Distinct Checkpoint Research Articles

Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model

Cancer immunotherapy has shown remarkable recent progress. Immune checkpoint blocking antibodies have become the most successful anti-cancer agent class ever developed, with six distinct agents approved since 2011 for a wide variety of cancers. Although age is the biggest risk factor for cancer (aside from selected early-onset pediatric cancers), these agents were tested pre-clinically in young hosts, and there is remarkably little published on the effects of host age on treatment outcomes in pre-clinical studies or human clinical trials. The three principal immune checkpoints against which blocking antibodies have been FDA-approved for human use are CTLA-4, PD-1 and PD-L1. We used a mouse model of transplantable, orthotopic B16 melanoma to test age effects of treatments with anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibodies. All three agents were highly effective in treating young tumor-bearing hosts as expected. Anti-PD-L1 as a single agent had no effect on tumor growth in aged hosts, anti-CTLA-4 had detectable, modest effects and anti-PD-1 was essentially as effective in aged as in young hosts, the first single agent we have identified not to lose efficacy with age in this model. Other important differences in young versus aged hosts included lack of anti-CTLA-4-mediated depletion of intratumor regulatory T cells in aged hosts and poorer ability of all three agents to activate T cells in aged versus young hosts. Anti-CTLA-4 efficacy appeared to improve when combined with anti-PD-L1. Regulatory T cell depletion with FDA-approved denileukin diftitox did not improve treatment by any single agent. Aged mice tolerated treatments as well as young mice without obvious toxicities at equivalent doses.

Clinical Development of PD-1 in Advanced Melanoma.

The development of new treatment options has dramatically improved the landscape for patients with advanced melanoma. Part of these advances emerged through the identification of the importance of factors that regulate the immune system, including proteins that negatively modulate T cell-mediated responses termed "immune checkpoints." Indeed, blockade of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) immune checkpoint served as a proof of principle that the manipulation of these molecules could induce robust anticancer effects, yet limited to a small percentage of patients. Targeting a distinct checkpoint, the PD-1 yielded improved outcomes and reduced toxicity compared with CTLA-4 blockade and, in separate studies, chemotherapy. More recently, combined CTLA-4/PD-1 blockade was shown to result in higher response rates, while accompanied by increased toxicity. In this article, we review the clinical development of anti-PD-1 monotherapy and combinations that may expand the benefit of immunotherapy for patients with advanced melanoma.

Abstract 2988: Immunogenomic analyses of tumor cells and microenvironment in patients with advanced melanoma before and after treatment with nivolumab

Abstract Background: Response to checkpoint blockade may be dependent on tumor mutational load and the presence of antigen-specific effector T cells in the tumor microenvironment; however, how blockade modulates these features during therapy is unclear. We assessed genomic changes in tumors from patients (pts) with advanced melanoma receiving nivolumab (nivo) who progressed on ipilimumab (ipi-P) or were ipi-naive (ipi-N). Methods: Tumor biopsies were collected pretreatment and 4 weeks post first nivo dose from ipi-N or ipi-P pts treated with nivo 3 mg/kg Q2W in the phase 1 open-label CA209-038 study (NCT01621490). Biopsies from 68 pts were analyzed by whole exome, transcriptome, and/or TCR sequencing (paired biopsies from 41, 42, and 34 pts, respectively). Results: Objective response rate (ORR) in the overall cohort (n=85) was 27% with similar ORR in ipi-N and ipi-P cohorts. In the genomic cohort (n=68), ORR was 23% with a similar number of complete or partial responses (CR/PR) in ipi-N and ipi-P pts (n=7 and n=8, respectively). Prior to treatment, mutational and neoantigen load were comparable, regardless of previous treatment. Following nivo treatment, both mutational and neoantigen load were reduced 5-fold in pts who responded (CR/PR; n=9) and 1.2-fold in pts with stable disease (SD, n=13) compared with a 1.1-fold increase in pts with progressive disease (PD, n=19). Intratumoral heterogeneity analysis before and after nivo demonstrated that CR/PR pts generally lost tumor mutation clones/subclones. Novel tumor mutation clones were observed in on-treatment samples from 2 CR/PR pts and all pts who progressed on nivo. Transcriptome analyses revealed significant increases in distinct tumor immune cell subsets (CD8+ T cells and NK cells) and immune checkpoint gene expression (LAG3, CTLA4, PCDC1, and CD274 [PD-L1]) following nivo, which were more pronounced in pts with CR/PR vs PD (log2 fold-changes of 1.24, 1.07, 1.71, and 0.74, respectively). Consistent with the transcriptome analyses, tumor-infiltrating lymphocytes, as assessed by immunohistochemistry, generally increased following nivo in pts who responded: 2.8 vs 1.9-fold change in CR/PR/SD vs PD in the ipi-P cohort; 4.8 vs 1.8-fold change in CR/PR/SD vs PD in the ipi-N cohort. Differences in treatment-related TCR repertoire diversity changes were apparent between pts who responded within the ipi-N and ipi-P cohorts: a decrease in the evenness of T-cell clonotype distribution was observed among pts with CR/PR/SD relative to pts with PD in the ipi-N cohort (P=0.036), but not in the ipi-P cohort. Conclusion: Nivo and ipi modulate T-cell repertoire and tumor mutational heterogeneity in pts with advanced melanoma, presenting potential mechanisms of action underlying successful nivo therapy. These data also show that prior ipi treatment may influence biological response to nivo, but further investigation is warranted. Citation Format: Timothy A. Chan, Nadeem Riaz, Jonathan J. Havel, Vladimir Makarov, Alexis Desrichard, Jennifer S. Sims, F. Stephen Hodi, Salvador Martín-Algarra, William H. Sharfman, Shailender Bhatia, Wen-Jen Hwu, Thomas F. Gajewski, Craig L. Slingluff, Sviatoslav M. Kendall, Han Chang, John-William Sidhom, Jonathan P. Schneck, Nils Weinhold, Christine E. Horak, Walter J. Urba. Immunogenomic analyses of tumor cells and microenvironment in patients with advanced melanoma before and after treatment with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2017-2988

Mechanisms Used by Plants to Cope with DNA Damage.

Because the genome stores all genetic information required for growth and development, it is of pivotal importance to maintain DNA integrity, especially during cell division, when the genome is prone to replication errors and damage. Although over the last two decades it has become evident that the basic cell cycle toolbox of plants shares several similarities with those of fungi and mammals, plants appear to have evolved a set of distinct checkpoint regulators in response to different types of DNA stress. This might be a consequence of plants' sessile lifestyle, which exposes them to a set of unique DNA damage-inducing conditions. In this review, we highlight the types of DNA stress that plants typically experience and describe the plant-specific molecular mechanisms that control cell division in response to these stresses.

Abstract PL02-01: What we learned from immunotherapy in previous years

Abstract PL02-01: What we learned from immunotherapy in previous years

DNA Break Site at Fragile Subtelomeres Determines Probability and Mechanism of Antigenic Variation in African Trypanosomes

Antigenic variation in African trypanosomes requires monoallelic transcription and switching of variant surface glycoprotein (VSG) genes. The transcribed VSG, always flanked by ‘70 bp’-repeats and telomeric-repeats, is either replaced through DNA double-strand break (DSB) repair or transcriptionally inactivated. However, little is known about the subtelomeric DSBs that naturally trigger antigenic variation in Trypanosoma brucei, the subsequent DNA damage responses, or how these responses determine the mechanism of VSG switching. We found that DSBs naturally accumulate close to both transcribed and non-transcribed telomeres. We then induced high-efficiency meganuclease-mediated DSBs and monitored DSB-responses and DSB-survivors. By inducing breaks at distinct sites within both transcribed and silent VSG transcription units and assessing local DNA resection, histone modification, G2/M-checkpoint activation, and both RAD51-dependent and independent repair, we reveal how breaks at different sites trigger distinct responses and, in ‘active-site’ survivors, different switching mechanisms. At the active site, we find that promoter-adjacent breaks typically failed to trigger switching, 70 bp-repeat-adjacent breaks almost always triggered switching through 70 bp-repeat recombination (∼60% RAD51-dependent), and telomere-repeat-adjacent breaks triggered switching through loss of the VSG expression site (25% of survivors). Expression site loss was associated with G2/M-checkpoint bypass, while 70 bp-repeat-recombination was associated with DNA-resection, γH2A-focus assembly and a G2/M-checkpoint. Thus, the probability and mechanism of antigenic switching are highly dependent upon the location of the break. We conclude that 70 bp-repeat-adjacent and telomere-repeat-adjacent breaks trigger distinct checkpoint responses and VSG switching pathways. Our results show how subtelomere fragility can generate the triggers for the major antigenic variation mechanisms in the African trypanosome.

A short adaptive path from DNA to RNA polymerases

DNA polymerase substrate specificity is fundamental to genome integrity and to polymerase applications in biotechnology. In the current paradigm, active site geometry is the main site of specificity control. Here, we describe the discovery of a distinct specificity checkpoint located over 25 Å from the active site in the polymerase thumb subdomain. In Tgo, the replicative DNA polymerase from Thermococcus gorgonarius, we identify a single mutation (E664K) within this region that enables translesion synthesis across a template abasic site or a cyclobutane thymidine dimer. In conjunction with a classic "steric-gate" mutation (Y409G) in the active site, E664K transforms Tgo DNA polymerase into an RNA polymerase capable of synthesizing RNAs up to 1.7 kb long as well as fully pseudouridine-, 5-methyl-C-, 2'-fluoro-, or 2'-azido-modified RNAs primed from a wide range of primer chemistries comprising DNA, RNA, locked nucleic acid (LNA), or 2'O-methyl-DNA. We find that E664K enables RNA synthesis by selectively increasing polymerase affinity for the noncognate RNA/DNA duplex as well as lowering the K(m) for ribonucleotide triphosphate incorporation. This gatekeeper mutation therefore identifies a key missing step in the adaptive path from DNA to RNA polymerases and defines a previously unknown postsynthetic determinant of polymerase substrate specificity with implications for the synthesis and replication of noncognate nucleic acid polymers.

DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization

Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.

Analysis of inhibition of topoisomerase IIα and cancer cell proliferation by ingenolEZ

We previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase inhibitory activity and/or inhibitory activity of cell proliferation. The inhibitory effects of 20-O-(2'E,4'Z-decadienoyl) ingenol and 3-O-(2'E,4'Z-decadienoyl)-ingenol among these compounds on topoisomerase II activity and on the cell proliferative activity and arrest phase of the cell cycle were studied using a mouse breast cancer (MMT) cell line. Although 20-O-ingenolEZ exerted inhibitory effects on both topoisomerase II activity and cell proliferative activity, 3-O-ingenolEZ exerted inhibitory activity on neither. The 20-O-ingenolEZ-induced cell arrest of MMT-cell proliferation led to a cell cycle arrest in the G2/M phase. Topoisomerase II inhibition can be divided into the poison and catalytic inhibitor types. A checkpoint mechanism is activated when cells are treated with these topoisomerase II inhibitors. Poison-type inhibition occurs via induction of the DNA damage checkpoint and the catalytic-type inhibition occurs via induction of the DNA-decatenation checkpoint, suggestive of distinct checkpoint reactions. 20-O-ingenolEZ inhibited topoisomerase IIalpha activity through inhibition of ATPase, and induced DNA-decatenation checkpoint without signaling for phosphorylation of H2AX.

A tale of two tails: Activation of DNA damage checkpoint kinase Mec1/ATR by the 9-1-1 clamp and by Dpb11/TopBP1

The DNA damage and replication checkpoint kinase Mec1/ATR is a member of the PI3-kinase related kinases that function in response to various genotoxic stresses. The checkpoint clamp 9-1-1 (Rad9-Rad1-Hus1 in S. pombe and mammals; Ddc1-Rad17-Mec3 in S. cerevisiae) executes two distinct checkpoint functions. In S. cerevisiae, DNA-bound 9-1-1 directly activates Mec1 kinase activity, a function that has not been demonstrated in other organisms. A second, conserved activity of 9-1-1 is that of TopBP1/Cut5/Dpb11 recruitment to stalled replication sites; subsequent activation of Mec1/ATR is carried out by TopBP1/Cut5/Dpb11. Biochemical studies indicate that the mode of Mec1/ATR activation by S. cerevisiae 9-1-1 is analogous to activation by S. cerevisiae Dpb11 or by vertebrate TopBP1: activation is mediated by the intrinsically disordered C-terminal tail of each activator. The relative contributions made by multiple activators of Mec1/ATR are discussed.

Defective in Mitotic Arrest 1/Ring Finger 8 Is a Checkpoint Protein That Antagonizes the Human Mitotic Exit Network

A molecular pathway homologous to the S. cerevisiae mitotic exit network (MEN) and S. pombe septation initiation network has recently been described in higher eukaryotes and involves the tumor suppressor kinase LATS1 and its subunit MOB1A. The yeast MEN/septation initiation network pathways are regulated by the ubiquitin ligase defective in mitotic arrest 1 (Dma1p), a checkpoint protein that helps maintain prometaphase arrest when cells are exposed to microtubule poisons. We identified here the RING domain protein ring finger 8 (RNF8) as the human orthologue of the yeast protein Dma1p. Like its yeast counterparts, human DMA1/RNF8 localized at the midbody and its depletion by siRNA compromised mitotic arrest of nocodazole-treated cells in a manner dependent on the MEN. Depletion of MAD2, a spindle checkpoint protein, also compromised mitotic arrest, but in a MEN-independent manner. Thus, two distinct checkpoint pathways maintain mitotic arrest in cells exposed to microtubule poisons.

Ipl1p-dependent phosphorylation of Mad3p is required for the spindle checkpoint response to lack of tension at kinetochores

The spindle checkpoint delays anaphase onset until all chromosomes are correctly attached to microtubules. Ipl1 protein kinase (Aurora B) is required to correct inappropriate kinetochore-microtubule attachments and for the response to lack of tension between sister kinetochores. Here we identify residues in the checkpoint protein Mad3p that are phosphorylated by Ipl1p. When phosphorylation of Mad3p at two sites is prevented, the cell's response to reduced kinetochore tension is dramatically curtailed. Our data provide strong evidence for a distinct checkpoint pathway responding to lack of sister kinetochore tension, in which Ipl1p-dependent phosphorylation of Mad3p is a key step.

CD4-Dependent Signaling Is Required for a Late Checkpoint during Th2 Development Associated with Resistance to Activation-Induced Cell Death

Previous studies have found that class II-restricted T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene have a specific defect in the development of Th2 effector cells; however, the reason for this defect was not clear. Following stimulation with a high potency peptide and exogenous IL-4, CD4-dependent signaling is required for optimal generation of a Th2 effector population. However, initial IL-4 and GATA-3 transcription is appropriately induced, suggesting that the initial stages of Th2 development are intact and independent of CD4 after priming with a strong agonist peptide. In addition to the defect in Th2 development, CD4 mutant T cells are also relatively resistant to activation-induced cell death (AICD). Furthermore, inhibition of AICD in wild-type T cells causes a defect in Th2 development similar to that seen in the CD4 mutant T cells. These data support the hypothesis that CD4-dependent signaling pathways regulate a distinct checkpoint in the expansion and commitment phase of Th2 development, which is related to dysregulation of AICD.

DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7–Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organsims.

The C-terminus of Bfa1p in budding yeast is essential to induce mitotic arrest in response to diverse checkpoint-activating signals.

During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit through mitotic checkpoints. In budding yeast, exit from mitosis is triggered by the activation of the small GTPase Tem1p. Bfa1p in association with Bub2p negatively regulates Tem1p in response to spindle damage, spindle misorientation, and DNA damage, resulting in cell cycle arrest. To delineate the Bfa1p domains that respond to distinct checkpoint signals, we constructed 13 Bfa1 deletion mutants. The C-terminal 184 amino acids of Bfa1p (Bfa1-D8(391-574)) contained the entire capacity of Bfa1p to generate mitotic arrest in response to spindle damage, spindle misorientation, and DNA damage. This domain was also enough to interact with the mitotic exit network proteins Tem1p, Bub2p, and Cdc5p, and to localize to the spindle pole body (SPB). Over-expression of Bfa1-D8(391-574) induced late anaphase arrest as efficient as the full-length Bfa1p in a Bub2p-dependent manner. In contrast, the N-terminal portion of Bfa1p (Bfa1-D16(1-376)) could not localize to SPB and did not block mitotic exit in response to diverse checkpoint signals. Bfa1-D16(1-376) interacted with Tem1p but not with Bub2p and its over-expression partially arrested cells in mitosis in the absence of Bub2p. By random mutagenesis of Bfa1-D8(391-574) with hydroxylamine, we isolated a point mutant of D8, D8(E438K), which interacts with both Tem1p and Bub2p but cannot respond to checkpoint signals. This mutant also showed reduced efficiency in the localization to SPB. Taken together, our study demonstrated that various checkpoint signals are transmitted to the C-terminal domain of Bfa1 (Bfa1-D8(391-574)) and that Bfa1p localization to SPB is necessary but not sufficient to regulate mitotic exit in response to various checkpoint signals.

The Arabidopsis 14-3-3 protein, GF14omega, binds to the Schizosaccharomyces pombe Cdc25 phosphatase and rescues checkpoint defects in the rad24- mutant.

The fission yeast (S. pombe) mitotic inducer gene, Spcdc25, interacts with the plant cell cycle to establish a small cell size phenotype compared with wild-type cells. We have investigated the nature of this interaction by yeast two-hybrid screening using Spcdc25 as bait in a cDNA library prepared from root tips of Arabidopsis thaliana (L.) Heynh. Three 14-3-3 proteins were detected: G-box Factor-like (GF)14kappa, lambda and omega; binding with Spcdc25 was confirmed by an independent immunoprecipitation assay. To test for cell cycle checkpoint function, GF14kappa, lambda and omega were transformed independently, using the strong nmt1+ promoter, into rad24-, a fission yeast mutant deficient in a 14-3-3 checkpoint protein. When exposed to UV irradiation or in the presence of 10 mM hydroxyurea, only cells transformed with GF14omega could fully rescue the defects in the DNA-damage and DNA-replication checkpoints of this mutant. Supporting evidence for a GF14omega cell cycle function was provided by semi-quantitative reverse transcription-polymerase chain reaction indicating that expression of this gene was elevated in regions of the plant that comprise dividing cells whereas GF14kappa and lambda expression was more evenly detected in all tissues examined. The data are consistent with the hypothesis that interaction between Spcdc25 and the plant cell cycle occurs at the level of a 14-3-3 protein with distinct checkpoint properties.

Amino Acids Regulate Hepatocyte Proliferation through Modulation of Cyclin D1 Expression

The mechanisms by which amino acids regulate the cell cycle are not well characterized. In this study, we examined the control of hepatocyte proliferation by amino acids and protein intake. In short-term culture, hepatocytes demonstrated normal entry into S phase and cell cycle protein expression in the absence of essential amino acids. However, deprivation of a set of nonessential amino acids (NEAA) potently inhibited cell cycle progression and selectively down-regulated the expression of proliferation-control proteins. Notably, NEAA withdrawal after the mitogen restriction point still inhibited entry into S phase, suggesting that these amino acids regulate a distinct checkpoint. Cyclin D1, an important mediator of hepatocyte proliferation, was markedly inhibited at the transcriptional level by NEAA deprivation, and transfection with cyclin D1 (but not cyclin E) overcame the cell cycle arrest. As previously shown, protein-deprived mice demonstrated impaired hepatocyte proliferation in vivo after 70% partial hepatectomy. The expression of cyclin D1 and downstream cell cycle proteins after partial hepatectomy was inhibited in these mice. Transfection with cyclin D1 in vivo triggered hepatocyte DNA synthesis and the expression of S phase proteins in the absence of dietary protein. Cyclin D1 also induced global protein synthesis in NEAA-deprived hepatocytes and promoted liver growth in vivo in the setting of protein deprivation. These results indicate that cyclin D1 is a key target of amino acid signaling in hepatocytes.

Human autoimmune sera as molecular probes for the identification of an autoantigen kinase signaling pathway.

Using human autoimmune sera as molecular probes, we previously described the association of phosphorylated serine/arginine splicing factors (SR splicing factors) with the U1-small nuclear ribonucleoprotein (U1-snRNP) and U3-small nucleolar RNP (snoRNP) in apoptotic cells. SR proteins are highly conserved autoantigens whose activity is tightly regulated by reversible phosphorylation of serine residues by at least eight different SR protein kinase kinases (SRPKs), including SRPK1, SRPK2, and the scleroderma autoantigen topoisomerase I. In this report, we demonstrate that only one of the known SRPKs, SRPK1, is associated with the U1-snRNP autoantigen complex in healthy and apoptotic cells. SRPK1 is activated early during apoptosis, followed by caspase-mediated proteolytic inactivation at later time points. SRPKs are cleaved in vivo after multiple apoptotic stimuli, and cleavage can be inhibited by overexpression of bcl-2 and bcl-xL, and by exposure to soluble peptide caspase inhibitors. Incubation of recombinant caspases with in vitro–translated SRPKs demonstrates that SRPK1 and SRPK2 are in vitro substrates for caspases-8 and -9, respectively. In contrast, topoisomerase I is cleaved by downstream caspases (-3 and -6). Since each of these SRPKs sits at a distinct checkpoint in the caspase cascade, SRPKs may serve an important role in signaling pathways governing apoptosis, alternative mRNA splicing, SR protein trafficking, RNA stability, and possibly the generation of autoantibodies directed against splicing factors.

A MAP kinase-dependent actin checkpoint ensures proper spindle orientation in fission yeast.

The accurate segregation of chromosomes at mitosis depends on a correctly assembled bipolar spindle that exerts balanced forces on each sister chromatid. The integrity of mitotic chromosome segregation is ensured by the spindle assembly checkpoint (SAC) that delays mitosis in response to defective spindle organisation or failure of chromosome attachment. Here we describe a distinct mitotic checkpoint in the fission yeast, Schizosaccharomyces pombe, that monitors the integrity of the actin cytoskeleton and delays sister chromatid separation, spindle elongation and cytokinesis until spindle poles have been properly oriented. This mitotic delay is imposed by a stress-activated mitogen-activated protein (MAP) kinase pathway but is independent of the anaphase-promoting complex (APC).

Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii

Tachyzoite endodyogeny is characterized by a three phase cell cycle comprised of major G1 and S phases with mitosis following immediately upon the conclusion of DNA replication. Cytokinesis, which begins with the formation of daughter apical complexes, initiates in late S phase and overlaps mitosis. There is no evidence to support an extended G2 period in these parasites. In all strains, parasites with a 2 N DNA content are a relatively small subpopulation and when tachyzoites expressing a fluorescent nuclear marker (green-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were observed by time-lapse microscopy, there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by flow cytometry demonstrated that the G1 and S periods were ≈60 and ≈30% of a single division cycle, although these phases were longer in strains that display a slower growth rate. The overall length of S phase was determined by [ 3H]-thymidine autoradiography using transgenic parasites expressing herpes simplex thymidine kinase and validated by Northern analysis of S phase specific genes during synchronous growth. The fraction of S phase parasites by flow cytometry paralleled autoradiography, however, within S phase, the distribution of parasites was bimodal in all strains examined. Parasites containing a 1–1.7 N DNA complement were a small fraction when compared to the major S phase population which contained a near-diploid (≈1.8 N) complement, suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missing G2, where checkpoints are thought to operate in other eukaryotes, the bimodal replication of tachyzoite chromosomes may represent a distinct premitotic checkpoint associated with endodyogeny.