Synchronized Cell Populations Research Articles

Connexin-based intercellular communication and astrocyte heterogeneity

This review gives an overview of the current knowledge on connexin-mediated communication in astrocytes, covering gap junction and hemichannel functions mediated by connexins. Astroglia is the main brain cell type that expresses the largest amount of connexin and exhibits high level of gap junctional communication compared to neurons and oligodendrocytes. However, in certain developmental and regional situations, astrocytes are also coupled with oligodendrocytes and neurons. This heterotypic coupling is infrequent and minor in terms of extent of the coupling area, which does not mean that it is not important in terms of cell interaction. Here, we present an update on heterogeneity of connexin expression and function at the molecular, subcellular, cellular and networking levels. Interestingly, while astrocytes were initially considered as a homogenous population, there is now increasing evidence for morphological, developmental, molecular and physiological heterogeneity of astrocytes. Consequently, the specificity of gap junction channel- and hemichannel-mediated communication, which tends to synchronize cell populations, is also a parameter to take into account when neuroglial interactions are investigated. This article is part of a Special Issue entitled Electrical Synapses.

A microfluidic “baby machine” for cell synchronization

Common techniques used to synchronize eukaryotic cells in the cell cycle often impose metabolic stress on the cells or physically select for size rather than age. To address these deficiencies, a minimally perturbing method known as the "baby machine" was developed previously. In the technique, suspension cells are attached to a membrane, and as the cells divide, the newborn cells are eluted to produce a synchronous population of cells in the G1 phase of the cell cycle. However, the existing "baby machine" is only suitable for cells which can be chemically attached to a surface. Here, we present a microfluidic "baby machine" in which cells are held onto a surface by pressure differences rather than chemical attachment. As a result, our method can in principle be used to synchronize a variety of cell types, including cells which may have weak or unknown surface attachment chemistries. We validate our microfluidic "baby machine" by using it to produce a synchronous population of newborn L1210 mouse lymphocytic leukemia cells in G1 phase.

Probabilistic RNA partitioning generates transient increases in the normalized variance of RNA numbers in synchronized populations of Escherichia coli

We explore the effects of probabilistic RNA partitioning during cell division on the normalized variance of RNA numbers across generations of bacterial populations. We first characterize these effects in model cell populations, where gene expression is modeled as a delayed stochastic process, as a function of the synchrony in cell division, the rate of division, and the RNA degradation rate. We further explore the additional variance that arises if the partitioning is biased. Next, in Escherichia coli cells expressing RNA tagged with MS2d-GFP, we measured the normalized variance of RNA numbers across several generations, with cell divisions synchronized by heat shock. We show that synchronized cell populations exhibit transient increases in normalized variance following cell divisions, as predicted by the model, which are not observed in unsynchronized populations. We conclude that errors in partitioning of RNA molecules generate diversity between the offspring of individual bacteria and thus constitute a form of reproductive bet-hedging.

Assembly of the Caulobacter cell division machine

Cytokinesis in Gram-negative bacteria is mediated by a multiprotein machine (the divisome) that invaginates and remodels the inner membrane, peptidoglycan and outer membrane. Understanding the order of divisome assembly would inform models of the interactions among its components and their respective functions. We leveraged the ability to isolate synchronous populations of Caulobacter crescentus cells to investigate assembly of the divisome and place the arrival of each component into functional context. Additionally, we investigated the genetic dependence of localization among divisome proteins and the cell cycle regulation of their transcript and protein levels to gain insight into the control mechanisms underlying their assembly. Our results revealed a picture of divisome assembly with unprecedented temporal resolution. Specifically, we observed (i) initial establishment of the division site, (ii) recruitment of early FtsZ-binding proteins, (iii) arrival of proteins involved in peptidoglycan remodelling, (iv) arrival of FtsA, (v) assembly of core divisome components, (vi) initiation of envelope invagination, (vii) recruitment of polar markers and cytoplasmic compartmentalization and (viii) cell separation. Our analysis revealed differences in divisome assembly among Caulobacter and other bacteria that establish a framework for identifying aspects of bacterial cytokinesis that are widely conserved from those that are more variable.

The Cell Cycle Regulated Transcriptome of Trypanosoma brucei

Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a “double-cut” elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.

Ubiquitin-dependent DNA damage bypass is separable from genome replication

Postreplication repair (PRR) is a pathway that allows cells to bypass or overcome lesions during DNA replication1. In eukaryotes, damage bypass is activated by ubiquitylation of the replication clamp PCNA through components of the RAD6 pathway2. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases3-5, polyubiquitylation is required for an error-free pathway that likely involves a template switch to the undamaged sister chromatid6. Both the timing of PRR events during the cell cycle and their location relative to replication forks, as well as the factors required downstream of PCNA ubiquitylation, have remained poorly characterised. Here we demonstrate that the RAD6 pathway normally operates during S phase. However, using an inducible system of DNA damage bypass in budding yeast, we show that the process is separable in time and space from genome replication, thus allowing direct visualisation and quantification of productive PRR tracts. We found that both during and after S phase ultraviolet radiation-induced lesions are bypassed predominantly via translesion synthesis, whereas the error-free pathway functions as a backup system. Our approach has for the first time revealed the distribution of PRR tracts in a synchronised cell population. It will allow an in-depth mechanistic analysis of how cells manage the processing of lesions to their genomes during and after replication.

Early Increase of Radiation-induced γH2AX Foci in a Human Ku70/80 Knockdown Cell Line Characterized by an Enhanced Radiosensitivity

A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (γH2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of γH2AX foci after irradiating synchronized cell populations with (60)Co γ-rays. Our results show statistically significant differences in the number of γH2AX foci between the repair-deficient and -proficient cell line, with a higher amount of γH2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.

The Aurora Kinase in Trypanosoma brucei Plays Distinctive Roles in Metaphase-Anaphase Transition and Cytokinetic Initiation

Aurora B kinase is an essential regulator of chromosome segregation with the action well characterized in eukaryotes. It is also implicated in cytokinesis, but the detailed mechanism remains less clear, partly due to the difficulty in separating the latter from the former function in a growing cell. A chemical genetic approach with an inhibitor of the enzyme added to a synchronized cell population at different stages of the cell cycle would probably solve this problem. In the deeply branched parasitic protozoan Trypanosoma brucei, an Aurora B homolog, TbAUK1, was found to control both chromosome segregation and cytokinetic initiation by evidence from RNAi and dominant negative mutation. To clearly separate these two functions, VX-680, an inhibitor of TbAUK1, was added to a synchronized T. brucei procyclic cell population at different cell cycle stages. The unique trans-localization pattern of the chromosomal passenger complex (CPC), consisting of TbAUK1 and two novel proteins TbCPC1 and TbCPC2, was monitored during mitosis and cytokinesis by following the migration of the proteins tagged with enhanced yellow fluorescence protein in live cells with time-lapse video microscopy. Inhibition of TbAUK1 function in S-phase, prophase or metaphase invariably arrests the cells in the metaphase, suggesting an action of TbAUK1 in promoting metaphase-anaphase transition. TbAUK1 inhibition in anaphase does not affect mitotic exit, but prevents trans-localization of the CPC from the spindle midzone to the anterior tip of the new flagellum attachment zone for cytokinetic initiation. The CPC in the midzone is dispersed back to the two segregated nuclei, while cytokinesis is inhibited. In and beyond telophase, TbAUK1 inhibition has no effect on the progression of cytokinesis or the subsequent G1, S and G2 phases until a new metaphase is attained. There are thus two clearly distinct points of TbAUK1 action in T. brucei: the metaphase-anaphase transition and cytokinetic initiation. This is the first time to our knowledge that the dual functions of an Aurora B homolog is dissected and separated into two clearly distinct time frames in a cell cycle.

Aurora B kinase inhibition in mitosis: Strategies for optimizing the use of Aurora kinase inhibitors such as AT9283

Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

Enzymology of Purine and Pyrimidine Antimetabolites Used in the Treatment of Cancer

Enzymology of Purine and Pyrimidine Antimetabolites Used in the Treatment of Cancer

Microfluidic Self-Sorting of Mammalian Cells to Achieve Cell Cycle Synchrony by Hydrophoresis

Cell cycle studies for examining regulatory mechanisms and progression invariably require synchronization of cell cultures at a specific phase of the cell cycle. Current implementations to produce synchronous cell populations, however, tend to perturb normal cellular progression and metabolism and typically require complex, time-consuming preparations. Thus, it is challenging for the development of a simple, noninvasive, and effective means for cell cycle synchronization. We demonstrate the use of hydrophoretic size separation to sort cells in target phases of the cell cycle entirely based on a hydrodynamic principle. With this method, we found that there is a linear relationship between a cell's size and its position distribution in the hydrophoretic device. We also demonstrate the robustness of the hydrophoretic method for practical applications by sorting cells in the G(0)/G(1) and G(2)/M phases out of the original, asynchronous cells with a high level of synchrony of 95.5% and 85.2%, respectively. These results show that the hydrophoretic size separation can be used in order to collect cells at the same phase of the cell cycle in a gentle, noninvasive way.

Identification of G1-Regulated Genes in Normally Cycling Human Cells

BackgroundObtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked.Methodology and FindingsWe used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates (fos, atf3 and tceb) by qPCR to further validate the newly identified genes.Conclusion and SignificanceGenome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease.

Identification of G1‐Regulated Genes in Normally Cycling Human Cells

Obtaining synchronous cell populations is essential for studies of the cell cycle. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle almost certainly alter cellular events upon re‐entry into the cell cycle. Here we use a robotic mitotic shake‐off apparatus to select cells in late telophase of mitosis. Using two separate microarray experiments we examined gene activity in early G1: one isolated RNA hourly and the second isolated RNA every 15 minutes. We have also included data obtained by a variety of experimental methods to verify synchrony of these cell populations, including BrdU uptake, FACS, and microarray analyses of histone gene activity as well as activity of stress response genes. Our approach enabled us to identify a unique set of genes whose functions are currently unknown, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease.

Selection of mammalian cells based on their cell-cycle phase using dielectrophoresis

An effective, noninvasive means of selecting cells based on their phase within the cell cycle is an important capability for biological research. Current methods of producing synchronous cell populations, however, tend to disrupt the natural physiology of the cell or suffer from low synchronization yields. In this work, we report a microfluidic device that utilizes the dielectrophoresis phenomenon to synchronize cells by exploiting the relationship between the cell's volume and its phase in the cell cycle. The dielectrophoresis activated cell synchronizer (DACSync) device accepts an asynchronous mixture of cells at the inlet, fractionates the cell populations according to the cell-cycle phase (G(1)/S and G(2)/M), and elutes them through different outlets. The device is gentle and efficient; it utilizes electric fields that are 1-2 orders of magnitude below those used in electroporation and enriches asynchronous tumor cells in the G(1) phase to 96% in one round of sorting, in a continuous flow manner at a throughput of 2 x 10(5) cells per hour per microchannel. This work illustrates the feasibility of using laminar flow and electrokinetic forces for the efficient, noninvasive separation of living cells.

Modeling synchronous growth of bacterial populations in phased cultivation

The phasing technique is a method for synchronizing cell populations in a bioreactor. Periodic changes of substrate supply and depletion can provoke a cell cycle phasing of originally stochastic scattered proliferation patterns. Synchronized cell populations characterized by changes in DNA content distribution can be monitored by flow cytometry. Thus, studies of the dynamics of single cells in specific cell cycle phases are facilitated. Here we present an age structured model framework investigating synchronized populations using delay differential equations. Applying the framework not only cell populations synchronously increasing under balanced growth conditions, but also synchronized cultures growing in continuous phasing experiments can be described. A process model developed for describing phased cultures was fitted to growth data obtained from a synchronous cultivation of Cupriavidus necator. Its potential utility is demonstrated by a quantitative process description and by its ability to identify ways in which the grade of synchrony could be improved.

An appreciation of Dr Steven Pfeiffer, 1940–2007

Soon after this special edition of the Journal of Neurochemistry went to press, we were informed of the sad news that Steve Pfeiffer had succumbed to cancer after a 2-year struggle. Steve was a loyal friend, a wonderful colleague, and his interactions with many scientists in the fields of glial cells and glycosphingolipids were always positive and stimulating. He will be greatly missed by his family and friends. A detailed obituary of Steve can be found on the ASN website, and also in the Hartford Courant on Monday, August 27. What follows are thoughts from two friends, who got to know Steve well over the past few years. One of us (AF) was a co-chair of the symposium to which this special issue is dedicated, and spent the year or so before the symposium working on a day-to-day basis with Steve, so much so that over 1300 e-mails were exchanged! It seems more than a little strange not to be consulting with Steve now about the contents of this short note. Steve had a productive research career, mainly in the field of myelin biology. He published his first paper, on selecting synchronous populations of mammalian cells, in Nature in 1967, and after that, moved to the study of Schwann cells, glial cells, and oligodendrocytes. This led him to the study of galactolipids and other glycolipids, and in the 1990s, he became an active member of the sphingolipid community and was a regular participant at many conferences in this field. He was particularly interested in the possible roles of lipid rafts in myelin function, and lectured on this issue at recent meetings. Apart from his scientific contributions, Steve was simply a great person to know and spend time with. By way of example, at a recent Gordon Conference, Steve was asked to chair a ‘poster session’, in which mainly young scientists were asked to give short 5-min talks. Steve was asked to chair this session, as we felt his easy-going manner, and excellent interactions with students and postdocs, would be perfectly suited for this task. At the ISN Neurochemistry Conference in Antigua, Steve was his usual bubbly self and maintained his sense of humor, despite the fact that at the time he was undergoing a number of unpleasant treatments for cancer. Steve was in particularly good form when he met the Prime Minister of Antigua, and when he chaired the closing session of the meeting! That was the last time that we saw him, though emails continued to flow back and forth till as recently as August 3, when Steve wanted to discuss the role of myelin proteins in a mutant mouse. Steve is sadly no longer with us. We will cherish the memories of all of our wonderful interactions with him.

13C-Labeled metabolic flux analysis of a fed-batch culture of elutriatedSaccharomyces cerevisiae

This study addresses the question of whether observable changes in fluxes in the primary carbon metabolism of Saccharomyces cerevisiae occur between the different phases of the cell division cycle. To detect such changes by metabolic flux analysis, a 13C-labeling experiment was performed with a fed-batch culture inoculated with a partially synchronized cell population obtained through centrifugal elutriation. Such a culture exhibits dynamic changes in the fractions of cells in different cell cycle phases over time. The mass isotopomer distributions of free intracellular metabolites in central carbon metabolism were measured by liquid chromatography-mass spectrometry. For four time points during the culture, these distributions were used to obtain the best estimates for the metabolic fluxes. The obtained flux fits suggested that the optimally fitted split ratio for the pentose phosphate pathway changed by almost a factor of 2 up and down around a value of 0.27 during the experiment. Statistical analysis revealed that some of the fitted flux distributions for different time points were significantly different from each other, indicating that cell cycle-dependent variations in cytosolic metabolic fluxes indeed occurred.

P276-00, a novel cyclin-dependent inhibitor induces G1-G2 arrest, shows antitumor activity on cisplatin-resistant cells and significant in vivo efficacy in tumor models

P276-00, a flavone that inhibits cyclin-dependent kinases, has been identified by us recently as a novel antineoplastic agent. In this study, we have selected a panel of human tumor cell lines and xenografts to allow determination of selectivity and efficacy of P276-00. When tested against a panel of 16 cisplatin-sensitive and cisplatin-resistant cell lines, the antiproliferative potential of P276-00 was found to be approximately 30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was approximately 26-fold more potent than cisplatin, and further, it was also found to be active against cisplatin-resistant tumors of central nervous system, melanoma, prostate, and renal cancers. Further, we studied the effects of P276-00 on cell cycle progression by flow cytometry using asynchronous and synchronous population of tumor and normal cells. Asynchronous population of human prostate carcinoma (PC-3) and human promyelocytic leukemia (HL-60) cells when exposed to P276-00 showed arrest of slow-growing PC-3 cells in G(2)-M with no significant apoptosis observed up to 72 h. Unlike PC-3, significant apoptosis was seen in fast-growing HL-60 cells at 6 h. However, synchronized human non-small cell lung carcinoma (H-460) and human normal lung fibroblast (WI-38) cells showed arrest of cells in G(1). H-460 cells undergo apoptosis, which increases with longer exposure to the compound and also after exposure to P276-00 for 48 h followed by recovery. In contrast, the normal cells (WI-38) remain arrested in G(1) with no significant apoptosis up to 72 h of exposure and also after 48 h of P276-00 treatment followed by recovery, confirming our previous results that P276-00 was less effective against normal cells compared with cancer cells. After promising in vitro results, P276-00 was checked for in vivo efficacy in murine tumor and human xenograft models. Growth inhibition of murine colon cancer (CA-51) was significant when P276-00 was administered i.p. at 50 mg/kg daily for 20 treatments. However, in murine lung carcinoma model (Lewis lung), an increased dose of 60 mg/kg (30 mg/kg twice daily) administered every alternate day i.p. for seven treatments showed significant inhibition in the growth. Further studies were undertaken to establish the efficacy profile of P276-00 in human tumor xenograft models. In the two xenograft models studied, P276-00 showed potent in vivo antitumor potential. Compound P276-00 at a dose of 35 mg/kg administered daily via the i.p. route for 10 days showed significant (P < 0.05) inhibition in the growth of human colon carcinoma HCT-116 xenograft. Furthermore, P276-00 at a dose of 50 mg/kg once daily and 30 mg/kg twice daily administered via i.p. route for 20 treatments significantly (P < 0.05) inhibited growth of human non-small cell lung carcinoma H-460 xenograft. Thus, the in vitro cellular potency, together with in vivo antitumor activity, confirms the potential of P276-00, a cyclin-dependent kinase inhibitor as an anticancer molecule.

Chemical Inactivation of Cdc7 Kinase in Budding Yeast Results in a Reversible Arrest That Allows Efficient Cell Synchronization Prior to Meiotic Recombination

Genetic studies in budding yeast have provided many fundamental insights into the specialized cell division of meiosis, including the identification of evolutionarily conserved meiosis-specific genes and an understanding of the molecular basis for recombination. Biochemical studies have lagged behind, however, due to the difficulty in obtaining highly synchronized populations of yeast cells. A chemical genetic approach was used to create a novel conditional allele of the highly conserved protein kinase Cdc7 (cdc7-as3) that enables cells to be synchronized immediately prior to recombination. When Cdc7-as3 is inactivated by addition of inhibitor to sporulation medium, cells undergo a delayed premeiotic S phase, then arrest in prophase before double-strand break (DSB) formation. The arrest is easily reversed by removal of the inhibitor, after which cells rapidly and synchronously proceed through recombination and meiosis I. Using the synchrony resulting from the cdc7-as3 system, DSB-dependent phosphorylation of the meiosis-specific chromosomal core protein, Hop1, was shown to occur after DSBs. The cdc7-as3 mutant therefore provides a valuable tool not only for understanding the role of Cdc7 in meiosis, but also for facilitating biochemical and cytological studies of recombination.

Regulation of Late G1/S Phase Transition and APCCdh1by Reactive Oxygen Species

Proliferating cells have a higher metabolic rate than quiescent cells. To investigate the role of metabolism in cell cycle progression, we examined cell size, mitochondrial mass, and reactive oxygen species (ROS) levels in highly synchronized cell populations progressing from early G1 to S phase. We found that ROS steadily increased, compared to cell size and mitochondrial mass, through the cell cycle. Since ROS has been shown to influence cell proliferation and transformation, we hypothesized that ROS could contribute to cell cycle progression. Antioxidant treatment of cells induced a late-G1-phase cell cycle arrest characterized by continued cellular growth, active cyclin D-Cdk4/6 and active cyclin E-Cdk2 kinases, and inactive hyperphosphorylated pRb. However, antioxidant-treated cells failed to accumulate cyclin A protein, a requisite step for initiation of DNA synthesis. Further examination revealed that cyclin A continued to be ubiquitinated by the anaphase promoting complex (APC) and to be degraded by the proteasome. This antioxidant arrest could be rescued by overexpression of Emi1, an APC inhibitor. These observations reveal an intrinsic late-G1-phase checkpoint, after transition across the growth factor-dependent G1 restriction point, that links increased steady-state levels of endogenous ROS and cell cycle progression through continued activity of APC in association with Cdh1.