The effect of growth-promoting agents on replication and cell cycle withdrawal in cultures of epidermal keratinocytes

Summary Epidermal keratinocytes grow in culture to form a stratified squamous epithelium. These cultures contain a replicating as well as a terminally differentiating population and undergo surface desquamation. Epidermal growth factor (EGF) and cholera toxin are usually employed as growth-promoting agents because they reduce the population doubling time; that is, the period required to increase the total cell number twofold. There are three ways in which this reduction in population doubling time could be achieved: (a) the time for one cell cycle or the cell cycle length may be shortened; (b) the number of cells that withdraw from the cell cycle and terminally differentiate may be reduced; or (c) the number of cells that desquamate into the medium over a set period of time may be reduced. We have explored these possibilities in growing cultures of epidermal keratinocytes using a newly developed double-label assay. This assay gives a measure of both cell length and cell cycle withdrawal. Results show that the growth enhancement induced by EGF and cholera toxin can be attributed primarily to a reduction in cell cycle withdrawal and, to a lesser degree, to a reduction in cell cycle length. EGF and cholera toxin have no significant effect on the rate of desquamation. A linear correlation was noted between cell cycle lengths and withdrawal, suggesting an interconnection between the rate of cell renewal and the likelihood of undergoing terminal differentiation. This research was supported by grant DE04511 from the National Institute of Dental Research, Bethesda, MD, and gifts from the University Hospital Auxilliary, Health Sciences Center, SUNY Stony Brook, and the Suffolk County Volunteer Firefighter Fund.     

Interactions between the developmental program and cell cycle regulation of Aspergillus nidulans

Aspergillus nidulans is a multicellular fungus being used to study developmental regulation and cell cycle regulation. Genetic and molecular mechanisms underlying both processes have been characterized. Two types of observations suggest that there is significant interaction between cell cycle and developmental regulatory mechanisms. First, A. nidulans development involves the formation of specialized cell types that contain different, but specific, numbers of nuclei that are differentially regulated for cell cycle progression. Second, mutations directly affecting nuclear division can have major affects on cell differentiation during development. In this essay we describe these interactions and point out potential mechanisms for the cross talk between morphogenesis and the cell cycle that are tractable for future experimental investigation.     

病毒感染对细胞周期调控影响的研究进展

大量研究表明,病毒感染细胞时,病毒编码的蛋白或DNA可以扰乱细胞周期通路:促进细胞向S期转化或者使细胞静息于G2/M期。在细胞内,细胞周期的调控机制十分复杂,其包含了由DNA损伤导致的细胞通路活化及其他方式。关于病毒对细胞周期的调控方式及细胞周期的改变对于病毒感染的研究已取得一定进展。对于病毒的此类研究可以揭示细胞活动中的关键调控因子及细胞周期检查点的具体分子机理。对病毒调控宿主细胞周期以达到自身最大化复制的机理进行综述。     

Apoptosis and the cell cycle

In this review, we consider apoptosis as a process intimately linked to the cell cycle. There are several reasons for thinking of apoptosis as a cell cycle phenomenon. First, within the organism, apoptosis is almost exclusively found in proliferating tissues. Second, artificial manipulation of the cell cycle can either prevent or potentiate apoptosis, depending on the point of arrest. Data from such studies have suggested that molecules acting late in G1 are required for apoptosis. Since passage through late G1 into S phase in mammalian cells is known to be regulated by p53 and by activation of cyclin-dependent kinases, we also examine recent studies linking these molecules to the apoptotic pathway.     

Clonal heterogeneity in specific growth rate of Succhuromyces cerevisiue cells

The cell volume increase in individual clones of cells of the yeast Saccharomyces cerevisiae has been measured using time lapse cinematography in populations showing steady state balanced exponential growth. There were significant differences in clonal specific growth rates within the population in each of 10 experiments using different strains on different media supporting different growth rates. The results suggest that specific growth rates of cells which are either genetically identical or very closely related can be different and this difference can be propagated over at least three generations. Since the proliferation rate in yeast is determined by growth rate, these observed differences provide an additional source of cell cycle variability for yeast cells that has not been considered before. The implications for the theoretical analysis of cell cycle kinetics are examined.     

Mouse 24p3 protein has an effect on L929 cell viability

It is well known that mouse uterine 24p3 protein, is an acute phase protein, secreted from the L929 cell line, and that it will be induced by the dexamethasone stimulation of the cell. We investigated the possible effects of 24p3 protein on the L929 cell line, by observing its morphological change, ROS increase and viability decrease, by the process of culturing in a 24p3 protein-supplemented medium. Following the L929 cells' exposure to the 24p3 protein supplement for a period of 72 hours, S-phase cells accumulated to a significant degree, suggesting that the entry into the G2/M phase from the S phase, in the cell cycle progression, was blocked. There was a significant decrease in cell numbers and increased DNA damage within the cells in the presence of 24p3 protein within the medium for 96 hours, implying that they have undergone pathway of cell death. After 96h incubation in low concentration of 24p3 protein, the result of PI/annexin V double staining showed cell death obviously. These results suggest that 24p3 protein-induced S phase arrest in the cell cycle, would cause DNA damage, followed by cell death in the L929 cells.     

Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint

The regulation of the Dictyostelium cell cycle has remained ambiguous owing to difficulties in long-term imaging of motile cells and a lack of markers for defining cell cycle phases. There is controversy over whether cells replicate their DNA during development, and whether spores are in G1 or G2 of the cell cycle. We have introduced a live-cell S-phase marker into Dictyostelium cells that allows us to precisely define cycle phase. We show that during multicellular development, a large proportion of cells undergo nuclear DNA synthesis. Germinating spores enter S phase only after their first mitosis, indicating that spores are in G2. In addition, we demonstrate that Dictyostelium heterochromatin is copied late in S phase and replicates via accumulation of replication factors, rather than recruitment of DNA to pre-existing factories. Analysis of variability in cycle times indicates that regulation of the cycle manifests at a single random transition in G2, and we present the first identified checkpoint in Dictyostelium, which operates at the G2-M transition in response to DNA damage.     

Cell cycle regulation in the postmitotic neuron: oxymoron or new biology?

Adult CNS neurons are typically described as permanently postmitotic but there is probably nothing permanent about the neuronal cell cycle arrest. Rather, it appears that these highly differentiated cells must constantly keep their cell cycle in check. Relaxation of this vigilance leads to the initiation of a cell cycle and entrance into an altered and vulnerable state, often leading to death. There is evidence that neurons which are at risk of neurodegeneration are also at risk of re-initiating a cell cycle process that involves the expression of cell cycle proteins and DNA replication. Failure of cell cycle regulation might be a root cause of several neurodegenerative disorders and a final common pathway for others.     

Synchronous cell growth occurs upon synchronizing the two regulatory steps of the Saccharomyces cerevisiae cell cycle

There are two known asynchronous steps in the budding yeast Saccharomyces cerevisiae cell cycle, where an asynchronous step is one which is completed in different lengths of time by different cells in an isogenic population. It is shown here that elimination of the asynchrony due to cell size by preincubation of cells with the mating pheromone alpha-factor, and decreasing the asynchrony in the cdc28 'start' step by lowering the pH, yields highly synchronous cell growth measured as the time period between the emergence of buds. In one experiment, cell budding for 92% of cells occurred within a 12-min period for at least two generations. Under identical conditions, cell number increase is not as synchronous as bud emergence indicating that there is a third asynchronous step, which is concluded to be at cell separation. These results are consistent with there being two--and only two--asynchronous steps in the cell cycle, measured from bud emergence to bud emergence. Surprisingly, these two steps are also the two major regulatory steps of the cell cycle. It is concluded that asynchrony may be a general feature of cell cycle regulatory steps. The asynchrony in the completion of the cdc28 'start' step which occurs in the first cell cycle after alpha-factor washout is shown here to be almost or entirely eliminated for the second passage through this step after alpha-factor washout. The 'true' time between the onset of budding and the point where 50% of cells have budded (called t50BE) is 17 and less than or equal to 2 min for the first and second budding, respectively, after alpha-factor washout. The cell cycle models requiring a transition probability, or asynchrony, at 'start' for every cell cycle are therefore incorrect.     

Integrated actions of progesterone receptor and cell cycle machinery regulate breast cancer cell proliferation

Multiple laboratories have investigated progesterone receptor (PR) involvement in breast cancer cell cycle progression. There is now a growing body of evidence demonstrating complex interactions between PR and cell cycle regulatory proteins. Here we review the current literature linking PR to cell cycle control and discuss gaps in the current knowledge. A more complete understanding of the relationships between PR and cell cycle regulatory molecules may reveal additional avenues for prevention and treatment of steroid receptor positive breast cancers.     

Cyclin and DNA Distributed Cell Cycle Model for GS-NS0 Cells

Mammalian cell cultures are intrinsically heterogeneous at different scales (molecular to bioreactor). The cell cycle is at the centre of capturing heterogeneity since it plays a critical role in the growth, death, and productivity of mammalian cell cultures. Current cell cycle models use biological variables (mass/volume/age) that are non-mechanistic, and difficult to experimentally determine, to describe cell cycle transition and capture culture heterogeneity. To address this problem, cyclins—key molecules that regulate cell cycle transition—have been utilized. Herein, a novel integrated experimental-modelling platform is presented whereby experimental quantification of key cell cycle metrics (cell cycle timings, cell cycle fractions, and cyclin expression determined by flow cytometry) is used to develop a cyclin and DNA distributed model for the industrially relevant cell line, GS-NS0. Cyclins/DNA synthesis rates were linked to stimulatory/inhibitory factors in the culture medium, which ultimately affect cell growth. Cell antibody productivity was characterized using cell cycle-specific production rates. The solution method delivered fast computational time that renders the model’s use suitable for model-based applications. Model structure was studied by global sensitivity analysis (GSA), which identified parameters with a significant effect on the model output, followed by re-estimation of its significant parameters from a control set of batch experiments. A good model fit to the experimental data, both at the cell cycle and viable cell density levels, was observed. The cell population heterogeneity of disturbed (after cell arrest) and undisturbed cell growth was captured proving the versatility of the modelling approach. Cell cycle models able to capture population heterogeneity facilitate in depth understanding of these complex systems and enable systematic formulation of culture strategies to improve growth and productivity. It is envisaged that this modelling approach will pave the model-based development of industrial cell lines and clinical studies.     

Expression of Cell Cycle-Related Genes During Neuronal Apoptosis: Is there a Distinct Pattern?

An emerging hypothesis considers the process of neuronal apoptosis as a consequence of unscheduled and unsynchronized induction of cell cycle mediators. Induction of several cell cycle genes precedes neuronal apoptosis and may be involved in determination of cell fate. We have now characterized changes in expression of cell cycle genes during apoptosis induced by oxidative stress in chick post-mitotic sympathetic neurons. Induction of cyclin B occurred prior to the commitment of neurons to both dopamine- and peroxide-triggered apoptosis. Both the neuronal death and the rise in cyclin B were inhibited by antioxidant treatment, suggesting a functional role for cyclin B induction during neuronal apoptosis. Induction of the cyclin dependent kinase CDK5 protein coincided with the time point when neurons were irreversibly committed to die. Expression of other cell cycle mediators such as cyclin D1 and the cyclin dependent kinases CDC2 and CDK2 was undetected and not induced by exposure to oxidative stress. Comparative analysis of the profile of cell cycle mediators induced during neuronal apoptosis of different neuronal cell populations revealed no distinct pattern of events. There are no cell cycle stage-specific mediators that are ultimately stimulated during neuronal apoptosis, suggesting that multiple pathways of re-activating the dormant cell-cycle, converge to determine entry into apoptosis. Nevertheless, the existence of some cell cycle mediators, that were not reported so far to be induced in post mitotic neurons during oxidative stress, substantiate them as part of the strong differentiating forces.     

Effects of growth phase,diel cycle and macronutrient stress on the quantification of Heterosigma akashiwo using qPCR and SHA

The development of molecular probe technologies over the last several decades has enabled more rapid and specific identification and enumeration of phytoplankton species compared to traditional technologies, such as light microscopy. Direct comparisons of these methods with respect to physiological status, however, are sparse. Here we directly compare quantitative real-time PCR (qPCR) and sandwich hybridization assay (SHA) for enumerating the raphidophyte Heterosigma akashiwo at several points during its growth phase, over a diel cycle and with macronutrient stress in laboratory cultures. To ensure consistency between comparisons, a single cellular homogenate was generated from each culture and split for analysis by qPCR and SHA. Since the homogenate was generated from the same number of cells during each experiment, results reflect changes in nucleic acid content (rRNA and DNA) at each time point or in response to environmental conditions relative to a reference sample. Results show a greater level of precision in SHA results which contributed to significant (2–3 fold) differences in rRNA content per cell in several of these analyses. There was significantly greater rRNA content during lag and exponential phases compared to stationary phase cultures, and a significant decrease in rRNA content during the light cycle compared to cells harvested in the dark. In contrast, there were no significant differences in DNA content per cell as determined by qPCR over a diel cycle or during different growth phases. There was also no decrease in either rRNA or DNA content for cultures under low P conditions compared to nutrient replete conditions. However, both rRNA and DNA content were significantly lower under N stress when compared to nutrient replete conditions. Results of this study suggest that growth stage, nutrient stress and cell cycle may impact molecular analyses, and that physiological status should be taken into account when using these methods for HAB monitoring.     

Microarrays and the relationship of mRNA variation to protein variation during the cell cycle

Microarray analyses have led to the postulated existence and identification of numerous genes that are believed to be expressed and presumably to act in a cell-cycle-specific manner because their expression varies during the cell cycle. It is important to see how protein variation can be produced from mRNA variation. We have calculated the protein content throughout the cell cycle resulting from cell-cycle-specific mRNA expression, and compared the result to protein content resulting from constant, cell-cycle independent, mRNA expression. For stable proteins, cell-cycle-specific mRNA expression leads to a maximum 2-fold change in protein content compared to proteins synthesized from constantly expressed mRNA. More realistic sinusoidal patterns of mRNA expression exhibit much smaller ratios of 1.25 or lower, even for extremely large amplitudes in mRNA expression. For unstable proteins that have a cycle-independent half-life, only at extremely short protein half-lives does mRNA variation have a significant impact on variation of protein content during the division cycle. We also apply these findings to proteins with a cycle-specific decay pattern. mRNA variations during the eukaryotic division cycle variation of mRNA during the cell cycle can have only a minimal affect on the variation of protein content during the cell cycle. We conclude that mRNA variations during the division cycle, as measured by microarrays, cannot by themselves, identify cycle-specific functions related to protein variations.     

On a heuristic point of view concerning the expression of numerous genes during the cell cycle

The current model of the eukaryotic cell cycle proposes that numerous genes are expressed at different times during the cell cycle. The existence of myriad control points for gene expression leads to theoretical and logical problems for cell cycle control. Each expressed gene requires a control element to appear in a cell-cycle specific manner; this control element requires another control element and so on, ad infinitum. There are also experimental problems with the current model based on ineffective synchronization methods and problems with microarray measurements of mRNA. Equally important, the efficacy of mRNA variation in affecting changes in protein content is negligible. An alternative view of the cell cycle proposes cycle-independent, invariant accumulation of mRNA during the cell cycle with decreases of specific proteins occurring only during the mitotic period of the cell cycle.     

Metformin Induces Cell Cycle Arrest,Reduced Proliferation,Wound Healing Impairment In Vivo and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients

BackgroundSeveral epidemiological studies in diabetic patients have demonstrated a protective effect of metformin to the development of several types of cancer. The underlying mechanisms of such phenomenon is related to the effect of metformin on cell proliferation among which, mTOR, AMPK and other targets have been identified. However, little is known about the role that metformin treatment have on other cell types such as keratinocytes and whether exposure to metformin of these cells might have serious repercussions in wound healing delay and in the development of complications in diabetic patients with foot ulcers or in their exacerbation.ResultsMetformin treatment significantly reduces cell proliferation; colony formation and alterations of the cell cycle are observed also in the metformin treated cells, particularly in the S phase. There is a significant increase in the area of the wound of the metformin treated animals at different time points (P<0.05). There is also a significant increase in the size and wound area of the patients with diabetic foot ulcers at the time of hospitalization. A protective effect of metformin was observed for amputation, probably associated with the anti inflammatory effects reported of metformin.ConclusionsMetformin treatment reduces cell proliferation and reduces wound healing in an animal model and affects clinical outcomes in diabetic foot ulcer patients. Chronic use of this drug should be further investigated to provide evidence of their security in association with DFU.