The relative effects of different types of growth factors on DNA replication, mitosis, and cellular enlargement

It was recently demonstrated that growth in cell size can be dissociated from DNA synthesis and mitosis. 3T3 cells starved to quiescence in low serum concentration can be stimulated to undergo DNA synthesis and one cell division without growing in size (unbalanced growth) (42-44). We report here that in cells stimulated to undergo unbalanced growth, the cell nucleus undergoes balanced growth, i.e., nearly doubles in size prior to mitosis. The reduced ability to grow in cell size under unbalanced growth conditions is thus mainly ascribable to the cytoplasm. Furthermore, the extent to which cells grow in size prior to mitosis is dependent on the serum concentration in the tissue culture medium (44). This data suggests that some macromolecular factor or factors in serum are required for growth in cell size prior to mitosis. We report in this study that epidermal growth factor (EGF) alone exerts a small but significant stimulatory influence on DNA synthesis and mitosis but does not affect cellular enlargement. In contrast, insulin added at supraphysiological concentrations does not stimulate quiescent cells to enter S phase but instead stimulates growth in cell size in the small fraction of dividing cells. Furthermore, cells stimulated to proliferate by EGF could be induced to undergo balanced growth when insulin was added concomitantly. Finally, platelet-derived growth factor (PDGF) stimulates quiescent sparse 3T3 cells to undergo DNA synthesis and mitosis. PDGF also exerts a limited but significant effect on cellular enlargement. However, PDGF alone could not induce a complete balanced growth, i.e., a doubling in cell size prior to mitosis.     

Instantaneous evaluation of mammalian cell culture growth rates through analysis of the mitotic index

Since a culture increases in cell number when dividing cells separate into two newborn cells, the fraction of mitotic cells in a growing cell population directly reflects the overall growth behavior of a cell culture. To rapidly assess the effects of growth conditions on the fraction of mitotic cells we have employed an antibody specific for the phosphorylated form of histone H3 for the identification of mitotic cells using flow cytometry. The phosphorylation of histone H3 closely correlates with the chromosomal condensation that accompanies the onset of mitosis, and, therefore, it represents a convenient marker for dividing cells. We have optimized the protocol for the staining of mitotic cells for both Chinese hamster ovary and hybridoma cell cultures. Fluorescence micrographs taken of stained cells show that cells in the various stages of mitosis can be detected based on the morphological characteristics of the chromosomes. The variation in the mitotic cell fraction has been determined throughout the batch growth phases of cultures under different growth conditions. The dynamics of the mitotic index show that balanced growth was never truly reached and that the growth rate is in fact quite variable for these cultures since large variations in the mitotic index are observed. In addition, a large increase in the fraction of mitotic cells just prior to the exponential growth phase for all cultures indicates that they are partially synchronized at the exit from the lag phase. According to a two-staged, age structured population balance model, the mitotic index is directly proportional to the growth rate of a culture. The proportionality constant for this case is shown to be the time required for cells to progress through mitosis. This time is believed to be constant for a particular cell line, as shown by experimental data. Thus, growth rates can be determined solely by measurement of the fraction of cells in mitosis. The mitotic index measurements were then used to calculate the growth in cell number of the cultures, and these simulations accurately reflect observed cell counts. Other simulations also show that changes in cell growth can be predicted before they are reflected in the cell count data. This technique can be used as a sensitive indicator of cell growth and could be useful as a process monitoring technique and for developing better feeding strategies for animal cell cultures.     

Modeling of self-organized avascular tumor growth with a hybrid cellular automaton

Pattern formation in multicellular spheroids is addressed with a hybrid lattice-gas cellular automaton model. Multicellular spheroids serve as experimental model system for the study of avascular tumor growth. Typically, multicellular spheroids consist of a necrotic core surrounded by rings of quiescent and proliferating tumor cells, respectively. Furthermore, after an initial exponential growth phase further spheroid growth is significantly slowed down even if further nutrient is supplied. The cellular automaton model explicitly takes into account mitosis, apoptosis and necrosis as well as nutrient consumption and a diffusible signal that is emitted by cells becoming necrotic. All cells follow identical interaction rules. The necrotic signal induces a chemotactic migration of tumor cells towards maximal signal concentrations. Starting from a small number of tumor cells automaton simulations exhibit the self-organized formation of a layered structure consisting of a necrotic core, a ring of quiescent tumor cells and a thin outer ring of proliferating tumor cells.     

A mitosis block links active cell cycle with human epidermal differentiation and results in endoreplication

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation.     

TPPII promotes genetic instability by allowing the escape from apoptosis of cells with activated mitotic checkpoints

Overexpression of TPPII correlates with accelerated growth and the appearance of centrosome and chromosome aberrations, suggesting that the activity of this enzyme may be critical for the induction and/or maintenance of genetic instability in malignant cells. We now find that the length of mitosis and of the entire cell cycle is significantly reduced in TPPII overexpressing HEK293 cells compared to untransfected and control transfected cells. Functional TPPII knockdown by shRNA interference caused a significant slowdown in cell growth and the accumulation of cells that delayed or failed to complete mitosis. TPPII overexpressing cells evade mitotic arrest induced by spindle poisons and display high levels of polyploidy despite the constitutively high expression of major components of the spindle checkpoint. TPPII overexpression correlated with upregulation of IAPs and with resistance to mitochondria dependent apoptosis induced by p53 stabilization. Thus, TPPII appears to promote malignant cell growth by allowing exit from mitosis and the survival of cells with severe mitotic spindle damage.     

Action of the head activator as a growth hormone in hydra.

At the cellular level the head activator from hydra acts as a mitogen or growth hormone. It shortens cell cycle times by stimulating cells arrested in the G2 period to go through mitosis. This is true for continuously proliferating cell types like epithelial cells, gland cells, and interstitial cells, and for differentiating interstitial cells including those undergoing a last mitosis before differentiating into nerves or nematocytes.     

The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.     

The elm1 kinase functions in a mitotic signaling network in budding yeast

In budding yeast, the Clb2 mitotic cyclin initiates a signaling network that negatively regulates polar bud growth during mitosis. This signaling network appears to require the function of a Clb2-binding protein called Nap1, the Cdc42 GTPase, and two protein kinases called Gin4 and Cla4. In this study, we demonstrate that the Elm1 kinase also plays a role in the control of bud growth during mitosis. Cells carrying a deletion of the ELM1 gene undergo a prolonged mitotic delay, fail to negatively regulate polar bud growth during mitosis, and show defects in septin organization. In addition, Elm1 is required in vivo for the proper regulation of both the Cla4 and Gin4 kinases and interacts genetically with Cla4, Gin4, and the mitotic cyclins. Previous studies have suggested that Elm1 may function to negatively regulate the Swe1 kinase. To further understand the functional relationship between Elm1 and Swe1, we have characterized the phenotype of Deltaelm1 Deltaswe1 cells. We found that Deltaelm1 Deltaswe1 cells are inviable at 37 degrees C and that a large proportion of Deltaelm1 Deltaswe1 cells grown at 30 degrees C contain multiple nuclei, suggesting severe defects in cytokinesis. In addition, we found that Elm1 is required for the normal hyperphosphorylation of Swe1 during mitosis. We propose a model in which the Elm1 kinase functions in a mitotic signaling network that controls events required for normal bud growth and cytokinesis, while the Swe1 kinase functions in a checkpoint pathway that delays nuclear division in response to defects in these events.     

A quantitative theory of liver regeneration in the rat. II: matching an improved mitotic inhibitor model to the data

A model of liver regeneration is put forward in which the rate of liver growth is controlled both by a liver-produced mitotic inhibitor and by the availability of parenchymal cells to enter the mitotic cycle. The model can be expressed as a pair of coupled differential equations, the first describing the dependance of inhibitor concentration on liver size and inhibitor decay and the second specifying the dependance of liver growth on inhibitor concentration and entry of cells into the mitotic cycle. The model is tested by comparing its solutions to the published data on mitotic indices following partial hepatectomy. For such a comparison, it is necessary to specify the cell-cycle time and the inhibitor dose-response function and half-life. If a negative exponential dose-response function, an inhibitor half-life of 11·4 h, and a cycle time of 18·25 h are postulated, the solutions match the data of Fabrikant (1968) who found that there were two waves of mitosis with a period of quiescence between them. The data of Grisham (1962), characterized by a single peak of mitosis, is matched by the theory using similar inhibitor properties but a shorter cell-cycle time (13·25 h); this causes the two peaks to overlap. In both cases, a better fit is obtained if the second cell cycle is longer than the first by 2–3 h. This suggests that cells enter a G₀ period after mitosis. A mechanism for littoral cell division, which occurs some 24 h after parenchymal cell division, is put forward in which the former cells depend on the enlargement of the latter for the stimulus to divide.     

Convergence of cell cycle regulation and growth factor signals on GRASP65

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.     

Cdc5 Interacts with the Wee1 Kinase in Budding Yeast

Development of a multicellular organism requires that mitosis and morphogenesis be coordinated. These processes must also be synchronized during the growth of unicellular organisms. In the yeast Saccharomyces cerevisiae, mitosis is dependent on the prior growth of a daughter cell in the form of a bud. Overexpression of wild-type Polo-like kinase Cdc5 or a catalytically inactive form resulted in the formation of multinucleate cells in budding yeast. Immunofluorescence analysis of these multinulceate cells showed that mitosis and bud formation were no longer linked. Others have shown that Swe1 is required for coupling mitosis to bud formation during a perturbed cell cycle. When the normal pathway of bud formation is perturbed, Swe1 functions to delay mitosis through negative regulation of Clb/Cdk. In cells lacking Swe1, multinucleate cells are formed in response to delays in bud formation. Affinity purification, two-hybrid analysis, and mutant characterization results suggested that Cdc5 and Swe1 interact. From these results, we conclude that multinucleate formation in response to Cdc5 overexpression is linked to titration of Swe1 function. These results also suggest that Cdc5 may be a negative regulator of Swe1.     

hyp loci control cell pattern formation in the vegetative mycelium of Aspergillus nidulans.

Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 microm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.     

Epidermal growth factor stimulates DNA synthesis while inhibiting cell multiplication of A-431 carcinoma cells

Epidermal growth factor (EGF) has been shown to inhibit the multiplication of the human epidermoid carcinoma cell line A-431. In the present report it is shown that, despite growth inhibition, EGF caused a marked synthesis of DNA and nonhistone proteins, without progression into mitosis. This event was associated with a retraction of the monolayer into colonies of cells. This suggests that the cell cycle of A-431 cells is controlled by two surface membrane signals: one generated by EGF stimulating the synthetic events of the G1 and S phases; a second signal, leading to progression into mitosis appears either not to be generated or to be inhibited by EGF.     

Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole

The dynamic balance between polymerization and depolymerization of microtubules is critical for cells to enter and exit mitosis, and drugs that disrupt this balance, such as taxol, colchicine, and nocodazole, arrest the cell cycle in mitosis. Although the Raf/MEK/MAPK pathway can be activated by these drugs, its role in mitosis has not been addressed. Here, we characterize activation of Raf/MEK/MAPK by nocodazole when mitosis is induced. We find that at early time points (up to 3 h) in nocodazole induction, Raf/MEK/MAPK is activated, and inhibition of MAPK activation by a MEK inhibitor, PD98059 or U0126, reduces the number of cells entering mitosis by creating a block at G(2). At later time points and in mitosis, activation of MEK/MAPK is severely inhibited, even though Raf-1 activity remains high and can be further increased by growth factor. This inhibition is reversed when cells are released from metaphase and enter G(0)/G(1) phase. In addition, we find that binding of Raf-1 to 14-3-3 is progressively induced by nocodazole, reaching a maximum in mitosis, and that this binding is necessary to maintain mitotic Raf-1 activity. Our present study indicates that activation of the Raf/MEK/MAPK pathway is necessary for the G(2)/M progression.     

Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding

In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed “mitotic nanotubes,” were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.     

Mechanokinetic model of cell membrane: theoretical analysis of plasmalemma homeostasis, growth and division

A theoretical model dealing with endocytosis, exocytosis and caveolae invagination, describing plasmalemma homeostasis during cell growth and division, is proposed. It considers transmembrane pressure, membrane tension and mechanosensitivity of membrane processes. Membrane hydraulic conductivity and the flux of transmembrane nonvesicular transport are taken into account. The developed mathematical analysis operates with a formulated set of constitutive equations describing the mechanical state and kinetics of changes in an open dynamic membrane system. The standard version of a model with adjusted parameters was implemented, and predictions including a discussion on the effect of possible parameter modifications were presented. Computer simulations indicate big changes in the magnitude of membrane tension and elasticity, and in the number of membrane buddings in young cells and during mitosis. They also show the extent of cell growth inhibition resulting from a decrease in transmembrane transport or an increase in the exerted difference in osmotic pressure. Moreover, the simulations reveal that exocytosis regulated during mitosis may not be as important for cell growth, as sometimes presumed. Finally, practical application and possible extension of the model are discussed.     

Serum starvation induces abnormal spindle location,RhoA delocalization,and extension of intercellular bridge with the midbody

Serum starvation induces binucleation in HeLa cells, but the effects of serum starvation on mitosis and the significance of binucleation remain unknown. We investigated the effect of serum starvation on mitosis and analyzed the growth of binucleated cells. The frequency of binucleation caused by cytokinesis failure in DMEM without FBS (0% medium) was higher than that in DMEM with FBS (10% medium). In 0% medium, the metaphase spindle location was off-center, and RhoA localization significantly lacked symmetry. The frequency of the extension of intercellular bridge with the midbody in 0% medium was significantly higher than that in 10% medium. Moreover, all mononucleated mitotic cells caused bipolar mitosis and produced only mononucleated daughter cells, but binucleated cells produced various nucleated cells by multipolar mitosis in 0% medium. These results suggest that serum starvation may have various effects on mitosis, and binucleated cells may be related to formation of aneuploidy.     

Analytic formulas for discrete stochastic models of cell populations with both differentiation and de-differentiation

Cell differentiation often appears to be a stochastic process particularly in the hemopoietic system. One of the earliest stochastic models for the growth of stem cell populations was proposed by Till et al. in 1964. In this model there are just two cell types: stem cells and specialized cells. At each time step there is a fixed probability that a stem cell differentiates into a specialized cell and a fixed probability that it undergoes mitosis to produce two stem cells. Even though this model is conceptually simple the myriad of possible outcomes has made it difficult to analyse. We present original closed-form expressions for the probability functions and a fast algorithm for computing them. Renewed interest in stem cells has raised questions about the effect de-differentiation has on stem cell populations. We have extended the stochastic model to include de-differentiation and show that even a small amount of de-differentiation can have a large effect on stem cell population growth.