Cell Cycle Transit Time Research Articles

Single-Cell Revelation of Extensive Heterogeneity in the Human Lymphocyte and Neutrophil/Monocyte Lineage Restriction Process Despite Highly Co-Regulated Changes in Cell Cycle Control

The production of mature blood lineages in humans has been viewed as a hierarchical process in which the primitive cells with multi-lineage potential generate a series of progenitors with increasingly reduced self-renewal abilities and restricted lineage potentials. This concept has been largely supported by phenotypic characterization of starting CD34+ subsets of human CD45+ cells isolated from different hematopoietic tissues and assayed for their mature cell outputs in supportive systems in vitro or post-transplant and more recently by tracking changes in their molecular composition. Such experiments have suggested that cells that give rise to the lymphocyte lineages (L) share a common origin almost exclusively with the neutrophil and monocyte (macrophage) lineages (NM). However, the mechanisms that regulate the subsequent separation of these lineages starting from cells with presumed restricted L+NM potential have remained elusive. To address this question, we first developed a culture system that supports outputs of differentiated B (CD19+) and NM (CD15+/CD14+) from ≥50% of single CD34+ cells plated on a stromal layer of MS-5 feeders in a medium containing human IL-7, SCF and FLT3L. Use of this clonal assay revealed that cells with B+NM bi-potential constitute 20% of the CD45RA-CLEC12A- (RA-C-) cell subset of the CB “P-Mix” phenotype (CD34+CD38medCD71-CD10-, Blood 133:927, 2019); with the CD45RA+CLEC12A- (RA+C-) and CLEC12A+ (C+) cells displaying largely restricted outputs of only B- (30%) or only NM- (50%) progeny phenotypes, respectively. These same 3 “progenitor” phenotypes (RA-C-, RA+C-, C+) were also found to be regenerated in xenografted immunodeficient mice as well as in a further optimized in vitro system and their clonal output types and numbers consistent with the outputs of those isolated from fresh CB. To analyze T-cell lineage (T) restriction in parallel, we then further modified this culture system to include adherent DLL4, that we found allows B, T, N and M progeny to be produced from single input CB cells at a 40-60% efficiency, with numerous clones derived from single RA+C- cells containing B+T+M, or B+M, or B+T or T-only; a few with B- or M- only, and none with N. In contrast, under the same conditions, half of the documented C+ outputs were exclusively N with much lower lymphoid outputs of T-only or T+N/M. Together, these findings reveal the unanticipated importance of the external stimuli in revealing lineage potentials when these are undergoing restrictive changes. We then asked if and how these identified early phenotypes might change their cycling properties during the course of their progression. For this, the rate and progeny content of sequential cell divisions during the stimulated expansion of the CFSE labelled RA-C- input cells were measured under a supportive liquid culture condition. Throughout a 6-day period, almost all of the C+ progeny and a slightly lower proportion of the RA+C- outputs were generated from cells that transit through the cell cycle within 24 hours, whereas cells maintaining an undifferentiated RA-C- phenotype divided at significantly slower rates, suggesting a simultaneously initiated shortening of the cell cycle transit time during both L- and NM- restriction. In agreement with these functional results, a recent single-cell CITE-seq dataset generated in parallel showed higher expression of genes involved in cycling activities in the early L- and NM-restricted subsets as compared to the unrestricted RA-C- cells. Taken together, these results reveal important and disconnected features in the cell cycle control and previously unrecognized complex patterns in the biological outputs and molecular changes during the steps of L and NM restriction. In addition, they set the stage to enable preferred sequences and molecular changes responsible for normal and regenerative needs to be identified and their potential relevance to the formation of human lymphoblastic and myeloid leukemias to be characterized.

Single-Cell Analysis of Human B Lymphoid and Neutrophil/Monocyte Lineage Restriction

Lifelong production of most types of mature blood cells is sustained by a small population of cells with extensive regenerative potential. However, the detailed steps that restrict multipotent or even bipotent human hematopoietic cells to any single lineage remain poorly understood. Those that segregate the human B-lymphoid and neutrophil/monocyte (NM) lineages are of particular interest as these appear to identify a stage that might control the different properties of human leukemias that display perturbed NM and/or B-cell programs. To undertake a refined analysis of this normal lineage restriction process in human cells, we first devised a culture system that permits it to be tracked clonally and efficiently (50%). Initial experiments showed this could be achieved using a combination of multiple stromal cell types and human growth factor-supplemented medium. To elucidate the intervening transitional steps we then used multiplexed flow cytometry to compare the progeny generated in this culture system over a 2-week time course from previously defined lymphoid progenitor-enriched (P-L), NM progenitor-enriched (P-NM) and less restricted P-mix cord blood (CB) subsets. The results suggested that gain of CD45RA (RA) expression and loss of CLEC12A (C) expression appeared to accompany the sequential restriction of early CD34+ progenitors first to cells with dual NM+B-lineage potential and then just to B-lineage potential.Subsequent tracking of the lineage outputs of CD34+ RA-C- cells initially produced in larger numbers from unfractionated CD34+ CB cells either in vitro or in xenografted immunodeficient mice, confirmed the CD34+ RA-C- subset to be highly enriched in cells with dual NM+B potential. In contrast, co-generated CD45RA+ (RA+C-) and RA+CLEC12A+ (RA+C+) phenotypes displayed separate B- and NM lineage-restricted activity, respectively, as indicated by their largely exclusive clonal outputs of CD19+ pre-B and CD14+/CD15+ NM precursors. In agreement with these phenotypically established separate NM and B-lineage outputs, RA+C- cells were found to contain higher levels of B-lineage-associated gene transcripts (e.g., DNTT, CD79A, and EBF1), whereas RA+C+ cells contained higher levels of the NM transcription factor mRNAs encoded by SPI1 and CEBPA. In a further optimized stroma-free liquid culture system, the RA-C- cells could be shown to produce continuously RA+C- and RA+C+ progeny after another 3-4 days, and also RA-C- progeny which are not produced from more restricted RA+C- and RA+C+ cells, suggesting that the acquired expression of RA precedes the separation of NM and B-lineage potential that is then marked by the differential activation of C expression in RA+ cells.To examine more precisely how the process of B+NM restriction to a single lineage might be related to successive cell cycles, we labeled RA-C- cells with carboxyfluorescein diacetate succinimidyl ester (CFSE) to enable the phenotypes and growth potential of the the progeny obtained after 6 days to be directly related to their prior division histories. This revealed extensive heterogeneity in the overall distribution of the initial progeny cell cycle times but with a clear segregation in the outputs of the faster and slower dividing cells. Notably, the faster dividers produced ultimately small M+B or uni-lineage clones whereas the initially slow dividers subsequently produced larger clones, 25% of which still contained CD34+ cells or N+M+B progeny.Taken together, these findings identify new hallmark phenotypic changes that identify a critical step in the lineage restriction of primitive human hematopoietic cells with dual NM+B-lineage potential and a previously unknown association of this process with a shortening of their cell cycle transit time. DisclosuresNo relevant conflicts of interest to declare.

Mathematical Model of Tumour Spheroid Experiments with Real-Time Cell Cycle Imaging

Three-dimensional (3D) in vitro tumour spheroid experiments are an important tool for studying cancer progression and potential cancer drug therapies. Standard experiments involve growing and imaging spheroids to explore how different conditions lead to different rates of spheroid growth. These kinds of experiments, however, do not reveal any information about the spatial distribution of the cell cycle within the expanding spheroid. Since 2008, a new experimental technology called fluorescent ubiquitination-based cell cycle indicator (FUCCI) has enabled real-time in situ visualisation of the cell cycle progression. Observations of 3D tumour spheroids with FUCCI labelling reveal significant intratumoural structure, as the cell cycle status can vary with location. Although many mathematical models of tumour spheroid growth have been developed, none of the existing mathematical models are designed to interpret experimental observations with FUCCI labelling. In this work, we adapt the mathematical framework originally proposed by Ward and King (Math Med Biol 14:39-69, 1997. https://doi.org/10.1093/imammb/14.1.39 ) to produce a new mathematical model of FUCCI-labelled tumour spheroid growth. The mathematical model treats the spheroid as being composed of three subpopulations: (i) living cells in G1 phase that fluoresce red; (ii) living cells in S/G2/M phase that fluoresce green; and (iii) dead cells that are not fluorescent. We assume that the rates at which cells pass through different phases of the cell cycle, and the rate of cell death, depend upon the local oxygen concentration. Parameterising the new mathematical model using experimental measurements of cell cycle transition times, we show that the model can qualitatively capture important experimental observations that cannot be addressed using previous mathematical models. Further, we show that the mathematical model can be used to qualitatively mimic the action of anti-mitotic drugs applied to the spheroid. All software programs required to solve the nonlinear moving boundary problem associated with the new mathematical model are available on GitHub. at https://github.com/wang-jin-mathbio/Jin2021.

Dynamic bistable switches enhance robustness and accuracy of cell cycle transitions

Bistability is a common mechanism to ensure robust and irreversible cell cycle transitions. Whenever biological parameters or external conditions change such that a threshold is crossed, the system abruptly switches between different cell cycle states. Experimental studies have uncovered mechanisms that can make the shape of the bistable response curve change dynamically in time. Here, we show how such a dynamically changing bistable switch can provide a cell with better control over the timing of cell cycle transitions. Moreover, cell cycle oscillations built on bistable switches are more robust when the bistability is modulated in time. Our results are not specific to cell cycle models and may apply to other bistable systems in which the bistable response curve is time-dependent.

Temperature-Induced uncoupling of cell cycle regulators

The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness we examined cell cycle progression in early Drosophila embryos at different temperatures. Our experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35​°C), the relative duration of individual steps varies with temperature. We find that the transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, we quantified the ratio of activities of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.

Dynamic Bistable Switches Enhance Robustness and Accuracy of Cell Cycle Transitions

Bistability is a common mechanism to ensure robust and irreversible cell cycle transitions. Whenever biological parameters or external conditions change such that a threshold is crossed, the system abruptly switches between different cell cycle states. Experimental studies indicate that the shape of the bistable response curve changes dynamically in time. Here, we show how such a dynamically changing bistable switch can provide a cell with better control over the timing of cell cycle transitions. Moreover, cell cycle oscillations built on bistable switches are more robust when the bistability is modulated in time.

Defects in base excision repair and homologous recombination, but not nucleotide excision repair, lead to altered cell cycle phase distributions in Saccharomyces cerevisiae

The DNA within cells is constantly subjected to chemical modifications resulting from exposure to endogenous and exogenous DNA damaging agents. Damaged or oxidized bases are primarily repaired by the base excision repair (BER) pathway, crosslinks induced by ultraviolet (UV) light and many bulky lesions are repaired by nucleotide excision repair (NER), mismatches are resolved by the mismatch repair system (MMR), and double‐strand breaks in DNA are repaired by either the homologous recombination (HR) pathway or the nonhomologous end‐joining (NHEJ) pathway. Inactivation of NER genes such as RAD2, RAD7 or RAD14 leads to extreme UV sensitivity in yeast cells. Mutants with defects in BER genes OGG1 or UNG1 are defective in repair of oxidized guanines and the removal of uracil bases from DNA, respectively. Yeast rad52 mutants are deficient in HR and are hypersensitive to ionizing radiation and chemicals that induce double‐strand breaks.Our laboratory has previously observed that rad52 haploid and diploid mutant cell cultures have high levels of distended large‐budded G2 phase cells and long cell cycle transit times (see poster by C. England et al.). This work also demonstrated that the high G2 phase cell phenotype of rad52 cells was abolished when any of seven known DNA damage checkpoint genes were co‐inactivated. The current project has extended these studies and asked whether defects in other DNA repair pathways also lead to accumulation of G2 phase cells and increases in doubling times. Initial studies revealed that log phase cultures of mutants deficient in NER (rad2, rad7 and rad14) or MMR (msh2, msh3 and msh6) do not have high levels of large‐budded cells. By contrast, such cells were strongly elevated in mutant cultures defective in the BER pathway (ogg1 and ung1 cells). Recent experiments are attempting to determine the fraction of these large‐budded cells that are G2 vs. M phase cells via DAPI staining and whether they are specifically associated with activation of the DNA damage checkpoint system. Cell cultures containing mutants defective in other DNA repair processes, such as Rad6‐mediated postreplication repair, translesion synthesis and nuclease processing of DNA ends, are also being analyzed using phase contrast and fluorescence microscopy to identify unusual cell morphology and cell cycle phase characteristics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tracking of Normal and Malignant Progenitor Cell Cycle Transit in a Defined Niche.

While implicated in therapeutic resistance, malignant progenitor cell cycle kinetics have been difficult to quantify in real-time. We developed an efficient lentiviral bicistronic fluorescent, ubiquitination-based cell cycle indicator reporter (Fucci2BL) to image live single progenitors on a defined niche coupled with cell cycle gene expression analysis. We have identified key differences in cell cycle regulatory gene expression and transit times between normal and chronic myeloid leukemia progenitors that may inform cancer stem cell eradication strategies.

Tracking Normal and Chronic Myeloid Leukemia Progenitor Cell Cycle Kinetics in a Defined Niche Using Live Time Lapse Confocal Imaging with Fucci2BL a Novel Lentiviral Bicistronic Reporter

Malignant reprogramming of progenitors into self-renewing cancer stem cells (CSCs) that have a predisposition for dormancy in protective niches has been implicated in therapeutic resistance of chronic myeloid leukemia (CML) and other CSC-driven malignancies. An unmet medical need for developing therapies that target niche dependent dormant human CSCs provides a compelling rationale for identifying key differences in gene expression at different cell cycle phases between normal and malignant progenitors in a CSC-supportive stromal co-culture system. Currently, few methods exist for quantifying cell cycle kinetics in live human leukemia stem cells (LSC). To date, efficient cell cycle transit time analysis in single live human leukemic progenitors derived from primary patient samples has been hampered by 1) decreased cell viability following transfection or transduction, 2) limited sample size, 3) dormancy of primitive progenitor populations thereby necessitating lentiviral rather than retroviral transduction and 4) increased apoptosis in the absence of a supportive microenvironment. To alleviate these challenges and improve transduction efficiency, we generated Fucci2BL, a lentiviral bicistronic reporter vector. Fucci2BL expresses mVenus-hGem(1/110) fused to mCherry-hCdt1(30/120) by the T2A peptide using an EF1 promoter that generates optimal levels of gene expression in progenitors. Initially, the reporter fidelity was characterized in 293A cells using flow cytometry and time-lapse confocal fluorescence microscopy. Time-lapse confocal fluorescence microscopy revealed normal cell morphology and distinct nuclear staining of either green or red fluorescence depending on the cell cycle stage. Once the fidelity of the Fucci2BL reporter was characterized, differences in gene expression levels between normal and malignant progenitors were analyzed. Whole transcriptome RNA-seq analysis revealed both cell cycle and DNA replication pathways were enriched in chronic phase CP (CML) compared to normal progenitors. Cell cycle kinetics between normal and chronic phase (CML) progenitors co-cultured in a niche were also analyzed using the Fucci2BL reporter. Normal progenitor cells on average transited the cell cycle within 26 hours while CP progenitor cells demonstrated a prolongation of transit through G1. In summary, the Fucci2BL system enables single transduction and single cell cycle tracking as well as gene expression changes in live primary progenitors in response to a niche. This robust lentiviral reporter can reproducibly distinguish cell cycle phases thereby providing an opportunity to quantitatively study the contribution of cell cycle kinetics to single cancer stem cell therapeutic resistance and to relapse. DisclosuresJamieson:J&J: Research Funding; GSK: Research Funding.

Abstract C159: Genome-wide CRISPR-Cas9 screens uncover therapeutic targets and tumor suppressor genes in glioblastoma multiforme

Abstract Precision oncology is currently based on the notion that genomic analysis of descriptive molecular signatures from patient tumors will lead to actionable therapeutic targets following the patient's arrival into the clinic. However, this approach has failed to deliver new effective treatments for glioblastoma multiforme (GBM), which is the most common and aggressive form of brain cancer; and thus, ∼90% of adult GBM patients receiving standard of care therapies continue to die within 2 years of diagnosis. In addition, this approach has neglected the importance for using functional genetics in precision oncology. To identify new therapeutic targets for GBM, we applied functional genetics to perform lethal genome-wide CRISPR-Cas9 knockout screens in patient-derived GBM stem-like cells (GSCs) and also non-transformed human neural stem cells (NSCs), non-neoplastic tissue of origin controls. Here, we present our latest findings from these screens, which include multiple novel GBM-specific lethal genes that were validated by both in vitro and in vivo preclinical studies. Knockout of GBM-specific lethal genes, including the WEE1-like kinase, PKMYT1/Myt1, lead to lethality in GSCs, but not NSCs. Focused mechanistic studies revealed that PKMYT1 acts redundantly with WEE1 to phosphorylate CDK1-Y15 and to promote timely completion of mitosis in NSCs, but that this redundancy is lost in most GBM isolates and in NSCs harboring activated alleles of EGFR and AKT1, which are commonly altered signaling pathways in GBM. Moreover, PKMYT1 depletion in GSCs and genetically altered NSCs requiring PKMYT1 lead to cytokinesis failure and cell death during mitosis. In addition to lethal genes, genes promoting in vitro expansion of NSCs upon knockout were examined. For this category, we validated multiple genes that are candidate tumor suppressors and involved in: the negative regulation of Hippo signaling, TP53 signaling, epigenetic regulation, promoting neural development, and other cellular functions. Knockout of these genes caused shortened cell cycle transit times and drastic growth advantages in NSCs, and in the case of CREBBP knockout, caused precious entry into S-phase and deregulation of cell cycle gene expression. The identification of these potential tumor suppressors here reveal new genetic drivers in glioma/GBM, which contributes to a growing body of work that will help redefine GBM gene signatures. Furthermore, we found that the molecular signatures of pathways and genes commonly altered in GBM are not ideal GBM therapeutic targets since most of these targets failed to score as GBM-specific lethal hits in our knockout screens and likely are non-essential or essential in both the GSCs and NSCs. Nonetheless, these common GBM alterations give rise to cancer-specific vulnerabilities, which then lead to genes, such as PKMYT1, that are required to overcome functional impairments in cancer cells. Taken together, we demonstrated here that genomics and functional genetics are equally important for precision oncology, as the combination of both tools can identify genetically altered genes, cancer-specific therapeutic targets, and tumor suppressors. These results are part of a rapidly growing body of work by the science community that will one day allow oncologists to tailor therapies for each patient based upon his or her tumor genetic profile and characteristics. Citation Format: Chad M. Toledo, Yu Ding, Pia Hoellerbauer, Ryan J. Davis, Ryan Basom, Emily J. Girard, Eunjee Lee, Philip Corrin, Hamid Bolouri, Jerry Davison, Qing Zhang, Do-Hyun Nam, Jeongwu Lee, Steven M. Pollard, Jun Zhu, Jeffery J. Delrow, Bruce E. Clurman, James M. Olson, Patrick J. Paddison. Genome-wide CRISPR-Cas9 screens uncover therapeutic targets and tumor suppressor genes in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C159.

ATPS-60COMPREHENSIVE IDENTIFICATION OF GLIOBLASTOMA MULTIFORME THERAPEUTIC TARGETS AND GROWTH SUPPRESSOR GENES USING GENE EDITING

Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults and is currently incurable. To identify new therapeutic targets for GBM, we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem cells (NSCs), non-neoplastic tissue of origin controls. These screens revealed thousands of gene candidates acting to promote or limit expansion of GSCs and NSCs. In vitro and in vivo validation studies revealed several novel therapeutic targets, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies revealed that PKMYT1 acts redundantly with WEE1 to facilitate mitosis in NSCs, but that this redundancy is lost in most GBM isolates and in NSCs harboring activated alleles of EGFR and AKT1. In addition to lethal genes, KOs promoting in vitro expansion of NSCs were also examined. For this category, we validated multiple genes, including: ARID1A, ARID1B, CREBBP, EP300, NF2, PDCD10, PTPN14, TAOK1, TGFBR2, and TP53, whose inhibition shortens NSC cell cycle transit times, causing dramatic growth advantages, and, in the case of CREBBP KO, dramatic deregulation of cell cycle gene expression and precocious S-phase entry. Taken together, these results demonstrate the use of CRISPR-Cas9 gene editing to identify novel cancer therapeutic targets and growth suppressor genes in human GBM isolates and NSCs. The results also inform precision medicine approaches for GBM, since most, if not all, candidate therapeutic targets could not be predicted from mutation, CNV, or gene expression analysis of the patient isolates alone.

Commentary: The Interleukin-2 T Cell System: A New Cell Growth Model.

Having created unique and novel cellular and molecular reagents, we could approach questions that had perplexed investigators interested in cell growth for over 50 years, i.e., the basis for variable cell cycle transit times of individual cells among genetically homogeneous cell populations (1). Studies of all cell populations, both prokaryote and eukaryote, revealed that within a cell population, the cell cycle times of individual cells follow a normal distribution when examined as a function of the division rate (the rate-normal distribution). Thus, some cells proliferate slowly while others proliferate faster, with most cells distributed about the mean on a log-linear plot. Prior to the discovery of the IL-2 molecule (2) and IL-2 receptors (IL-2R) (3), T cell populations were known to follow this rate-normal distribution, common to all living cells. Mathematical analysis of proliferating cell populations indicated that individual cell variability in cell cycle transit times was stochastic, depending upon a “hidden variable.” However, once the “hidden” molecular variables of T cell cycle proliferation had been identified (the IL-2 concentration, IL-2R density, affinity of the IL-2/IL-2R interaction, and the duration of the IL-2/IL-2R interaction), experiments could be preformed for the first time revealing that as long as these crucial characteristics of T cell cycle progression were known, then the variability of cell cycle transit times were entirely predictable and deterministic, not probabilistic or left to chance. These experiments and approaches were possible because of painstaking attention to the creation of critical cell clones and homogeneous purified IL-2 molecules, as well as the development of the radiolabeled IL-2 binding assay, and monoclonal antibodies reactive with both IL-2 and its receptor. Moreover, the employment of the flow cytometer allowed us to proceed beyond studies of cell populations to quantify the intermolecular interactions of individual cells for the first time. Doreen Cantrell, a postdoctoral fellow skilled in flow cytometry, was critical to our experimental approach. Because the growth characteristics of all known cell populations are identical to those of T cell populations, it followed that individual cells of all other cell populations would demonstrate the same type of molecular determinants. Thus, the title of this article: “The interleukin-2 T cell system: A new cell growth model,” a new universal paradigm in cell and molecular biology (1). The significance of these findings was obvious. As cells of all tissues, especially of metazoans, have identical growth characteristics of T cells, it followed that the cells of all metazoans are regulated in the same molecular fashion, i.e., cells are directed to undergo all-or-none (quantal) cell fate decisions by critical molecular concentrations. Furthermore, as all malignant cells arise from a single cell, so that all malignancies are clonal in origin, a gain of function mutation of one of the genes encoding the molecular determinants of cell cycle progression could obviate the strict cytokine/receptor/signaling pathways normally controlling the decision to divide, thereby resulting in a neoplastic cell growth (4). In other words, a critical “driver mutation” can put the cell on “autopilot.” From the viewpoint of the immune system, Burnet’s “Clonal Selection Theory,” derives from the observation that antigen-reactive clones of immune cells are selected by antigen, but subsequent to selection, each clone undergoes a proliferative clonal expansion (5). It is this clonal proliferative expansion that is determined by the molecular parameters discovered in this series of experiments focused on IL-2 and T cells. Accordingly, the regulatory cells and other cytokine molecules that influence the concentrations of the IL-2 molecules, their receptors, and the molecules of their signaling pathways ultimately determine the tempo, magnitude, and duration of immune responses. That is, internally derived hormones and their receptors regulate the immune system, just as every other bodily system is regulated, and immunity is not regulated solely by environmental antigens from without, a previous central dogma of immunology. For immunologists, it is especially noteworthy that the lymphocyte antigen receptors function in an identical fashion to convert TCR signals to a digital control of cytokine gene expression (6–10). These findings and considerations, led me to propose “The Quantal Theory of Immunity” (11–14). Thus, the immune system is regulated at the systemic (population) level by the number of clones responding and the extent of the proliferative expansion of each clone as originally stated by Burnet. However, at the level of individual cells, participation in an immune response is regulated in a quantal (all-or-none) fashion, and this quantal decision is determined by the absolute number of intermolecular interactions, which the cells somehow “count.” Once the crucial number has been surpassed, the cell responds in a quantal fashion. It goes without mentioning that all of the molecules and cells that participate in an immune response obey identical principles.

Atomic structure of the APC/C and its mechanism of protein ubiquitination

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex and interphase early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of future experiments to investigate APC/C functions in vivo.

Saccharomyces cerevisiaedeletion strains with complex DNA content profiles

To identify Saccharomyces cerevisiae genes required for the proper timing of cell cycle transitions, we previously reported a systematic examination of the DNA content of homozygous diploid deletion strains. However, deletion strains with complex DNA content profiles were not examined in that study. Here, we report S.cerevisiae genes that when deleted give rise to DNA content profiles consistent with roles of the corresponding gene products during DNA replication. We also identified a set of genes whose deletion leads to increased DNA content, consistent with defects in mitosis, cytokinesis, or cell separation. Finally, we examined known interactions between the gene products of each group, placing these gene products in functional networks. Taken together, the data we present further validate the roles of the corresponding gene products in these processes, facilitating efforts to delineate gene function critical for genome replication, maintenance, and segregation.

Systematic Analysis of Cell Cycle Effects of Common Drugs Leads to the Discovery of a Suppressive Interaction between Gemfibrozil and Fluoxetine

Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, in most cases, it is not known whether the compounds tested alter the timing of particular cell cycle transitions. Here, we evaluated an FDA-approved drug library to identify pharmaceuticals that alter cell cycle progression in yeast, using DNA content measurements by flow cytometry. This approach revealed strong cell cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. Based on their effects on cell cycle progression, we also examined cell proliferation in the presence of both compounds. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. Combinations of interest among diverse pharmaceuticals are difficult to identify, due to the daunting number of possible combinations that must be evaluated. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation.

Molecular networks and system-level properties

Molecular systems biology aims to describe the functions of complex biological processes through recursive integration of molecular analysis, modeling, simulation and theory. It focuses on networks that originate from interconnection of genes, proteins and metabolites whose dynamic interactions generate, as an emergent property of the system, the corresponding function. Although evolutionary optimized, intracellular biochemical parameters, such as the expression level of gene products or the affinity between two or more proteins, must have a permissible range that gives robustness against perturbations to the system. Using the yeast G 1-to-S transition network as an example we show that sophisticated relations exist among network structure, emergent property and robustness. Different emergent properties are generated from the same network by changing the strength of its interactions, not only by altering expression level, but also through mono and multi-site phosphorylation/dephosphorylation. Besides, multi-site protein phosphorylation modules, widespread in cell cycle, may ensure robust and coherent timing of cell cycle transitions as it happens for the onset of DNA replication. In conclusion, the modulation of biological function/emergent property by modifying interaction strength provides an efficient, highly tunable device to regulate biological processes. Furthermore, the principles outlined herein may provide new insight to network analysis in drug discovery.

Securin regulates entry into M-phase by modulating the stability of cyclin B

Timely progression into mitosis is necessary for normal cell division. This transition is sensitive to the levels of cyclin B, the regulatory subunit of the master mitotic kinase, Cdk1. Cyclin B accumulates during G2 and prophase when its rate of destruction by the anaphase promoting complex (APC) is low. Securin is also an APC substrate and is known for its role in inactivating the cohesin-cleaving enzyme, separase, until the metaphase to anaphase transition. Here we show that securin has an additional role in cell-cycle regulation, that of modulating the timing of entry into M-phase. In mouse oocytes, excess securin caused stabilization of cyclin B and precocious entry into M-phase. Depletion of securin increased cyclin B degradation, resulting in delayed progression into M-phase. This effect required APC activity and was reversed by expression of wild-type securin. These data reveal a role for securin at the G2-M transition and suggest a more general mechanism whereby physiological levels of co-competing APC substrates function in modulating the timing of cell-cycle transitions.

Steel factor responsiveness regulates the high self-renewal phenotype of fetal hematopoietic stem cells

AbstractFetal hematopoietic stem cells (HSCs) regenerate daughter HSCs in irradiated recipients more rapidly than do adult HSCs. However, both types of HSCs divide in vitro with the same cell-cycle transit times, suggesting different intrinsically determined self-renewal activities. To investigate the mechanism(s) underlying these differences, we compared fetal and adult HSC responses to Steel factor (SF) stimulation in vitro and in vivo. These experiments were undertaken with both wild-type cells and W41/W41 cells, which have a functionally deficient c-kit kinase. In vitro, fetal HSC self-renewal divisions, like those of adult HSCs, were found to be strongly dependent on c-kit activation, but the fetal HSCs responded to much lower SF concentrations in spite of indistinguishable levels of c-kit expression. Fetal W41/W41 HSCs also mimicked adult wild-type HSCs in showing the same reduced rate of amplification in irradiated adult hosts (relative to fetal wild-type HSCs). Assessment of various proliferation and signaling gene transcripts in fetal and adult HSCs self-renewing in vitro revealed a singular difference in Ink4c expression. We conclude that the ability of fetal HSCs to execute symmetric self-renewal divisions more efficiently than adult HSCs in vivo may be dependent on specific developmentally regulated signals that act downstream of the c-kit kinase.

The human synMuv-like protein LIN-9 is required for transcription of G2/M genes and for entry into mitosis

Regulated gene expression is critical for the proper timing of cell cycle transitions. Here we report that human LIN-9 has an important function in transcriptional regulation of G2/M genes. Depletion of LIN-9 by RNAi in human fibroblasts strongly impairs proliferation and delays progression from G2 to M. We identify a cluster of G2/M genes as direct targets of LIN-9. Activation of these genes is linked to an association between LIN-9 and B-MYB. Chromatin immunoprecipitation assays revealed binding of both LIN-9 and B-MYB to the promoters of G2/M regulated genes. Depletion of B-MYB recapitulated the biological outcome of LIN-9 knockdown, including impaired proliferation and reduced expression of G2/M genes. These data suggest a critical role for human LIN-9, together with B-MYB, in the activation of genes that are essential for progression into mitosis.

Prolonged Cell Cycle Transit Is a Defining and Developmentally Conserved Hemopoietic Stem Cell Property

Adult mouse hemopoietic stem cells (HSCs) are typically quiescent and enter and progress through the cell cycle rarely in steady-state bone marrow, but their rate of proliferation can be dramatically enhanced on demand. We have studied the cell cycle kinetics of HSCs in the developing fetal liver at a stage when they expand extensively. Despite that 100% of fetal liver HSCs divide within a 48-h period, their average cell cycle transit time (10.6 h) is twice that of their downstream progenitors, translating into a prolonged G(1) transit and a period of relative quiescence (G(0)). In agreement with their prolonged G(1) transit when compared with hemopoietic progenitors, competitive transplantation experiments demonstrate that fetal HSCs are highly enriched in G(1) but also functional in S-G(2)-M. This observation combined with experimental data demonstrating that adult HSCs forced to expand ex vivo also sustain a uniquely prolonged cell cycle and G(1) transit, demonstrate at least in part why purified HSCs at any state of development or condition are highly enriched in the G(0)-G(1) phases of the cell cycle. We propose that a uniquely prolonged cell cycle transit is a defining stem cell property, likely to be critical for their maintenance and self-renewal throughout development.