Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

Frequency doubling in the cyanobacterial circadian clock

Organisms use circadian clocks to generate 24‐h rhythms in gene expression. However, the clock can interact with other pathways to generate shorter period oscillations. It remains unclear how these different frequencies are generated. Here, we examine this problem by studying the coupling of the clock to the alternative sigma factor sigC in the cyanobacterium Synechococcus elongatus. Using single‐cell microscopy, we find that psbAI, a key photosynthesis gene regulated by both sigC and the clock, is activated with two peaks of gene expression every circadian cycle under constant low light. This two‐peak oscillation is dependent on sigC, without which psbAI rhythms revert to one oscillatory peak per day. We also observe two circadian peaks of elongation rate, which are dependent on sigC, suggesting a role for the frequency doubling in modulating growth. We propose that the two‐peak rhythm in psbAI expression is generated by an incoherent feedforward loop between the clock, sigC and psbAI. Modelling and experiments suggest that this could be a general network motif to allow frequency doubling of outputs.     

Analyzing cell fate control by cytokines through continuous single cell biochemistry

Cytokines are important regulators of cell fates with high clinical and commercial relevance. However, despite decades of intense academic and industrial research, it proved surprisingly difficult to describe the biological functions of cytokines in a precise and comprehensive manner. The exact analysis of cytokine biology is complicated by the fact that individual cytokines control many different cell fates and activate a multitude of intracellular signaling pathways. Moreover, although activating different molecular programs, different cytokines can be redundant in their biological effects. In addition, cytokines with different biological effects can activate overlapping signaling pathways. This prospect article will outline the necessity of continuous single cell biochemistry to unravel the biological functions of molecular cytokine signaling. It focuses on potentials and limitations of recent technical developments in fluorescent time‐lapse imaging and single cell tracking allowing constant long‐term observation of molecules and behavior of single cells. J. Cell. Biochem. 108: 343–352, 2009. © 2009 Wiley‐Liss, Inc.     

Reappraisal of G1-phase arrest and synchronization by lovastatin

It has been proposed that lovastatin arrests cells in the G1-phase of the division cycle, and that release from lovastatin inhibition produces a synchronized culture. A new method of methocel time-lapse-videography has been used to analyse cell division patterns following lovastatin treatment. Release of L1210 cells from lovastatin inhibition failed to produce synchronized divisions. Moreover, contrary to earlier proposals, lovastatin did not arrest cells with a G1-phase amount of DNA. Analysis of previous reports of 'synchronization' and growth-arrest support these findings. It is concluded that lovastatin neither synchronizes cells, nor arrests cells in the G1-phase of the division cycle.     

Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy

Raman micro-spectroscopy is a laser-based technique which enables rapid and non-invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro-spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell-specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G(0)/G(1), S, and G(2)/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G(0)/G(1) to G(2)/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G(0)/G(1)-phase cells and G(2)/M-phase cells. The non-invasive analysis of live cell cycle dynamics with Raman micro-spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels.     

N‐nitrosomelatonin enhances photic synchronization of mammalian circadian rhythms

Most physiological processes in mammals are synchronized to the daily light:dark cycle by a circadian clock located in the hypothalamic suprachiasmatic nucleus. Signal transduction of light‐induced phase advances of the clock is mediated through a neuronal nitric oxide synthase‐guanilyl cyclase pathway. We have employed a novel nitric oxide‐donor, N‐nitrosomelatonin, to enhance the photic synchronization of circadian rhythms in hamsters. The intraperitoneal administration of this drug before a sub‐saturating light pulse at circadian time 18 generated a twofold increase of locomotor rhythm phase‐advances, having no effect over saturating light pulses. This potentiation was also obtained even when inhibiting suprachiasmatic nitric oxide synthase activity. However, N‐nitrosomelatonin had no effect on light‐induced phase delays at circadian time 14. The photic‐enhancing effects were correlated with an increased suprachiasmatic immunoreactivity of FBJ murine osteosarcoma viral oncogene and period1. Moreover, in vivo nitric oxide release by N‐nitrosomelatonin was verified by measuring nitrate and nitrite levels in suprachiasmatic nuclei homogenates. The compound also accelerated resynchronization to an abrupt 6‐h advance in the light:dark cycle (but not resynchronization to a 6‐h delay). Here, we demonstrate the chronobiotic properties of N‐nitrosomelatonin, emphasizing the importance of nitric oxide‐mediated transduction for circadian phase advances.

     

The circadian mutation PER2(S662G) is linked to cell cycle progression and tumorigenesis

Circadian oscillation and cell cycle progression are the two most essential rhythmic events present in almost all organisms. Circadian rhythms keep track of time and provide temporal regulation with a period of about 24 h. The cell cycle is optimized for growth and division, but not for time keeping. Circadian gated cell divisions are observed in nearly all organisms. However, the implications of this coupling to the physiology of mammals are unknown. A mutation (S662G) in the clock protein PERIOD2 (PER2) is responsible for familial advanced sleep phase syndrome in which sleep onset occurs in the early evening and wakefulness occurs in the early morning. Here, we provide evidence that the PER2^S662 mutation leads to enhanced resistance to X-ray-induced apoptosis and increased E1A- and RAS-mediated oncogenic transformation. Accordingly, the PER2^S662 mutation affects tumorigenesis in cancer-sensitized p53^R172H/+ mice. Finally, analyzing the clock-controlled cell cycle genes p21, c-Myc, Cyclin D1 and p27, we found that the relative phases between p21 and Cyclin D expression profiles have been changed significantly in these Per2 allele mutant mouse embryonic fibroblasts. This key role of the Per2-mediated phase alteration of p21 provides what we believe to be a novel mechanism in understanding cell cycle progression, its plasticity and its resistance to interference.     

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal‐derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum‐free, protein‐free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single‐cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD‐CHO? and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Circadian clock,cell cycle,and breast cancer: an updated review

Some key elements are common to two fundamental periodic regulatory processes; the circadian cycle and the cell cycle. Underlying mechanisms of coordination between the two processes are critical for proper cellular functioning and physiology. Disruption in the mechanisms of one process may affect the role of other that may direct critical physiological changes and may cause severe diseases like cancer, etc. More or less persuasive evidences evolve from the breast cancer research. In this mini review, we highlighted the molecular coordination’s of the elements of circadian cycle and the cell cycle and their altered expressions associated with the genesis and progression of breast cancer.     

Fluorescence kinetics in HeLa cells after treatment with cell cycle arrest inducers visualized with Fucci (fluorescent ubiquitination-based cell cycle indicator)

Fucci (fluorescent ubiquitination-based cell cycle indicator) is able to visualize dynamics of cell cycle progression in live cells; G1- and S-/G2-/M-phase cells expressing Fucci emit red and green fluorescence, respectively. This system could be applied to cell kinetic analysis of tumour cells in the field of cancer therapy; however, it is still unclear how fluorescence kinetics change after various treatments, including exposure to anticancer agents. To explore this, we arrested live HeLa cells expressing the Fucci probes at various cell cycle stages and observed the fluorescence, in conjunction with flow cytometric analysis. X-irradiation, HU (hydroxyurea) and nocodazole arrest cells at G2/M boundary, early S-phase and early M-phase, respectively. Although X-irradiation and HU treatment induced similar accumulation kinetics of green fluorescent cells, nocodazole treatment induced an abnormal red fluorescence at M phase, followed by accumulation of both red and green fluorescent cells with 4N DNA content. We conclude that certain agents that disrupt normal cell cycle regulation could cause unexpected fluorescence kinetics in the Fucci system.     

Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal‐fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse‐chase LC‐MS‐based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine‐derived fluxes: Oxidation of glucose‐derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation.     

Screening of five specific cell cycle inhibitors using a T Cell lymphoma cell line synchrony/release assay

To obtain different cell populations at specific cell cycle stages, we used a cell culture synchronization protocol. Effects of five different cell cycle inhibitors acting throughout the cell cycle were examined by DNA flow cytometric analysis of a synchrony/release lymphoma cell line (CEM). The screening synchronized protocol showed that staurosporine, mimosine and aphidicolin are reversible G1 phase inhibitors that act at different times. Staurosporine acted in early G1, exhibited the strongest cytotoxic effect, and induced apoptosis. Mimosine and aphidicolin acted in late G1 and at the G1/S boundary, respectively. Hydroxyurea arrested CEM cells in early S phase, but later than the aphidicolin arrest point. Nocodazole synchronized CEM cells in M phase. All the inhibitors examined in this study can be used to synchronize cells at different phases of the cell cycle and were reversible with little toxicity except for staurosporine which is highly toxic. Because the regulatory mechanism of the cell cycle is disrupted by their effects on protein synthesis, however, these drugs must be used with caution.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G₁ period of the HeLa cell cycle. HeLa cells synchronized in the G₁ period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N₂O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G₁ period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G₁ period, when DNA synthesis is initiated.     

Characterization of the KG1a cell line for use in a cell migration based screening assay

High-throughput screening has become a popular method used to identify new “leads” for potentially therapeutic compounds. Further screening of these lead compounds is typically done with secondary assays which may utilize living, functioning cells as screening tools. A problem (or benefit) with these cell-based assays is that living cells are very sensitive to their environment. We have been interested in the process of stem cell migration and how it relates to the cellular therapy of bone marrow transplantation. In this study we describe a secondary, cell-based assay for screening the effects of variousin-vitro conditions on Immature Hematopoietic Cell (IHC) migration. Our results have revealed many subtle factors, such as the cell's adhesive characteristics, or the effect of a culture's growth phase, that need to be accounted for in a screening protocol. Finally, we show that exponentially growing KG1a cells (a human IHC cell line) were 10 times more motile than those in the lag or stationary phases. These data strongly suggest that KG1a cells secrete a chemokinetic factor during the exponential growth phase of a culture.