CELL KILLING BY ACTINOMYCIN D IN RELATION TO THE GROWTH CYCLE OF CHINESE HAMSTER CELLS

Using Chinese hamster cells in culture, we have measured the effectiveness of actinomycin D to suppress division as a function of the position, or age, of a cell in its growth cycle. Cells were first exposed to millimolar concentrations of hydroxyurea in order to produce a synchronized population just before the onset of DNA synthesis. Thereafter, the survival response after 30 min exposures to actinomycin D was measured. Cells become resistant as they enter the S phase and then sensitive again in the latter part of S. When they reach G₂ (or G₂-mitosis) they are maximally resistant; at 1.0 µg/ml, for example, the survival in G₂ is 30-fold greater than it is in G₁. These results, plus measurements reported earlier on the interaction of damage in S cells due to actinomycin D and X-irradiation, suggest that the age-response pattern of the toxic effects of this drug probably reflects both the functional capacity of DNA-actinomycin complexes and the ability of this antibiotic to penetrate chromatin and bind to DNA.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

DIFFERENT LETHAL EFFECTS OF MITOMYCIN C AND ACTINOMYCIN D DURING THE DIVISION CYCLE OF HELA CELLS

The lethal actions of mitomycin C and actinomycin D were followed during the division cycle of HeLa cells. The cells were most susceptible to a 2 hr pulse of mitomycin C during the G₁ phase, whereas their sensitivity to actinomycin D was most pronounced in the S phase. Posttreatment of the cells with acetoxycycloheximide, a potent inhibitor of protein synthesis, increased the survival (colony-forming ability) of cells treated with mitomycin C but had very little effect on the survival of cells treated with actinomycin D. The significance of these findings is discussed.     

Dynamics of cell cycle phase perturbations by trabectedin (ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematical simulation approach

Abstract. Objectives: Trabectedin (ET‐743, Yondelis^®) is a natural marine product, with antitumour activity, currently in phase II/III clinical trials. Previous studies have shown that cells hypersensitive to ultraviolet (UV)‐rays because of nucleotide excision repair (NER) deficiency, were resistant to trabectedin. The purpose of this study was to investigate whether this resistance was associated with different drug‐induced cell cycle perturbations. Materials and Methods: An isogenic NER‐proficient cellular system (CHO‐AA8) and a NER‐deficient one (CHO‐UV‐96), lacking functional ERCC‐1, were studied. Flow cytometric assays showed progressive accumulation of cells in G₂ + M phase in NER‐proficient but not in NER‐deficient cells. Applying a computer simulation method, we realized that the dynamics of the cell cycle perturbations in all phases were complex. Results: Cells exposed to trabectedin during G₁ and G₂ + M first experienced a G₁ block, while those exposed in S phase were delayed in S and G₂ + M phases but eventually divided. In the presence of functional NER, exit from the G₁ block was faster; then, cells progressed slowly through S phase and were subsequently blocked in G₂ + M phase. This G₂ + M processing of trabectedin‐induced damage in NER‐proficient cells was unable to restore cell cycling, suggesting a difficulty in repairing the damage. Conclusions: This might be due either to important damage left unrepaired by previous G₁ repair, or that NER activity itself caused DNA damage, or both. We speculate that in UV‐96 cells repair mechanisms other than NER are activated both in G₁ and G₂ + M phases.     

Effect of hypertonicity and freezing on survival of unprotected synchronized mammalian cells

Survival of unprotected Chinese hamster cells frozen to −196 °C in tissue culture medium or exposed to a 6X NaCl solution is a function of the time in the cell cycle at which freezing or the exposure to hypertonicity takes place. In both cases, the cells are sensitive to exposure in M and G₂, and are resistant in late S. The survival curves are different in late G₁ or early S. This indicates that damage due to hypertonicity is not the only mode of injury present when cells are frozen to −196 °C.     

Changes in the content of condensed chromatin during the cell cycle in antheridial filaments ofChara vulgaris L. as related to DNA and RNA synthesis,3H actinomycin D binding and RNA polymerase activity

Summary Using light microscopic autoradiography it was found that in the middle S and G₂ phases of the cell cycle in antheridial filaments (S + G₂ + M type) ofChara vulgaris L., an intensified RNA synthesis took place. The process was correlated with enhanced binding of³H actinomycin D to the DNA template, increased RNA polymerase activity, and enhanced chromatin decondensation.This work was supported by the Polish Academy of Sciences within the project 09.7.3.1.4.     

Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KGl), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G₁ were resistant; the sensitivity of G₂ or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G₂ or M, to these inhibitors. The data show that γ-irradiation (0.5–15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G₁ cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G₂ either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G₁, G₂ or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.     

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.     

Variation In G1 Transit Time Relative to the Cycloheximide and Actinomycin D Drug Restriction Points

The transit time distribution at various points in the cell cycle of synchronized Chinese hamster ovary cells was determined from the mitotic index, [³H]thymidine labeling index and increase in cell number monitored at regular intervals after mitotic selection. Variation in G₁ transit time compared with that for the total cell cycle indicates that variation in cell cycle transit time occurs mainly during G₁ phase. the cycloheximide (5.0 μg/ml) and actinomycin D (3.0 μg/ml) restriction points occur 0.2 and 1.7 hr prior to entry into S phase, respectively. the transit time distributions are further characterized by the moments of the distributions. the variance (2nd moment about the mean) of the transit time distribution at the actinomycin D restriction point is similar to the variance of the transit time distribution at the G₁/S border, thus variation in cell cycle transit time originates earlier than 1.7 hr prior to entry into S phase (i.e., the first 3/4 of G₁). If G₁ transit time variability and cell cycle control are related, then the results presented here indicate that the major regulatory events do not occur during late G₁ phase.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea. Received: 16 June 1998 / Accepted: 13 July 1998     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Cell cycle-dependent gene expression in V point-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells stimulated to proliferate in an amino acid-deficient medium arrest in mid-G₁ at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G₁ and enter S phase. As data presented here show that mRNA synthesis is needed for 2–3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid-G₁ arrest protocol to analyze gene expression in late G₁. We found that although stimulation of cells in amino acid-deficient medium did not inhibit the induction of genes expressed in early G₁, genes normally expressed in late G₁ were expressed only after release from the V point. The expression of late G₁ genes in cells released from the V point was temporally similar, in respect to G₁ location, as was seen in stimulation of quiescent G_o cells. As this protocol effectively divides gene expression into early (pre-V point) and late (post-V point) categories, it should be useful in studies of growth factor-modulated events that regulate traverse of late G₁ and commitment to DNA synthesis. In addition, we used c-myb antisense oligonucleotides to show that c-myb expression, which occurs in late G₁, is required for BALB/c-3T3 fibroblasts to traverse late G₁ and initiate DNA synthesis. © 1993 Wiley-Liss, Inc.     

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G₁ and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G₁ phase was enriched in origin sequences in comparison with matrix-attached DNA from early G₁ phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G₁ phase and dissociate after initiation of DNA replication in S phase.     

Delayed inhibition of DNA synthesis in mouse jejunum by low doses of actinomycin D

Actinomycin D (0.008 μg/gm of body weight) injected intraperitoneally every two hours, produced a prompt 50% inhibition of RNA synthesis in the jejunum of mice, and a delayed inhibition of DNA synthesis, that reached its maximum inhibition (68% of control values) 4.5 hours after the first injection of actinomycin D. Autoradiographic studies indicated that this low level of actinomycin D inhibited a step in the G₁ phase of the cell cycle, preventing the initiation, but not affecting the continuation, of DNA biosynthesis. The activity of DNA polymerase was not affected under these conditions. The results are substantially similar to those previously obtained with Ehrlich ascites cells growing in the peritoneal cavity of mice and can be interpreted as indicating that in the G₁ phase of dividing cells there is an actinomycin sensitive step whose inhibition prevents the entrance of cells into the DNA-synthesis phase.     

STUDIES ON THE DIVISION CYCLE OF MAMMALIAN CELLS : V. Modifications of Time Parameters by Different Steady-State Culture Conditions

In cultures of murine neoplastic mast cells, the duration of different phases of the division cycle (G₁, S, G₂, and mitosis [M]) was determined under optimal and several well-defined suboptimal growth conditions. Two methods of evaluation were applied to the same culture system: first, the relative number of G₁, S, G₂, and M cells was determined by pulse labeling of samples with thymidine-³H and subsequent radioautography in conjunction with a microfluorometric technique permitting rapid measurements of cellular DNA content; second, after pulse labeling with thymidine-³H, the variations with time of the mitotic labeling index were analyzed. Suboptimal culture conditions were obtained by reducing the concentration of single essential medium components (leucine, glucose, or serum) or by the addition of specific metabolic inhibitors (actinomycin D, amethopterin). Growth-limiting culture conditions resulted in increased generation times. Even under control conditions, the cell number doubling time exceeded the generation time, and this difference was more pronounced in suboptimal media. Under most of the suboptimal conditions tested, the increase in generation time was attributable primarily to an extended duration of the G₁ phase. Under certain growth-limiting conditions, however, other phases were also prolonged. In addition, the variabilities of the generation time and of certain cell cycle phases were increased under suboptimal culture conditions. Results obtained by the two methods of evaluation were, in general, in good agreement with each other. Some differences were, however, observed and interpreted in terms of cell death and/or asymmetric frequency distributions of cell cycle parameters.     

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.     

Detection of replication initiation by a replicon family in DNA of synchronized pea (Pisum sativum) root cells using benzoylated naphthoylated DEAE-cellulose chromatography

Fractionated replicating DNA from pea was obtained from both synchronized cells just starting replication and from carbohydrate-starved cells ending replication. Benzoylated naphthoylated DEAE-cellulose chromatography of pulse-labeled DNA digested with EcoR I gave evidence that a family of replicons initiated replication 45 to 60 min after synchronized cells were released from the G₁/S phase boundary. DNA from cells labeled in late S phase, on the other hand, showed no signs of additional replication initiations before entering G₂ phase. Results with DNA from both early and late S phase cells comply with a model based on the premise that with short pulses of [³H]-thymidine the isotope is localized at replication forks and that longer pulses label both replication forks and recently replicated segments of double-stranded DNA. The model applies only to DNA subjected to fragmentation before chromatography.The results also suggest that benzoylated naphthoylated DEAE-cellulose chromatography is a useful means to isolate origins and replication forks from synchronized plant cells.     

DNA damage-induced S phase arrest in human breast cancer depends on Chk1, but G2 arrest can occur independently of Chk1, Chk2 or MAPKAPK2

Checkpoint kinases Chk1 and Chk2 are two key components in the DNA damage-activated checkpoint signaling pathways. To distinguish the roles of Chk1 and Chk2 in S and G₂ checkpoints after DNA damage, derivatives of the human breast cancer cell line MDA-MB-231 were established that express short hairpin RNAs to selectively suppress Chk1 or Chk2 expression. DNA damage was induced with the topoisomerase I inhibitor SN38 which arrests cells in S or G₂ phase depending on concentration. Depletion of Chk1 resulted in loss of S phase arrest upon incubation with SN38, but the cells still arrested in G₂. Suppression of Chk2 had no impact on cell cycle arrest, while cells concurrently suppressed for both Chk1 and Chk2 still arrested primarily in G₂ suggesting the presence of an alternate checkpoint regulator. One critical target for Chk1 is Cdc25A which is phosphorylated and degraded to prevent cell cycle progression. Cells arrested in G₂ in the absence of Chk1/Chk2 still showed regulation of Cdc25A consistent with the action of an alternate kinase. One candidate for an alternate checkpoint kinase is MAPKAPK2 (MK2), yet this kinase was minimally activated by DNA damage and its inhibition did not facilitate either S or G₂ progression. Furthermore, we were unable to substantiate the recent observation that the Chk1 inhibitor UCN-01 inhibits MK2. These results show that Chk1, but neither Chk2 nor MK2, is an important regulator of S phase arrest, and suggest that an additional kinase can contribute to the G2 arrest.