalone on DNA synthesis in partially synchronized RTE-2 keratinocytes in vitro

The specific action of a pig skin fraction enriched in epidermal G₁-chalone, a tissuespecific inhibitor of epidermal DNA synthesis, was investigated by means of flow cytofluorometry. The results indicate that G₁-chalone inhibits progression of partially synchronized rat tongue epithelial cells (line RTE-2) through the cell cycle at a point 2 h prior to the beginning of the S-phase. Approximately 8 h after chalone addition, the cells can overcome the inhibition and begin to enter the S-phase. The duration of this delay is concentrationindependent, but the fraction of cells affected is proportional to the chalone concentration. The progression of cells which already have entered S-phase is not affected. In contrast to the G₁-chalone preparation, aphidicolin, a potent inhibitor of DNA polymerase α, clearly shows S-phase-specific inhibition. These results indicate that the epidermal G₁-chalone inhibits epidermal cell proliferation in a fully reversible manner by a highly specific effect on cell cycle traverse.     

EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE RADIATIONS ON CELL CYCLE KINETICS IN EXCISED ROOT MERISTEMS OF PISUM SATIVUM

Near-ultraviolet and visible radiations increased the duration of the mitotic cycle in excised pea root meristems primarily by lengthening the duration of the pre-DNA synthetic period (G₁). All radiations tested shortened the duration of the post-DNA synthetic period (G₂). The most pronounced effects were exhibited by green radiation, which lengthened the duration of the cell cycle, G₁, DNA synthesis (S), and mitosis (M), and shortened the duration of G₂. Progression of cells arrested by starvation in G₁ and G₂ into DNA synthesis and mitosis was also affected by light treatments. Green radiation appeared to arrest a group of cells in DNA synthesis as well as in G₁ and G₂. Meristems receiving green and near-ultraviolet radiations exhibited the most rapid progression of G₁ cells through S and G₂.     

Nucleic acids methylation of synchronized BHK 21 HS 5 fibroblasts during the mitotic phase

The methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G₁ phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl-¹⁴C] methionine and [methyl-³H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis. A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G₁ phase. In both conditions it suppressed ³H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments. The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post-synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.     

CID755673 enhances mitogenic signaling by phorbol esters, bombesin and EGF through a protein kinase D-independent pathway

Recently, CID755673 was reported to act as a highly selective inhibitor of protein kinase D (PKD). In the course of experiments using CID755673, we noticed that it exerted unexpected stimulatory effects on [³H]thymidine incorporation and cell cycle progression in Swiss 3T3 cells stimulated by bombesin, a Gq-coupled receptor agonist, phorbol 12,13-dibutyrate (PDBu), a biologically active tumor promoting phorbol ester and epidermal growth factor (EGF). These stimulatory effects could be dissociated from the inhibitory effect of CID755673 on PKD activity, since enhancement of DNA synthesis was still evident in cells with severely down-regulated PKD1 after transfection of siRNA targeting PKD1. A major point raised by our study is that CID755673 can not be considered a specific inhibitor of PKD and it should be used with great caution in experiments attempting to elucidate the role of PKD family members in cellular regulation, particularly cell cycle progression from G₁/G_o to 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.     

SOME EFFECTS OF BLEOMYCIN ON THE PROLIFERATION, MATURATION TIME and PROTEIN SYNTHESIS OF HAIRLESS MOUSE EPIDERMIS

Hairless mice were given 2 mg Bleomycin i.p. in 1-0 ml saline on two successive days. By a stathmokinetic method, by micro-flow fluorometry and by autoradiography certain kinetic parameters were measured during 10 days after the last injection. Cell counts were made and the turnover time of the differentiating cells estimated. Protein synthesis was estimated by the uptake of radioactive histidine, and dry cell mass measured by weighing. Bleomycin affected cell proliferation in the epidermis by depressing biphasically both the number of cells in, and the passage of cells through, the cell cycle phases: S, G₂ and M, most probably by directly affecting late Gj cells and cells in mitosis. The time between the two minima of depressed DNA synthesis corresponded to the mean generation time of the basal cells. Histidine uptake and dry cell mass were slightly affected, but the turnover time of the differentiating cells was prolonged. Bleomycin thus had a strong long-lasting inhibitory effect on epidermal cell proliferation and a marked inhibitory effect on epidermal cell maturation in mice.     

Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G₁, most virus transformed or tumor cells are unaffected in G₁ but arrest in G₂ phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nucleolar p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 μM SSP.MOLT-4 cells did not arrest in G₁ or G₂ phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G₁ phase at higher DNA ploidy (tetraploidy; G_1T), and then progressed through S_T (rereplication) into G_2T and M_T. The rates of entrance to G₂ and G_2T were essentially identical, indicating that the rates of cell progression through S and S_T as well as through G₂ and G_2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 μM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis. © 1994 Wiley-Liss, Inc.     

THE ACTION OF HYDROXYUREA ON MOUSE L-CELLS*

The toxic and inhibitory properties of hydroxyurea (HU) have been studied in asynchronous and synchronized populations of mouse L-cells. Hydroxyurea is a potent growth inhibitor and appears to be specifically lethal for cells which are in the early part of S phase at the time the compound is introduced. Cells in late S phase, G₂, mitosis and G₁ appear to progress normally around the cycle in the presence of the compound until they reach the G₁/S boundary. There are indications that at least some G₁ cells are able to enter the S phase even in the presence of the drug; however their flow into S is much slower than that of control cells and therefore they are killed at a slow rate. Upon prolonged exposure to the drug a second phase of more rapid killing is observed, beginning at about the time division would occur in uninhibited cells. Hydroxyurea exhibits a rapid and marked inhibition on DNA synthesis but its effect on RNA synthesis is much less pronounced and may be a consequence of the inhibition of DNA synthesis. The effects of hydroxyurea on cell viability and DNA synthesis can be partially prevented by the addition of deoxyribonucleosides which in sufficient concentration appear to compete temporarily with the drug. The fact that the protection is only temporary would appear to rule out the hypothesis that the primary mode of action of the drug is the inhibition of the reduction which converts ribonucleotides to deoxyribonucleotides. The data presented in this communication taken together with observations of other workers would appear to suggest that the effect of the drug may be directly on the DNA molecule.     

A novel mutation of ALK2, L196P, found in the most benign case of fibrodysplasia ossificans progressiva activates BMP-specific intracellular signaling equivalent to a typical mutation, R206H

Naphthoquinone derivatives have been reported to possess various pharmacological activities, such as antiplatelet, anticancer, antifungal, and antiviral properties. In this study, we investigated the effects of a newly-synthesized naphthoquinone derivative, 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (2-decylamino-DMNQ), on VSMC proliferation and examined the molecular basis of the underlying mechanism. In a dose-dependent manner, 2-decylamino-DMNQ inhibited PDGF-stimulated VSMC proliferation with no apparent cytotoxic effect. While 2-decylamino-DMNQ did not affect PDGF-Rβ or Akt, it did inhibit the phosphorylation of Erk1/2 and PLCγ1 induced by PDGF. Moreover, 2-decylamino-DMNQ suppressed DNA synthesis through the arrest of cell cycle progression at the G₀/G₁ phase, including the suppression of pRb phosphorylation and a decrease in PCNA expression, which was related to the downregulation of cell cycle regulatory factors, such as cyclin D1/E and CDK 2/4. It was demonstrated that both U0126, an Erk1/2 inhibitor, and U73122, a PLCγ inhibitor, increased the proportion of cells in the G₀/G₁ phase of the cell cycle. Thus, these results suggest that 2-decylamino DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and the mechanism of this action is through cell cycle arrest at the G₀/G₁ phase. This may be a useful tool for studying interventions for vascular restenosis in coronary revascularization procedures and stent implantation.     

Existence of a commitment program for mitosis in early G1 in tumour cells

The proliferation of normal non-tumourigenic mouse fibroblasts is stringently controlled by regulatory mechanisms located in the postmitotic stage of G₁ (which we have designated G₁ pm). Upon exposure to growth factor depletion or a lowered de novo protein synthesis, the normal cells leave the cell cycle from G₁ pm and enter G₀. The G₁ pm phase is characterized by a remarkably constant length (the duration of which is 3 h in Swiss 3T3 cells), whereas the intercellular variability of intermitotic time is mainly ascribable to late G₁ or pre S phase (G₁ ps) (Zetterberg & Larsson (1985) Proc. Natl. Acad. Sci. USA 82 , 5365). As shown in the present study two tumour-transformed derivatives of mouse fibroblasts, i.e. BPA31 and SVA31, did not respond at all, or only responded partially, respectively, to serum depletion and inhibition of protein synthesis. If the tumour cells instead were subjected to 25-hydroxycholesterol (an inhibitor of 3-hydroxy-3 methyglutaryl coenzyme A reductase activity), their growth was blocked as measured by growth curves and [³H]-thymidine uptake. Time-lapse analysis revealed that the cells were blocked specifically in early G₁ (3-4h after mitosis), and DNA cytometry confirmed that the arrested cells contained a G₁ amount of DNA. Closer kinetic analysis revealed that the duration of the postmitotic phase containing cells responsive to 25-hydroxycholesterol was constant. These data suggest that transformed 3T3 cells also contain a ‘G₁ pm program’, which has to be completed before commitment to mitosis. By repeating the experiments on a large number of tumour-transformed cells, including human carcinoma cells and glioma cells, it was demonstrated that all of them possessed a G₁ pm-like stage. Our conclusion is that G₁ pm is a general phenomenon in mammalian cells, independent of whether the cells are normal or neoplastic.     

THE EFFECTS OF LOW CONCENTRATIONS OF ACTINOMYCIN D ON THE PROGRESS OF CELLS THROUGH THE CELL CYCLE

The progress of L-cells through the cell cycle in asynchronous and in synchronous culture has been studied at a concentration of actinomycin D which mediates an apparently‘nucleolar-specific’inhibition of RNA synthesis. Under such conditions, cells may be blocked or seriously delayed in the G₁ and G₂ phases of the cycle whilst the processes of DNA synthesis and mitosis once initiated, can still occur at control rates. The results show that the sensitivity of a cell to these blocks depends critically upon the position of that cell within the cycle at the time of drug addition. The possible mechanisms of the drug's action are discussed.     

Effects of inhibition of RNA or protein synthesis on CHO cell cycle progression

Chinese hamster ovary (CHO) cells, synchronized by selective detachment at mitosis, were treated with various concentrations of actinomycin D (AMD) or cycloheximide (CHX) either immediately, or 1, 2, or 3 hr after mitosis. Since the minimum duration of G₁ phase in these cultures was 3.4 hr, the addition of RNA or protein synthesis inhibitors took place at the beginning, first third, second third, or end (G₁–S boundary) of G₁ phase. The kinetics of exit from G₁ phase, the rate and extent of traverse of S phase, and the reaccumulation of RNA were estimated under each set of growth conditions by flow cytometry of acridine orange-stained cells. A mathematical model was constructed to describe the trajectories of the cell populations with respect to their increase in RNA and DNA content in the absence or presence of the inhibitor. The chronologic synchrony imposed on the CHO cell population began to decay within 3 hr, resulting in stochastic entrance of cells into S phase in the absence of inhibitor. Addition of AMD or CHX at 0, 1, 2, or 3 hr after mitosis, regardless of the inhibitor concentration, did not provide evidence of a critical restriction point in G₁ beyond which cells were committed to enter S phase and were no longer sensitive to moderate suppression of RNA or protein synthesis. The observed kinetics of cell entrance into and traverse of S phase were consistent with an inherently heterogenous response to serum stimulation occurring at or just after cell division.     

DNA SYNTHESIS AND MITOSIS IN MERISTEMS: REQUIREMENTS FOR RNA AND PROTEIN SYNTHESIS

Following provision of sucrose to starved, stationary phase pea root meristems, G₁ and G₂ cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G₁ and G₂ cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8-12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.     

RNA synthesis in synchronously growing populations of HeLa S3 cells. I. Rate of total RNA synthesis and its relationship to DNA synthesis

The rate of RNA synthesis in synchronously growing HeLa S3 cells was determined as a function of position in the cell generation cycle. Measurements throughout the cycle of both the rate of incorporation of radioactively-labeled uridine and of the total amount of RNA indicate that (1) the rate of RNA synthesis is constant (or increases only slightly) during G₁, approximately doubles during the first half of S, and then remains constant during the remainder of S and G₂, and (2) cells attain the average G₁ rate of RNA synthesis very early in G¹, and maintain the average G₂ rate until mitosis. If the initiation of DNA synthesis is blocked, the acceleration of RNA synthesis is markedly reduced or eliminated. Further experiments in which DNA synthesis was inhibited at different times in S, or to varying degrees from the beginning of S, suggest that the extent to which RNA synthesis is accelerated depends on the amount of DNA duplicated. These data also indicate that duplication of the first half, and in particular the first few per cent, of the DNA complement results in a disproportionate acceleration of RNA synthesis. The possibility that fluctuations in the sizes of precursor pools may lead to misinterpretation of labeled-uridine incorporation data was examined. Experiments indicate that in this system pool fluctuations do not cause invalid measures of RNA synthesis. It is concluded that RNA synthesis occurs throughout interphase, but undergoes a two-fold increase in rate which is dependent on the duplication of DNA.     

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G₁ phase to G₂ and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G₁ cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.     

Autocrine regulation of cell cycle progression in normal human keratinocytes

Summary Removal of competence factors insulin and pituitary extract from the culture medium, concomitant with the addition of picomolar concentrations of the late-G₁ inhibitor transforming growth factor-beta, effectively arrested cell cycle progression of normal human keratinocytes prior to their entry into the DNA synthesis phase; arrest continued for a minimum of 36 h following removal of unbound inhibitor and subsequent addition of factor-deficient medium. To demonstrate the reversibility of transforming growth factor-beta-induced arrest, two dissimilar cell populations were recruited to synthesize DNA in a predictable and reproducible manner; whereas the reinstatement of omitted competence factors induced noncycling cells to begin synthesizing DNA within 24 h, addition of keratinocyte-conditioned medium prompted an immediate progression of late-G₁ cells into S phase. Studies to determine the extent that autocrine signaling regulates cell cycle progression revealed that nontransformed keratinocytes produce an endogenous factor required for DNA replication and that production of this progression factor required competence factors insulin and pituitary extract. Keratinocyte progression factor recruited late-G₁ cells into S phase within 1–2 h, reversed transforming growth factor-beta-induced arrest in the presence of bound inhibitor, and elicited a calcium mobilization response consistent with receptor-mediated signaling. Hence, these studies demonstrate that G₁ progression of nontransformed keratinocytes into S phase requires an endogenous progression factor and suggest that this factor may direct G₁ progression by modulating the activity of a calcium-dependent kinase.     

Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells.

Both DNA and the centriole pairs are replicated once in each cell generation. The cyclic changes in both must be coordinated so that the two centriole pairs can participate in mitosis when the genetic material is to be partitioned to the two daughter cells. One of the centriole pairs also forms a primary (“9 + 0”) cilium sometime during the cell cycle. In this study, we asked whether some aspects of the coordination of the DNA and centriole cycles occur in G₁, a part of the cell cycle when non-neoplastic cells become irreversibly committed to DNA synthesis. We used indirect immunofluorescence with antitubulin antibody to reveal the centriole pairs as a microtubule organizing center with or without a cilium. Quiescent Balb/c and Swiss 3T3 cells in low serum or at high cell density stopped in G₁ with ciliated, probably unduplicated centrioles. When these quiescent 3T3 cells were stimulated to enter DNA synthesis, the centriole's ciliation changed in three phases: first, an initial but transient deciliation within 1–2 hr; second, a return of the cilium by 6–8 hr; and third, a subsequent final deciliation of the centriole coincident with the initiation of DNA synthesis at 12–24 hr.The deciliated and duplicated centrioles subsequently separated in preparation for mitosis. Together with other information, these results imply that centrioles in growing mammalian cells are primarily ciliated in a part of G₁ during which the cells can arrest in suboptimal environmental conditions. Arrests in low serum or at high cell density also occur before centriole replication. These results suggest that deciliation and duplication of the centriole may occur near the time that quiescent cells become irreversibly committed to DNA synthesis. Certain centriole events may therefore be necessary before DNA synthesis can be initiated in 3T3 cells.