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C L Mummery

Publications and source records attributed to C L Mummery.

86 records · Page 5Linked to original sources
PubMed1984

The cell cycle, cell death, and cell morphology during retinoic acid-induced differentiation of embryonal carcinoma cells.

Time-lapse films were made of PC13 embryonal carcinoma cells, synchronized by mitotic shake off, in the absence and presence of retinoic acid. Using a method based on the transition probability model, cell cycle parameters were determined during the first five generations following synchronization. In undifferentiated cells, cell cycle parameters remained identical for the first four generations, the generation time being 11-12 hr. In differentiating cells, with retinoic acid added at the beginning of the first cycle, the first two generations were the same as controls. The duration of the third generation, however, was increased to 15.7 hr while the fourth and fifth generation were approximately 20 hr, the same as in exponentially growing, fully differentiated cells. The increase in generation time of dividing cells was principally due to an increase in the length of S phase. Cell death induced by retinoic acid also occurred principally in the third and subsequent generations. Cell population growth was then significantly less than that expected from the generation time derived from cycle analysis of dividing cells. Cells lysed frequently as sister pairs suggesting susceptibility to retinoic acid toxicity determined in a generation prior to death. Morphological differentiation, as estimated by the area of substrate occupied by cells, was shown to begin in the second cell cycle after retinoic acid addition. These results demonstrate that as in the early mammalian embryo, differentiation of embryonal carcinoma cells to an endoderm-like cell is also accompanied by a decrease in growth rate but that this is preceded by acquisition of the morphology characteristic of the differentiated progeny.

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PubMed1983

Loss of EGF binding and cation transport response during differentiation of mouse neuroblastoma cells.

Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown tht exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+-K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.

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PubMed1983

Membrane regulation of the Na+,K+-ATPase during the neuroblastoma cell cycle: correlation with protein lateral mobility.

The pumping activity of the plasma membrane-bound Na+,K+-ATPase shows considerable variation during the cell cycle of mouse neuroblastoma Neuro-2A cells. Addition of external ATP at millimolar concentrations, which selectively enhances the plasma membrane permeability of Neuro-2A cells for sodium ions, stimulates the Na+,K+-ATPase pumping activity at all phases of the cell cycle from a factor of 1.05 in mitosis up to 2.2 in G1 phase. Determination of the number of Na+,K+-ATPase copies per cell by direct 3H-ouabain binding studies in the presence of external ATP shows a gradual increase in the number of pump sites on passing from mitosis to the late S/G2-phase by approximately a factor of 2. From these data the pumping activity per copy of Na+,K+-ATPase, optimally stimulated with respect to its various substrate ions, has been determined during the various phases of the cell cycle. This optimally stimulated pumping activity per enzyme copy, which is a reflection of the physicochemical state of the plasma membrane, is high in mitosis, almost twofold lower in early G1 phase, and increases gradually again during the other phases of the cell cycle. This shows that the observed regulation of Na+,K+-ATPase activity during the cell cycle is caused by a combination of three independent factors--namely variation in intracellular substrate availability (Na+), changes in number of enzyme copies per cell, and modulation of the plasma membrane environment of the protein molecules. The modulation of the optimal pumping activity per enzyme copy shows a good correlation (rho = 0.96) with the known modulation of protein lateral mobility during the cell cycle, such that a high protein lateral mobility correlates with a low enzyme activity. It is concluded that changes in plasma membrane properties take place during the Neuro-2A cell cycle that result in changes in the rate of protein lateral diffusion and Na+,K+-ATPase activity in directly correlated way.

Animals↗
PubMed1983

Screening for cytotoxicity in neuroblastoma cells. I. Dependence of growth inhibition on the presence of serum.

The growth-inhibitory effects of a variety of potentially toxic compounds on neuroblastoma cells in defined, serum-free medium were compared with those in serum-containing medium. For 13 of 21 compounds tested, concentrations between 2 and 10(5) times higher were required for 50% inhibition of growth in serum-containing medium. The ranking of substances for their potency in inhibiting growth was thereby different in the two different culture conditions. The presence of bovine serum albumin (BSA) in the medium had similar effects as serum on the dose-response curves.

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PubMed1983

Screening for cytotoxicity in neuroblastoma cells. II. Growth inhibition: cell death or impaired cell cycle progression?

Time-lapse films were made of neuroblastoma cells in chemically defined, serum-free medium in the presence of a variety of potentially toxic compounds. Using a simple method of analysis, based on the transition probability model and the increase in the number of cells per frame with time, growth inhibition in cell cultures could be specifically attributed to cell death or delayed progression through the cell cycle. For 4 of 8 compounds growth inhibition was the result of almost all cells showing an impaired rate of cell cycle progression while for 4 compounds there were equal or greater contributions from cell death and/or detachment.

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PubMed1981

Characterization of 42K+ and 86Rb+ transport and electrical membrane properties in exponentially growing neuroblastoma cells.

For measuring K+ efflux from exponentially growing neuroblastoma cells (clone Neuro-2A), two methods were used, a sampling method and a washing method. Both methods indicated that K+ efflux kinetics were as from a two-compartment system, but the two compartments could only be resolved completely using the washing method. A fast compartment, containing 143 +/- 16 nmol K+/10(6) cells, was found to be associated to the cell surface, and a slow compartment, containing 151 +/- 7 nmol K+/10(6) cells, was found to represent the intracellular K+. The rate constant of the slow compartment was 0.0164 +/-0.0005 min-1, and the K+ efflux rate was 2.46 +/- 0.14 nmol K+/10(6) cells per min. Using the appropriate conditions to measure K+ influx, the kinetics of influx were equal to the kinetics of efflux, indicating steady-state conditions. In addition a comparison was made between 42K+ and 86Rb+ as radioactive tracers for K+ flux. It was found that 86Rb+ was specifically bound on both the inside and the outside of the cells, and for this reason was not a suitable tracer for studying K+ flux kinetics in neuro-2A cells. A membrane potential of -42.9 +/- 1.3 mV and intracellular K+ activity of 108.1 +/- 3.0 mM were measured using conventional and ion-selective microelectrodes. A correlation was made between the K+ flux and electrophysiological data, using the equations of electrodiffusion theory. Thus, the permeabilities of K+ and Na+ were calculated as (3.9 +/- 0.4) . 10(-8) cm/s and (0.6 and 0.1) . 10(-8) cm/s respectively, together with K+ conductance of (2.8 +/- 0.3) . 10(-6) omega-1/cm2.

Animals↗
PubMed1981

Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells.

Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.

Animals↗
PubMed1981

Cation transport and growth regulation in neuroblastoma cells. Modulations of K+ transport and electrical membrane properties during the cell cycle.

Cation transport and membrane potential were studied during the cell cycle of neuroblastoma cells (clone Neuro-2A) to investigate the role of these parameters in growth regulation. The cells were synchronized by selective detachment of mitotic cells. The membrane potential and intracellular K+ activity were measured with conventional and K+-selective microelectrodes respectively. Both the membrane potential and K+ activity were high in mitosis, decreased to half maximal in G1 phase, and rose again during S phase. K+ efflux across the plasma membrane was studied with 42K+ as a radioactive tracer using a washing method for cells grown in monolayer and a continuous efflux method for mitotic cells in suspension. The intracellular K+ content and unidirectional K+ efflux rate obtained from these measurements showed modulations during the cell cycle similar to those of the membrane potential. Using equations of electrodiffusion theory the membrane permeabilities to K+ and Na+ were calculated. These permeabilities were high in mitosis, decreased rapidly in G1 phase and increased during S phase, followed by a transient decrease in G2 phase. A rapid increase was observed between G2 phase and the next mitosis. A similar pattern was obtained for the K+ conductance. K+ resistance changes during the cell cycle were similar to changes in the specific membrane resistance, measured by microelectrodes, except for the early cell cycle phases (mitosis and G1). These studies clearly demonstrate large modulations of the passive membrane permeability properties during the cell cycle. These modulations can be correlated with physicochemical membrane variations during the cell cycle, such as membrane fluidity and lateral mobility of lipids.

Animals↗
PubMed1981

Rapid ionic events and the initiation of growth in serum-stimulated neuroblastoma cells.

Rapid effects of serum stimulation on electrical and ionic membrane properties and their relationship to the initiation of DNA synthesis and cell division have been investigated in mouse N1E-115 neuroblastoma cells. Addition of 10% fetal calf serum to serum-deprived N1E-115 cells results in the initiation of DNA synthesis after a lag of approximately 10 hr. The earliest events following serum addition include: transient membrane potential and resistance changes, detectable within seconds and lasting 5--10 min; a persistent increase in the initial rate of 22Na+ influx, the major part of which is not of electrodiffusional origin, and which is potentiated by weak acid anions; and an external Na+-dependent increase in the rate of the Na+, K+ pump. In the absence of serum the stimulation of the Na+, K+ pump can be mimicked by increasing net Na+ influx with monensin or neurotoxins. Growth-depleted serum fails to induce any of the electrical and ionic events. The diuretic amiloride (0.4 mM) inhibits serum-induced Na+ influx, Na+, K+ pump stimulation and DNA synthesis, but does not affect the electrical response or the basal influx rates. The results suggest that serum growth factors act, at least in part, by stimulating an electroneutral, amiloride-sensitive Na+/H+ exchange mechanism. The enhanced Na+ influx then results in the observed stimulation of the Na+, K+ pump, while the simultaneous efflux of protons may raise the intracellular pH.

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PubMedDate not supplied

Monovalent cation transport during the cell cycle (review).

This paper reviews the properties and regulation of cation transport during the cell cycle. Extensive modulations occur in both electro-diffusional and non-electro-diffusional cation transport, most prominently during mitosis and G1 phase. These modulations can be related to compositional, structural and dynamic membrane properties. The implications of modulated cation transport for progression through the cell cycle are discussed.

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