CELL-CYCLE ANALYSIS OF PERTURBED CELL POPULATIONS: COMPUTER SIMULATION OF SEQUENTIAL DNA DISTRIBUTIONS

A mathematical model is presented that permits simulation of a time sequence of DNA distributions with a single set of cell-cycle parameters. The method is particularly suited to the quantitative analysis of sets of sequential DNA distributions from perturbed cell populations. The model permits determination of the durations and associated dispersions of the phases of the cell cycle as well as the point in the cell cycle at which the perturbing agent exerts its effect. The mathematical details of the simulation technique are presented, and the technique is applied to the analysis of DNA distributions from perturbed cell populations. Three cell populations are modeled: CHO-line cells released from a block at the interface of the G₁ and S-phases, 3T3 cells released from a G₁-phase block produced by serum starvation, and S49 mouse lymphoma cells responding to a block in the G₁-phase produced by N⁶,0²′-dibutyryl adenosine 3′:5′-cyclic monophosphate (Bt₂cAMP).     

New insights into the kinetic resistance to anticancer agents

Kinetic resistance plays a major role in the failure of chemotherapy towards many solid tumors. Kinetic resistance to cytotoxic drugs can be reproduced in vitro by growing the cells as multicellular spheroids (Multicellular Resistance) or as hyperconfluent cultures (Confluence-Dependent Resistance). Recent findings on the cell cycle regulation have permitted a better understanding why cancer cells which arrest in long quiescent phases are poorly sensitive to cell-cycle specific anticancer drugs. Two cyclin-dependent kinase inhibitors (CDKI) seem particularly involved in the cell cycle arrest at the G1 to S transition checkpoint: the p53-dependent p21^cip1 protein which is activated by DNA damage and the p27^kip1 which is a mediator of the contact inhibition signal. Cell quiescence could alter drug-induced apoptosis which is partly dependent on an active progression in the cell cycle and which is facilitated by overexpression of oncogenes such as c-Myc or cyclins. Investigations are yet necessary to determine the influence of the cell cycle on the balance between antagonizing (bcl-2, bcl-X_L...) or stimulating (Bax, Bcl-X_S, Fas...) factors in chemotherapy-induced apoptosis. Quiescent cells could also be protected from toxic agents by an enhanced expression of stress proteins, such as HSP27 which is induced by confluence. New strategies are required to circumvent kinetic resistance of solid tumors: adequate choice of anticancer agents whose activity is not altered by quiescence (radiation, cisplatin), recruitment from G1 to S/G2 phases by cell pretreatment with alkylating drugs or attenuation of CDKI activity by specific inhibitors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Temporal structure of S phase analyzed using an automated synchrony system

An automated system for cell cycle analysis is described in which synchronous populations of greater than 95% mitotic cells can be selected and subcultured without resorting to inhibitors or altered media. Selection of mitotic cells from roller bottle cultures generates synchronous populations of up to 5 × 10⁷ cells, as often as every 30 min, under conditions of constant temperature. A detailed analysis of the temporal pattern of DNA synthesis in the S phase of several Chinese hamster cells is presented. Several potential artefacts and uncertainties regarding the cell cycle blockage point in S phase synchronization are discussed.     

Patterns of DNA Synthesis and Cell Division in Marine Dinoflagellates1

Changes in mean cell size, DNA and cell density were monitored at 6-h intervals for 72 h in populations of six species (eight clones) of marine dinoflagellates to determine the temporal relationships between the cell cycle events of DNA replication and cytokinesis. Batch cultures were maintained at 15 or 20°C on a 12-h light: 12-h dark photoperiod. Cell densities and size frequency distributions were determined conductimetrically and the amount of DNA within populations was measured fluorometrically. A variety of intra- and interspecific relationships were observed, ranging from parallel phasing of cell cycle processes to variations which involved the temporal uncoupling of DNA synthesis from the phased pattern of cell division which is characteristic of dinoflagellate cell cycles. Daily growth rates of individual populations varied from 0.05 (Gymnodinium nelsoni) to 2.08 (Amphidinium carteri) cell divisions day^-1 and DNA doubling rates ranged from 0 to 1.14 day^-1. Mean doubling rates for DNA were usually 30–40% lower than those for cells. The degree of difference in these rates and the amount of variability evident in cell cycle sequences may be major factors in determining the rate and extent of development of dinoflagellate populations in nature.     

Separation of Plasmacytoid Dendritic Cells from B-Cell-Biased Lymphoid Progenitor (BLP) and Pre-Pro B Cells Using PDCA-1

B-cell-biased lymphoid progenitors (BLPs) and Pre-pro B cells lie at a critical juncture between B cell specification and commitment. However, both of these populations are heterogenous, which hampers investigation into the molecular changes that occur as lymphoid progenitors commit to the B cell lineage. Here, we demonstrate that there are PDCA-1⁺Siglec H⁺ plasmacytoid dendritic cells (pDCs) that co-purify with BLPs and Pre-pro B cells, which express little or no CD11c or Ly6C. Removal of PDCA-1⁺ pDCs separates B cell progenitors that express high levels of a Rag1-GFP reporter from Rag1-GFP^low/neg pDCs within the BLP and Pre-pro B populations. Analysis of Flt3-ligand knockout and IL-7Rα knockout mice revealed that there is a block in B cell development at the all-lymphoid progenitor (ALP) stage, as the majority of cells within the BLP or Pre-pro B gates were PDCA-1⁺ pDCs. Thus, removal of PDCA-1⁺ pDCs is critical for analysis of BLP and Pre-pro B cell populations. Analysis of B cell potential within the B220⁺CD19⁻ fraction demonstrated that AA4.1⁺Ly6D⁺PDCA-1⁻ Pre-pro B cells gave rise to CD19⁺ B cells at high frequency, while PDCA-1⁺ pDCs in this fraction did not. Interestingly, the presence of PDCA-1⁺ pDCs within CLPs may help to explain the conflicting results regarding the origin of these cells.     

CELL CYCLE TIME DETERMINATIONS BASED ON LIQUID SCINTILLATION COUNTING OF 3H-TdR LABELED MITOTIC CELLS

Following a 10 min pulse labeling with ³H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G₁, S, G₂ and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.     

Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis

Abstract We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2–3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (<2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found that the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.     

Cdk pathway: Cyclin-dependent kinases and cyclin-dependent kinase inhibitors

Many mechanisms either activate or inhibit the cdks and thereby either promote or arrest progression through the mitotic cell cycle. Since the signal transduction pathways emanating from extracellular mitogens and the agents controlling these pathways are complicated there may yet be novel mechanisms of cell cycle regulation remaining to be elucidated. In this article we outline the different techniques used to study the cell cycle and its regulation. These include: establishing that the cell cycle is arrested by propidium iodide staining followed by FACS analysis or by measuring ³H-thymidine incorporation into DNA; measuring the amount of cyclin/cdk associated kinase activity; assessing the steady-state expression profiles of cyclins, cdks and ckis by immunoblotting; and investigating the formation of complexes between these proteins by coimmunoprecipitations. Caveats and advantages of each technique are discussed. Following this paradigm yielded the discovery of the cell cycle inhibitors p27^Kip1 and p21^Cip1 and could very well lead to the discovery or novel cell cycle regulatory mechanisms.     

A Method For Measuring the Generation Time and Length of Dna Synthesizing Phase of Clonogenic Cells In A Heterogenous Population

Information on the cell cycle of progenitor cells in haemopoietic tissue is useful for understanding population control under physiological and abnormal conditions. Unfortunately, methods that have been developed for measuring cell cycle parameters are applicable only to cells of homogenous populations and not to morphologically non-recognizable progenitor cells such as colony forming units (CFU) that are present at low frequency in a heterogenous population. to circumvent this difficulty, a method was developed to measure CFU cell cycle parameters based on specific killing of cells in S phase by [³H]thymidine ([³H]TdR). This was done by estimating the number of CFU killed following exposure of the cell suspension to [³H]TdR for various time periods. Since cycling CFU are continuously entering S phase, a linear curve relating the percentage CFU-kill to the length of exposure of the cells to [³H]TdR in culture can be obtained. the slope of the curve (percentage kill/hr) indicates the rate that CFU enter the S phase and travel through the cell cycle. the inverse of this value will then represent a time period for CFU to move through a complete cell cycle (generation time). the length of S phase can then be obtained by multiplying generation time by the fraction of cells in S phase at time zero. This method has been used to measure generation time and length of S phase of three kinds of haemopoietic progenitor cells: mouse granulocyte-macrophage CFU, human T lymphocyte CFU and CFU from regenerating mouse spleens. This method should be applicable to any normal or neoplastic clonogenic cell populations and the latter could be either of haematological or of solid tumour origin.     

Membrane dielectric responses of bufalin-induced apoptosis in HL-60 cells detected by an electrorotation chip

A non-invasive electrorotation (ROT) technique was used to monitor the apoptosis-induced changes in HL-60 cells. The specific membrane capacitance of the cells fell from 15.6 ± 0.9 mF/cm² to 6.4 ± 0.6 mF/cm² after 48 h treatment with 10 nM bufalin, a component of bufadienolides in traditional Chinese medicine, Chan Su. However, the average membrane conductance remained almost constant during the first 24 h of treatment and then increased afterwards. Apoptosis was verified by a DNA fragmentation assay and scanning electron microscopy. The results demonstrate that the ROT technique gives a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the testing and development of new pharmaceuticals and active cell agents. Chengjun Huang and Ailiang Chen contributed equally to this work.     

Flow cytometry analysis of cell population dynamics and cell cycle during HIV-1 envelope-mediated formation of syncytia in vitro

Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell–cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4⁺ cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4⁺ cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env⁺ cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6 h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4⁺ cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell–cell fusion can be performed by flow cytometry.     

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

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

Dynamic aspects of radiorespirometry during the cell cycle of Candida utilis: Some observations in phased culture under carbon-, nitrogen-, and phosphorus-limited growth

Many changes that occur in a cell during the cell cycle can be demonstrated in synchronous cultures and can reveal dimensions of cell metabolism not attainable by the study of balanced growth of asynchronous populations in batch cultures or the steady state in chemostat cultures. The release of ¹⁴CO₂ from specifically labeled glucose by phased (continuously synchronized) cultures follows a characteristic pattern (profile) that depends upon the stage in the cell cycle and the period of labeling used. Successive profiles throughout a cycle showed differences that were altered under different nutrient-limiting growth conditions. Profiles obtained with glucose-1-¹⁴C, glucose-2-¹⁴C, glucose-3,4-¹⁴C, and glucose-6-¹⁴C and phased cells of Candida utilis under N-, P-, and C-limited growth demonstrated the variable character of the metabolic activity that occurred in the cells while contour changes within the profiles across the cycle indicated possible correlations with activities of the hexose monophosphate, Embden-Meyerhof-Parnas, and tricarboxylic acid cycle pathways during the cell cycle. The basis of these changes and their use as elementary parameters for study of problems of physiological changes in vivo are considered.     

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The formation of a soluble H³-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H³-thymidine during G1 or G2. Uptake of H³-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H³-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H³-thymidine, the pool does not turn over during non-S periods.     

Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis

We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2-3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (less than 2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

The Proliferative State of Clonogenic Cells In the Lewis Lung Tumour After Treatment With Cytotoxic Agents

The proportion of clonogenic cells from the Lewis lung carcinoma which are in S-phase of the cell cycle has been measured as the fraction killed by a short exposure to hydroxyurea in vitro. Estimates of the proportions of Sphase cells before and 30 min after doses of γ-radiation of 1000–2000 rad suggest no alternation in the cell cycle age distribution due to these doses of radiation. As the survivors of these high doses of radiation are predominantly hypoxic, the results imply that hypoxic cells have the same cell cycle age distribution as oxygenated cells in Lewis lung tumours. After treatment with cyclophosphamide or CCNU, the proportion of S-phase cells among the survivors exceeds the faction of S-phase cells in untreated populations. This increase is consistent with a relative resistance of S-phase cells to alkylating agents and nitrosoureas.     

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry¹ offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture⁵. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.     

PROLIFERATION RATE OF HAEMOPOIETIC STEM CELLS AFTER DAMAGE BY SEVERAL CYTOSTATIC AGENTS

The haemopoietic tissue of mice was damaged by different cell-cycle-stage specific and cell-cycle-stage non-specific cytostatic agents. The proliferation rate among the surviving pluripotential stem cells, i.e. those cells forming colonies in spleens of lethally irradiated mice (CFUs), was then investigated. The results suggest that, at least in the CFUs population, the cells which synthesize DNA in the S phase of the cell cycle inhibit the entry of the non-proliferating G₀ cells into cell cycle. This evidence was based on the ability of three cytostatic agents, hydroxyurea, cytosine arabinoside and methotrexate, which are toxic specifically to the S phase cells to increase the proliferation in the CFUs population. This increase was quite out of proportion to the small amount of damage they caused to the population. Colchicine, which kills cells in mitosis, and ionizing irradiation, damaging cells in all stages, proved to be much weaker stimulators of proliferation. It has been suggested that a mechanism for the control of cellular proliferation might be based on the negative feedback in the cell cycle. In this feedback control loop the cells which are preparing for cell division in the S phase of the cell cycle inhibit the entry of the non-proliferating G₀ cells into cell cycle.