DNA damage-dependent cell cycle checkpoints and genomic stability

In response to genotoxic stress, which can be caused by environmental or endogenous genotoxic insults such as ionizing or ultraviolet radiation, various chemicals and reactive cellular metabolites, cell cycle checkpoints which slow down or arrest cell cycle progression can be activated, allowing the cell to repair or prevent the transmission of damaged or incompletely replicated chromosomes. Checkpoint machineries can also initiate pathways leading to apoptosis and the removal of a damaged cell from a tissue. The balance between cell cycle arrest and damage repair on one hand and the initiation of cell death, on the other hand, could determine if cellular or DNA damage is compatible with cell survival or requires cell elimination by apoptosis. Defects in these processes may lead to hypersensitivity to cellular stress, and susceptibility to DNA damage, genomic defects, and resistance to apoptosis, which characterize cancer cells. In this article, we have noted recent studies of DNA damage-dependent cell cycle checkpoints, which may be significant in preventing genomic instability.     

Establishment and Maintenance of Cell Polarity During Leukocyte Chemotaxis

The term polarity refers to the differential distribution of the macromolecular elements of a cell, resulting in its asymmetry in function, shape and/or content. Polarity is a fundamental property of all metazoan cells in at least some stages, and is pivotal to processes such as epithelial differentiation (apical/basal polarity), coordinated cell activity within the plane of a tissue (planar cell polarity), asymmetric cell division, and cell migration. In the last case, an apparently symmetric cell responds to directional cues provided by chemoattractants, creating a polarity axis that runs from the cell anterior, or leading edge, in which actin polymerization takes place, to the cell posterior (termed uropod in leukocytes), in which acto-myosin contraction occurs. Here we will review some of the molecular mechansisms through which chemoattractants break cell symmetry to trigger directed migration, focusing on cells of the immune system. We briefly highlight some common or apparently contradictory pathways reported as important for polarity in other cells, as this suggests conserved or cell type-specific mechanisms in eukaryotic cell chemotaxis.     

Early development of the granule cell in the cerebellum of the chick embryo

The sequence of differentiation of the cerebellar granule cell in chick embryos from the eighth to the 15th days of incubation has been studied in Golgi-stained celloidin sections. In the germinal-cell phase, the presumptive granule cell sends out one or two horizontal processes which may originate either in the body of the cell or in the extension which attaches it to the pial surface. Thus the germinal cell may be converted into either a monopolar or a bipolar presumptive granular cell. Bipolar cells may have two processes of the same length (symmetrical cells) or of unequal length (asymmetrical cells). In the symmetrical as well as asymmetrical bipolar cells the leading process is formed, by means of which the perikaryon emigrates until it situates itself definitely in the internal granular layer. Thus, symmetrical and asymmetrical bipolar cells give rise to a granule cell with parallel fibers of equal or different lengths. The monopolar element may originate a second process or may remain in the monopolar phase until it reaches the internal granular layer. Once there, it completes the formation of the parallel fibers.     

Establishment and Maintenance of Cell Polarity During Leukocyte Chemotaxis

The term polarity refers to the differential distribution of the macromolecular elements of a cell, resulting in its asymmetry in function, shape and/or content. Polarity is a fundamental property of all metazoan cells in at least some stages, and is pivotal to processes such as epithelial differentiation (apical/basal polarity), coordinated cell activity within the plane of a tissue (planar cell polarity), asymmetric cell division, and cell migration. In the last case, an apparently symmetric cell responds to directional cues provided by chemoattractants, creating a polarity axis that runs from the cell anterior, or leading edge, in which actin polymerization takes place, to the cell posterior (termed uropod in leukocytes), in which acto-myosin contraction occurs. Here we will review some of the molecular mechanisms through which chemoattractants break cell symmetry to trigger directed migration, focusing on cells of the immune system. We briefly highlight some common or apparently contradictory pathways reported as important for polarity in other cells, as this suggests conserved or cell type-specific mechanisms in eukaryotic cell chemotaxis.Key Words: chemotaxis, polarization, lipid rafts, signaling, cytoskeleton     

On the constancy of cell division rate in the root meristem

This review examines under what circumstances the rate of cell division among cells of the root meristem is known to vary. First, methods are compared that have been used to quantify cell division rate. These can be grouped as being either cytological, in which the rate of accumulation of cells in a particular phase of the cell cycle is determined based on some form of cytological labeling, or kinematic, in which the rate of cell accumulation is determined from the net movement of cells. Then, evidence is reviewed as to whether cell division rates vary between different tissues or cell types, between different positions in the root, or finally between different environments. The evidence is consistent with cells dividing at a constant rate, and well documented examples where cell division rate changes substantially are rare. The constancy of cell division rate contrasts with the number of dividing cells, which varies extensively, and implies that a major point for cell cycle control is governing the exit from the proliferative state at the basal boundary of the meristem.     

Modulating L-type calcium current affects discontinuous cardiac action potential conduction.

We have used pairs of cardiac cells (i.e., one real guinea pig ventricular cell and a real-time simulation of a numerical model of a guinea pig ventricular cell) to evaluate the effects on action potential conduction of a variable coupling conductance in combination with agents that either increase or decrease the magnitude of the L-type calcium current. For the cell pairs studied, we applied a direct repetitive stimulation to the real cell, making it the "leader" cell of the cell pair. We have demonstrated that significant delays in action potential conduction for a cell pair can occur either with a decreased value of coupling conductance or with an asymmetry in size such that the follower cell is larger than the leader cell. In both conditions we have shown that isoproterenol, applied to the real cell at very low concentrations, can reversibly decrease the critical coupling conductance (below which action potential conduction fails) for a cell pair with fixed cell sizes, or, for a fixed value of coupling conductance, increase the maximum allowable asymmetry in cell size for successful conduction. For either of these effects, we were able to show that treatment of the real cell with BayK 8644, which more specifically increases the magnitude of the L-type calcium current, was able to mimic the actions of isoproterenol. Treatment of the leader cell of the cell pair (the real cell) with nifedipine, which selectively lowers the magnitude of the L-type calcium current, had effects opposite those of isoproterenol or BayK 8644. The actions of nifedipine, isoproterenol, and BayK 8644 are all limited to conditions in which the conduction delay is on the order of 5 ms or more, whether this delay is caused by limited coupling conductance or by asymmetry in size of the cells. This limitation is consistent with the time course of the L-type calcium current and suggests that the effects of calcium channel blockers or beta-adrenergic blocking drugs, in addition to being selective for regions of the heart that depend on the L-type calcium current for the upstroke of the action potential, would also be somewhat selective for regions of the heart that have discontinuous conduction, either normally or because of some pathological condition.     

The effects of interleukin 1 on human B cell activation and proliferation

The precise role of B cell surface immunoglobulin (slg) in the activation of B cells is unclear at present. In particular, it is uncertain whether ligands interacting with the B cell slg suffice to induce proliferation, or simply induce a state of activation in which the B cell becomes responsive to growth factors made by accessory cells. We have examined the effects of two ligands, Staphylococcus aureus Cowan strain I (SAC) and antihuman mu chain (anti-mu), which interact with B cell slg on highly purified human peripheral blood and tonsillar B cells cultured at low cell concentrations. The effects on B cell proliferation of these ligands alone or in combination with highly purified interleukin 1 (IL 1) or a supernatant of a human T-T hybridoma containing a B cell growth factor (BCGF) were studied. SAC with its high cell wall content of protein A triggered maximal B cell proliferation which was not increased further by IL 1 or BCGF. High concentrations of soluble F(ab')2 fragments of goat anti-mu chain also induced significant B cell proliferation. Lower concentrations of anti-mu resulted in little or no B cell proliferation but activated the B cell to a state of responsiveness to both IL 1 and BCGF. IL 1 by itself had no effect on the proliferation of unstimulated B cells or on the proliferation of in vivo-activated B cells which responded to BCGF in vitro, but demonstrated clear synergy with low concentrations of anti-mu antibody. BCGF alone augmented the proliferation of unstimulated B cells, presumably by acting on B cells which had undergone some degree of activation in vivo. In addition, it showed marked synergy with anti-mu antibody, which resulted in proliferation similar in magnitude to that induced by SAC. This synergy was far greater than that seen between anti-mu antibody and IL 1, and the resulting proliferative response was only slightly increased by the presence of IL 1. We conclude that the importance of accessory cell factors for the initial rounds of B cell proliferation depends on the strength of the initial slg-mediated activation signal. When this is strong, the response is maximal and independent of accessory cells or accessory cell factors. When it is suboptimal, a moderate synergy is seen with IL 1 and a dramatic synergy with BCGF.     

Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage

In neuroscience, combining patch-clamping with protein identification in the same cell is becoming increasingly important to define which subtype or developmental stage of a neuron or glial cell is being recorded from, and to attribute measured membrane currents to expressed ion channels or receptors. Here, we describe a protocol to achieve this when studying cells in acute brain slices, which antibodies penetrate poorly into and for which detergent permeabilization cannot be used when using antibodies that recognize lipid components such as O4 sulfatide. The method avoids the need for resectioning of the electrophysiologically recorded slices. It employs filling of the cell with a fluorescent dye during whole-cell recording, to allow subsequent localization of the cell, followed by fixation and free-floating section labeling with up to three antibodies, which may recognize membrane, nuclear or cytosolic proteins. With practice, approximately 80% of patch-clamped cells can be retrieved and have their proteins identified in this way. The entire protocol can be completed in 3-4 d.     

Properties of cell lines derived from altered-cell foci in baby mouse skin cultures

Long-term cell lines were readily developed from a proportion of either baby mouse skin (BMS) cultures passing through colchicine-induced crisis or altered-cell foci selected from BMS cultures exposed to light and/or oxygen followed by colchicine. The developing cell lines behaved as though they passed through a continuing, profound genetic reshuffling process, which was usually lethal but which in some cases eventually yielded a gene set that favored long-term survival. Some cell lines have passed 120 cell doublings, and none has shown a second crisis or signs of senescence. As soon after isolation as measurement was possible (19–50 days) the cell lines were predominantly or entirely tetraploid or subtetraploid. Although BMS cells and all of the cell lines were density inhibited, the BMS, C14, C21 and C23 cells overgrew (formed colonies on) monolayers of the same cells in most combinations. The cell lines retained a variety of neoplastic morphological characters, although their morphology was more normal than in the original focus. No cell line, however, showed anchorage-independent growth or formed tumors in syngeneic hosts. The cell lines may all, therefore, be regarded as preneoplastic.     

Myogenic stem cell commitment probability remains constant as a function of organismal and mitotic age

Chicken myogenic stem cells can undergo symmetric and asymmetric cell divisions. Symmetric divisions produce two stem cells or two cells committed to terminal muscle differentiation. Asymmetric divisions produce one stem cell and one committed cell. Committed cells undergo four divisions, and their progeny differentiate into postmitotic, biochemically distinct muscle cells, which can be identified immunocytochemically. The control of stem cell commitment was investigated in vitro by means of cell cloning and subcloning experiments, and computer modeling. We found that stem cell commitment is a process which can be modeled as a stochastic event, with a central tendency or probability of 0.2 +/- 0.1. This value is independent of organismal or mitotic age of the stem cells, cell density, or growth in a mitogen-poor environment. Myogenic stem cells stop dividing after approximately 30 divisions in vitro. Since the probability of commitment to terminal differentiation remains below 0.5, clonal senescence and terminal differentiation are separate processes in this system.     

A mathematical model of cellular apoptosis and senescence through the dynamics of telomere loss

The shortening of telomeric repeats as a cell replicates has long been implicated as a determinant of cell viability. However, recent studies have indicated that it is not telomere length, but rather whether telomeres have bound a telomere-related protein, which in mammals is TTAGGG repeat binding factor-2 (TRF2), that determines whether a cell undergoes apoptosis (programmed cell death), enters senescence (a quiescent, non-replicative state), or continues to proliferate. When bound to a telomere, TRF2 allows a cell to recognize the telomere as the point where a chromosome ends rather than a break in DNA. When telomeres are not bound by TRF2, the cell can either immediately trigger senescence or apoptosis via the DNA damage response pathway, or indirectly trigger it by attempting to repair the chromosome, which results in chromosomal end joining. We model the ability of telomeres to bind TRF2 as a function of telomere length and apply the resulting binding probability to a model of cellular replication that assumes a homogeneous cell population. The model fits data from cultured human fibroblasts and human embryonic kidney cells for two free parameters well. We extract values for the percent of telomere loss at which cell proliferation ceases. We show, in agreement with previous experiments, that overexpression of TRF2 allows a cell to delay the senescence setpoint. We explore the effect of oxidative stress, which increases the rate of telomere loss, on cell viability and show that cells in the presence of oxidative stress have reduced lifespans. We also show that the addition of telomerase, an enzyme that maintains telomere length, is sufficient to result in cell immortality. We conclude that the increasing inability of TRF2 to bind telomeres as they shorten is a quantitatively reasonable model for a cause of either cellular apoptosis or senescence.     

Trypsin sensitivity of mammalian cells grown in a serum-free medium

Summary Two mammalian cell lines which multiply in vitro in culture medium devoid of serum were investigated for sensitivity to twice crystallized trypsin. The minimum trypsin concentration which showed a concomitant increase in cell numbers and detachment from the surface of the culture vessel in one cell line was 0.01 μg per ml or approximately 10⁻⁸ μg per cell. These effects could be neutralized by normal human or calf serum at a dilution of 1∶500. The second cell line, which normally grows in suspension, was unaffected by these concentrations of trypsin.     

Characterization of the cell wall and cell wall proteins of Chromatium vinosum.

Highly purified cell walls of Chromatium vinosum were isolated by differential centrifugation, with or without Triton X-100 extraction. The isolated material had a protein composition similar to that of cell walls obtained by sucrose density gradient centrifugation. Twenty-two proteins were reproducibly detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 42-kilodalton protein was shown to account for 65% of the total cell wall protein. The majority of cell wall proteins were solubilized in sodium dodecyl sulfate at room temperature; however, they existed as high-molecular-weight complexes unless heated to 45 degrees C or above. The cell wall contained one heat-modifiable protein which migrated with an apparent molecular weight of 37,400 when solubilized at 70 degrees C or below, but which migrated with an apparent molecular weight of 52,500 if solubilized at 100 degrees C. The electrophoretic mobility of three proteins was modified by 2-mercaptoethanol. The majority of C. vinosum cell wall proteins had isoelectric points between pH 4.5 and 5.5, and the 42-kilodalton protein focused at pH 4.9. No proteins were detected which were analogous to the lipoprotein or peptidoglycan-associated proteins of the Enterobacteriaceae. Nearest-neighbor analysis with a reducible, cross-linking reagent indicated that three proteins, including the 42-kilodalton protein, associated with themselves. Most of the cell wall proteins were partially accessible to proteases in both intact cells and isolated cell walls. Protease treatment of the whole cell or isolated cell wall digested approximately an 11,000-molecular-weight portion of the 42-kilodalton protein.     

Some comments regarding chromosome pulverization (premature chromosome condensation or PCC, prophasing).

Premature chromosome pulverization (PCC) or prophasing is a much misunderstood cytological entity. It must be separated from chromosome damage caused by a number of chemical, physical and biological agents. Prophasing is observed in fused cells in which one of the constituent cells must be in metaphase and another in interphase. The morphology of the "pulverized" interphase nucleus will depend on the phase of the cell cycle in which the interphase cell was in when exposed to a substance present in the cytoplasm of the metaphase cell leading to "prophasing". Prophasing is a normal cellular phenomenon occurring prematurely or under abnormal conditions (fusion of cells) and its demonstration in human cells or tumors may be indicative of the presence of a virus (or its products) which leads to cell fusion, but does not play a role in prophasing.     

Automated analysis and survival selection of anchorage-dependent cells under normal growth conditions

An instrument is described that can automatically analyze and select for a subpopulation of anchorage-dependent cells in tissue culture. Cells that label with fluorescently tagged antibodies or demonstrate structural variations are saved from exposure to a destructive high-intensity argon laser beam. The surviving population may then be cloned. The cell selection may occur in a tissue culture plate or in a microflow incubator which is designed to maintain a constant flow of media at 37 degrees C across cells growing on a glass coverslip. This incubator sits on an inverted microscope which focuses the laser beam to a diameter as small as 1 micron. A high-speed computer-controlled two-dimensional stage moves the cells past the beam for analysis, the results of which determine the fate of each cell: whether it is to be destroyed by radiant energy or selected for survival and subsequent proliferation. Another selection strategy performed by the instrument involves growing the cells on a thin, blackened polyester film which can be cut by the argon laser beam. Cells selected for cloning are then circumscribed. The heat of cutting welds the circumscribed film to a plastic coverslip surface or tissue culture chamber bottom. Nonselected cells may be removed by pulling the unattached polyester sheet from the attachment surface. The selected cells remain on polyester film disks welded to the plastic. Selections may be done automatically under computer control or manually by operator direction of stage movements. This instrument extends the art of automated cell selection and analysis to normal cell lines that must maintain cell-substratum contact (anchorage dependence) for differentiated cell function, e.g., neurons, fibroblasts, or kidney cells.     

A viscoelastic model of blood capillary extension and regression: derivation, analysis, and simulation

This work studies a fundamental problem in blood capillary growth: how the cell proliferation or death induces the stress response and the capillary extension or regression. We develop a one-dimensional viscoelastic model of blood capillary extension/regression under nonlinear friction with surroundings, analyze its solution properties, and simulate various growth patterns in angiogenesis. The mathematical model treats the cell density as the growth pressure eliciting a viscoelastic response from the cells, which again induces extension or regression of the capillary. Nonlinear analysis captures two cases when the biologically meaningful solution exists: (1) the cell density decreases from root to tip, which may occur in vessel regression; (2) the cell density is time-independent and is of small variation along the capillary, which may occur in capillary extension without proliferation. The linear analysis with perturbation in cell density due to proliferation or death predicts the global biological solution exists provided the change in cell density is sufficiently slow in time. Examples with blow-ups are captured by numerical approximations and the global solutions are recovered by slow growth processes, which validate the linear analysis theory. Numerical simulations demonstrate this model can reproduce angiogenesis experiments under several biological conditions including blood vessel extension without proliferation and blood vessel regression.     

Hepatic proliferation inhibitor

Summary Although a long held tenet of biology has been that endogenous inhibitors can modulate cell proliferation, little progress was made in purifying any such inhibitor. This was largely due to the rarity of non-malignant cell cultures in which regulation of cell division was still operative, and to problems in separating cytotoxic and cytostatic effects in the complex biological extracts which were being studied. During the last decade, hepatic proliferation inhibitors of varying degrees of purity have been isolated using regenerating rat liver or hepatoma cell cultures as test systems. In these early studies, a number of inhibitors with differing molecular weights, physicochemical properties and biological responses were purified from liver cytosol and/or serum. Some of them could inhibit DNA synthesis or mitosis and thus were considered to be G1 or G2 inhibitors. However, experiments which could give precise answers about mechanisms of action could not be done until an inhibitor purified to homogeneity was available.Using well-characterized rat liver diploid epithelial cell cultures, which maintain a number of liver properties and which do not possess any transformation markers or malignant properties, we recently purified an hepatic proliferation inhibitor to a homogenous protein. It has a molecular weight of 26 000 daltons and an isoelectric point of 4.65. It specifically inhibits cell division and DNA synthesis in a number of non-malignant rat liver epithelial cell types, and has no effect on transformed liver cells, or hepatoma cells, in culture. Its effect is not mediated through destruction or sequestration of essential nutrients or calcium ions. Nor have preliminary experiments shown the hepatic proliferation inhibitor to interfere with the binding of epidermal growth factor to its receptors. The majority of the cells treated with the inhibitor are blocked in the G1 phase. Further experiments to study its mechanism of action and the inter-relationship, if any, between the cell cycle block induced by serum or nutrient deprivation, and the inhibitor-induced cycle block are in progress.     

The surface potential of and double-layer interaction force between surfaces characterized by multiple ionizable groups.

At the beginning of cleavage, a cell develops the form of a short central cylinder capped at the ends with ellipsoids. The strain pattern produced by this shape, with the application of constricting forces, guides the cell either to divide into two spherical cells or to take the shape of a long, thin cell which may remain a single cell or divide into two thin cells.     

Study of pectin esterase and changes in pectin methylation during normal and abnormal peach ripening

Peach fruit ( Prunus persica cv. Hermosa) were allowed to ripen immediately after harvest or after 30 days of 0°C storage. The fruits lost 75–80% of their firmness after 5 days at 20°C. During ripening after harvest there was a loss of both uronic acid and methyl groups from the cell wall. Cell wall labelling with JIM 7, a monoclonal antibody which recognized pectins with a high degree of methylation, was lower in ripe fruits than in freshly harvested fruits. However, ripe fruit cell walls did not cross-react with JIM 5, which recognizes pectins with low methylation. During storage, de-methylation occurred and in fruit ripened after storage there was little further change in pectin methylation or pectin content in the cell walls. The labelling of stored or stored plus ripened cell walls with JIM 7 was similar, but the cell walls of fruit ripened after storage showed some low cross-reactivity with JIM 5. The in vitro activity and mRNA abundance of pectin esterase (EC 3.1.1.11) was not correlated with the amount of de-esterification as measured chemically or by immuno-labelling in the cell walls. Eighty percent of the fruits which ripened after storage developed a woolly texture. It is suggested that woolliness is due to de-esterification of pectins, not accompanied by depolymerization, which leads to the formation of a gel-like structure in the cell wall.