Sulfhydryl groups during the S phase: comparison of cells from G 1 , plateau-phase G 1 , and G 0

The non-protein sulfhydryl (NPSH) content of cells moving into S from G₁, plateau phase G₁, and G₀ was measured. Chinese hamster ovary (CHO) cells accumulated in G₁ by growth into plateau phase contain only one-fourth the NPSH concentration of cycling C₁ cells or G₁ cells accumulated by brief growth in isoleucine-deficient medium. Upon dilution of plateau cultures with fresh medium, cellular NPSH content increases rapidly, reaching the same level as that in cycling cells within four hours. This increase is prevented by cycloheximide but not by actinomycin D or hydroxyurea. Neither CHO cells cycling in vitro nor salivary gland G₀ cells stimulated with isoproterenol in vivo show significant changes in intracellular NPSH concentrations during S phase. This suggests that the concentration of intracellular NPSH (glutathione) remains constant during the cell cycle except when cells are grown to plateau phase in exhausted or deficient medium, in which case normal degradation exceeds synthesis and the gross level falls until fresh medium is provided and synthesis, apparently on preexisting RNA templates, accelerates.     

The mechanism of regulation of fibroblastic cell replication. I. Properties of the system

Sixty to eighty per cent of the cells in a culture of human diploid fibroblasts may be stimulated from the state of density dependent inhibition of replication to active DNA synthesis and division. The maximum response is effected by 50% serum within the pH range 7.2–8.0. The proportion of cells responding depends on the concentration of serum protein in the medium which may be effectively substituted by crystalling serum albumin. There is a differential sensitivity to the stimulus of cells in the densely packed centers of whorls and in the less dense areas between the whorls. The cell response is parasynchronous and the median durations of the various phases of the cell cycle are: G₁I 6 β ?æ^® ¿ ∞ 8 hours, G₂ = 6 hours and doubling time = 30 hours. The stimulatory effect of fresh medium is lost during contact with dense cultures so that it has only 50% of its initial capacity after 14 hours. It can be restored by dialysis against serum-free medium. The stimulus must be applied for at least ten hours to be effective in inducing DNA synthesis. During the latter half of ten hour induction period subsequent DNA synthesis becomes exquisitely sensitive to actinomycin D. After this time an increasing number of cells become irreversibly committed to replicate. The data are interpreted to indicate that during contact with serum proteins (including albumin) changes in the cell surface, if continued long enough, trigger a mechanism which involves the synthesis of a unique RNA species during the fifth to tenth hours. After this RNA has been synthesized the cells are then committed to DNA synthesis.     

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.     

Decreased epidermal growth factor binding in cells growth arrested in G1 by nutrient deficiency

Previous studies have shown that the nontransformed AKR-2B mouse embryo derived cell line may growth arrest by two separate mechanisms in the G₁ phase of the cell cycle-growth factor deficiency arrest (G₀) and low molecular weight nutrient deficiency arrest. An examination of epidermal growth factor (EGF) receptors under the different resting or growth conditions has shown that rapidly growing cells or cells arrested due to growth factor deficiency have the expected amount of ¹²⁵I-EGF binding with approximately 10⁵ receptors per cell being present in G₀ arrested cells. In contrast, cells arrested due to nutrient deficiency show a reduction in ¹²⁵I-EGF binding to 10--20% of that observed under the other conditions. This effect appears to be due to decreased receptor number and not to a change in the affinity of the receptor. Stimulation of DNA synthesis by nutrient replenishment causes a tenfold increase in EGF binding 20 hours later, with some increase in binding being detectable as early as six hours. The increase in binding is inhibited by cycloheximide and actinomycin D. This suggests that new mRNA synthesis as well as increased protein synthesis is required for the increase in EGF binding.     

Lipoxygenase metabolism is required for interleukin-3 dependent proliferation and cell cycle progression of the human M-07e cell line

The cell line M-07e requires either Interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF) for proliferation in vitro. Cells deprived of growth factor for up to 48 hours remain viable but no longer divide. The growth-factor-deprived M-07e cells begin to divide within 48 hours of reexposure to IL-3. Flow cytometric analysis of M-07e cells labeled with hypotonic propidium iodide demonstrates that the percentage of cells undergoing DNA synthesis decreases from 24%, in a log phase population of IL-3 stimulated cells, to 1% when cells are deprived of IL-3 for 24 hours. IL-3-deprived cells accumulate predominantly in a flow cytometry peak representative of G₀/G₁. DNA synthetic activity, as determined by tritiated thymidine uptake and flow cytometry, resumes between 12 and 18 hours after reexposure to IL-3, reaching a peak of up to 40% by 24 hours and returning to log phase levels by 72 hours. Prior to initiation of DNA synthesis, increases are seen in mRNA levels for five-lipoxygenase-activating protein (FLAP). Following reexposure to IL-3, a rapid time-dependent biosynthesis of leukotriene D4 (LTD4) is induced by M-07e cells. When IL-3 is added in the presence of any of three lipoxygenase inhibitors tested (Piriprost, caffeic acid, nordihydroguiaretic acid) or FLAP inhibitor, MK-886, there is dose-dependent inhibition of the resumption of proliferation and of DNA synthesis. Flow cytometric cell cycle analysis demonstrates that the inhibited cells remain in the G₀/G₁ population and do not progress through the cell cycle. These results are consistent with our previous observation that an intact lipoxygenase pathway is necessary for hematopoietic growth-factor-stimulated colony formation of normal bone marrow myeloid progenitors and suggest that the induction of a lipoxygenase metabolite or metabolites is necessary for myeloid cells to progress through the cell cycle when stimulated by a hematopoietic growth factor. J. Cell. Physiol. 170:309–315, 1997. © 1997 Wiley-Liss, Inc.     

Effects of rifampicin, streptolydigin and actinomycin D on the replication of Col E1 plasmid DNA in Escherichia coli

We recently reported (Clewell et al., 1972) on an inhibitory effect of rifampicin on Col E1 plasmid replication. The present study represents a further characterization of this phenomenon as well as a study of the effects of two other known inhibitors of RNA synthesis, Streptolydigin and actinomycin D.During treatment of cells with chloramphenicol the colicinogenic factor E₁ (Col E1) continues to replicate for more than ton hours. During this time 4 to 5 S RNA is also synthesized. When varying concentrations of rifampicin were included during chloramphenicol treatment, inhibition of plasmid DNA synthesis correlated very closely with inhibition of cellular RNA synthesis. Similar experiments testing the effects of Streptolydigin and actinomycin D (during chloramphenicol treatment) showed that cellular RNA synthesis was at least 100 times more sensitive to these drugs than was plasmid DNA synthesis.When actively growing cells (i.e. cells not treated with chloramphenicol) were treated with a high concentration of rifampicin (250 μg/ml), chromosomal DNA synthesis continued to an extent representing about a 50% increase in DNA, while plasmid DNA synthesis appeared to stop abruptly.     

Reversible accumulation of plant suspension cell cultures in g(1) phase and subsequent synchronous traverse of the cell cycle

The induction of DNA synthesis in Datura innoxia Mill. cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G₂ phase, and underwent cell division simultaneously. Only 10% of the cells remained in G₀ or G₁. Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G₀ + G₁/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G₂ phase. However, reversal of G₁ arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G₀ + G₁ phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G₀. The frequency of cells entering G₀ was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

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.     

AGE-DEPENDENT TOXIC PROPERTIES OF ACTINOMYCIN D AND X-RAYS IN CULTURED CHINESE HAMSTER CELLS

A comparative study was made of the toxic properties of actinomycin D and X-rays using synchronized populations of Chinese hamster cells cultured in vitro. X-irradiated cells are most resistant in the latter half of the DNA synthetic period (late S). While cells treated with actinomycin D appear to go through a survival maximum at the same age, they are most resistant after the completion of DNA synthesis; i.e. in G₂ (or G₂-mitosis). In spite of these differences, we found that actinomycin D damage in late S cells interacts with X-ray damage. Thus, a common locus for the site of actions of both agents is suggested which may be in or around the genome of a cell in view of the well-known DNA binding properties of actinomycin D.     

Effects of 1000 R whole-body X-irradiation on DNA synthesis and mitosis in the duodenal crypts of the BCF1 mouse

Summary Effects of 1000 R, whole-body X-irradiation on the proliferative cells of the mouse duodenal crypts, in the four phases of the generation cycle; namely, the DNA synthesis phase, S; the pre-mitotic gap, G ₂; the division phase or mitosis, M; and the pre-synthesis gap, G ₁. As pointed out by Whitmore and Till (1964) G₁ and G₂ are characterized only by the fact that no DNA synthesis is taking place in these phases.In the intestinal crypts of BCF₁ mice, a 1000 R whole-body X-ray exposure blocks cells in G₂ for approximately 18 hours, and reduces the number of cells in S to less than 1/2 that observed in control animals during the first 12 hours after exposure. Cells synthesizing DNA, and undergoing division, remain few in number for more than 48 hours. Between 48 and 72 hours a compensatory reaction begins, and the number of cells in M and S increases from 28 at 48 hours to 150 at 72 hours and reaches a mean value of 482 at 96 hours.Work supported under the auspices of the US Atomic Energy Commission.     

EFFECT OF METABOLIC INHIBITORS ON NUCLEAR PORE FORMATION DURING THE HELA S3 CELL CYCLE

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S₃ cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G₁ phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3–4 h after mitosis, i.e., about 1–2 h before S phase begins.     

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.     

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.     

Reduced inhibition of DNA synthesis and G2 arrest during the cell cycle of resistant HeLa cells in response tocis-diamminedichloroplatinum

The influence of cisplatin, an anticancer agent, on DNA synthesis and cell cycle progression of a cisplatin-resistant cell line was investigated. Cell cycle analysis using flow cytometry showed that cytotoxic concentrations of cisplatin caused a transient inhibition of parental HeLa cells at S phase, followed by accumulation at G₂ phase. In contrast, the resistant cells progressed through the cell cycle without being affected by the same treatment. However, cell cycle distributions were the same in the resistant and the parental cells at IC₅₀, the drug concentration inhibiting cell growth by 50%. Studies using a [³H]thymidine incorporation technique also demonstrated a transient inhibition of DNA synthesis in HeLa cells by cisplatin; such inhibition was greatly reduced in the resistant cells. These data argue for the hypothesis that the inhibition of DNA synthesis is important in determining cisplatin-induced cytotoxicity. In addition, the accumulation of cells at G₀/G₁ by serum starvation was not effective in the resistant cells compared to the parental cells, suggesting that the control of cell cycle exiting is also altered in the resistant cells. Taken together, these results support the notion that alterations in cell cycle control, in particular G₂ arrest, are important in determining the sensitivity or resistance of mammalian cells to cisplatin and may have a role in clinical protocols.     

Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G₁/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU-treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU-treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR-treated cultures cells slowly progressed into mid S-phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G₂ phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G₁/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.     

RELATIONSHIPS BETWEEN NUCLEIC ACID SYNTHETIC PATTERNS AND ENCYSTMENT IN AGING UNAGITATED CULTURES OF ACANTHAMOEBA CASTELLANII

Changes in the levels of DNA and RNA syntheses have been studied in unagitated cultures of Acanthamoeba castellanii during the phases of logarithmic multiplication (LM) and population growth deceleration (PGD). Pulse-labeling experiments show that the rate of DNA synthesis decreases at the same time that DNA per cell is known to drop by 50%. The drop in DNA content has been explained by demonstrating with hydroxyurea that the majority of LM amebas can replicate once when DNA synthesis is inhibited and, therefore, must be in G₂, whereas the PGD amebas cannot multiply in the presence of inhibitor and, therefore, must be in G₁. The inhibition of DNA synthesis in LM or PGD cells has been shown to induce encystment. The rate of RNA synthesis, as illustrated by pulse-labeling experiments, increases 25% in late LM-early PGD while RNA per cell increases 75%. The rate of synthesis then decreases 65%. The majority of accumulated RNA has been demonstrated to be ribosomal by disc electrophoresis. By using actinomycin D at different stages during the RNA build-up, the ability of the amebas to encyst has been shown to depend on the presence of this RNA. The observations on DNA and RNA are discussed with respect to the occurrence of cysts in the cultures during PGD.     

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.     

Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up-regulation of oncogenes in S-49 cyc− cells

Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c-AMP in prostaglandin mediated cell cycle arrest, we use the-49 lymphoma variant (cyc^?) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A₁ (dmPGA₁) inhibits DNA synthesis and cell growth in cyc^? cells. DNA synthesis is inhibited 42% by dmPGA₁ (50 μM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the α,β unsaturated ketone ring. Dimethyl PGA₁ is most effective in inhibiting DNA synthesis in cyc^? cells, with prostaglandins PGE₁ and PGB₁ being less potent inhibitors of DNA synthesis. DmPGE₂ caused a significant stimulation of DNA synthesis. S-49 cyc^- variant cells exposed to (30–50 μm) dmPGA₁, arrested in the G₁ phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S-49 cyc^? cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G₁, S, G₂, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S-phase DNA synthesis from the G₁ cell cycle block. The S-49 cyc^? cells are known to have a G₁/S boundary through M phase transition time of 14.8 h, making the location of the prostaglandin cell cycle arrest at or very near the G₁/S interface. The oncogenes, c-fos and c-myc which are normally expressed during G₁ in proliferating cells have a 2–3 fold enhanced expression in prostaglandin G₁ arrested cells. These data using the S-49 variants demonstrate that dmPGA₁ inhibits DNA synthesis and arrests the cell cycle independent of cAMP-mediated effects. The prostaglandin arrested cells maintain the gene expression of a G₁ synchronous cell which suggests a unique molecular mechanism for prostaglandin action in arresting cell growth. These properties indicate that this compound may be an effective tool to study molecular mechanisms of regulation of the cell cycle.     

Phosphorylation of cyclin D1 regulated by ATM or ATR controls cell cycle progression

Cyclin D1 is required at high levels for passage through G₁ phase but must be reduced to low levels during S phase to avoid the inhibition of DNA synthesis. This suppression requires the phosphorylation of Thr286, which is induced directly by DNA synthesis. Because the checkpoint kinase ATR is activated by normal replication as well as by DNA damage, its potential role in regulating cyclin D1 phosphorylation was tested. We found that ATR, activated by either UV irradiation or the topoisomerase IIβ binding protein 1 activator, promoted cyclin D1 phosphorylation. Small interfering RNA against ATR inhibited UV-induced Thr286 phosphorylation, together with that seen in normally cycling cells, indicating that ATR regulates cyclin D1 phosphorylation in normal as well as stressed cells. Following double-stranded DNA (dsDNA) breakage, the related checkpoint kinase ATM was also able to promote the phosphorylation of cyclin D1 Thr286. The relationship between these checkpoint kinases and cyclin D1 was extended when we found that normal cell cycle blockage in G₁ phase observed following dsDNA damage was efficiently overcome when exogenous cyclin D1 was expressed within the cells. These results indicate that checkpoint kinases play a critical role in regulating cell cycle progression in normal and stressed cells by directing the phosphorylation of cyclin D1.