Jean-Marc Dubois

The influence of cell volume changes on tumour cell proliferation.

Ion channels and cell volume control participate in a wide variety of cellular functions, including cell proliferation. According to the “pump-leak model” or the “double Donnan system”, the cell volume is constant in physiological medium so long as the cell metabolism and the Na-K pump are not inhibited and the passive Na+ permeability is not dramatically increased. At short term, this model has been supported by a large number of experiments made on different cell types. However, at long term, it may be insufficient to describe the volume control because it does not take into account the fact that cells possess a large number of membrane transporters and interconnected volume regulatory mechanisms. In this review, we present recent results indicating that, in physiological conditions, ion channels may have important roles in cell volume control. Furthermore, we emphasize that cell proliferation and volume are phenomenologically correlated. On the basis of the macromolecular crowding theory, the possibility that the cell osmolyte and water content mediates this correlation is discussed.

Cell size-proliferation relationship in rat glioma cells

The homeostasis of the central nervous system is highly controlled by glial cells and is dramatically altered in the case of glioma. In this respect, the complex connection between cell size and division is of particular importance and needs clarifying. In order to investigate this connection, cell number and volume were measured in C6 rat glioma cells under different experimental conditions, including continuous cell culture, Cl− channel blockade, and anisotonicity, and in the presence of an inhibitory conditioned medium collected from cell cultures or in a medium containing a low level of fetal calf serum. The rate of cell proliferation changed with cell volume in a bell‐shaped manner, so that it is optimal within a cell volume window and appears to be controlled by low and high cell size checkpoints. The cell size‐proliferation relationship can be defined by Boltzmann‐like equations, which may reflect the effects of macromolecular crowding on proteins controlling the cell cycle progression. Altogether, these observations indicate that glioma cell proliferation is controlled predominantly but not exclusively by cell size‐dependent mechanisms.

Silencing of hSlo potassium channels in human osteosarcoma cells promotes tumorigenesis

Potassium channels, the most diverse superfamily of ion channels, have recently emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large conductance Ca2+‐activated K+ channels, often referred to as BK channels, are at the crossroads of several tumor‐associated processes such as cell proliferation, survival, secretion and migration. Despite the high BK channel expression in osteosarcoma (OS), their function has not yet been investigated in this malignant bone pathology. Here, using stable RNA interference to reduce the expression of hSlo, the human pore‐forming α‐subunit of the BK channel, in human Cal72 OS cells, we show that BK channels play a functional role in carcinogenesis. Our results reveal for the first time that BK channels exhibit antitumoral properties in OS in vivo and affect the tumor microenvironment through the modulation of both chemokine expression and leukocyte infiltration.

Functional expression of V-ATPases in the plasma membrane of glial cells.

Vacuolar H+ ATPase (V‐ATPase) activity is essential for many cellular processes, including intracellular membrane traffic, protein processing and degradation, and receptor‐mediated endocytosis. Proton transport by V‐ATPases could also play a role during cell transformation, tumorigenesis, and cell metastasis, and V‐ATPase c‐subunit overexpression was reported to be correlated with invasiveness of pancreatic tumors (Ohta et al., 1996 ). In the present work, we found that mRNAs encoding V‐ATPase subunits are not overexpressed in C6 tumoral glioma cells when compared with immortalized astrocytes DI TNC1 and astrocytes in primary cultures. Accordingly, V‐ATPase subunit mRNA levels are similar in human gliomas (grade II or IV) and in peritumoral tissues. A significant proportion (25%) of V‐ATPase is present in the plasma membrane of both the C6 and the DI TNC1 astrocytic cells in culture. A bafilomycin‐sensitive hyperpolarizing pump current through the plasma membrane was detected and measured after ionic channel inhibition, which corresponds most probably to an electrogenic transport of protons. This suggests that the plasma membrane V‐ATPase is active. It could contribute to cytoplasmic pH regulation in astrocytic cells. GLIA 37:365–373, 2002.

Cell size-dependent and independent proliferation of rodent neuroblastoma x glioma cells.

For decades, the connection between cell size and division has been the subject of controversy. While in yeast, cell size checkpoints coordinate cellular growth with cell‐cycle progression, it has been recently shown that large and small Schwann cells proliferate at the same rate (Conlon and Raff, 2003, J Biol 2:7). From this point of view, it is important to know whether normal and tumoral cells are similar. During continuous culture of NG108‐15 neuroblastoma x glioma cells, the rate of proliferation, cell size, and external pH changed in parallel. At constant pH, the cell size‐proliferation relationship followed a bell‐shaped curve, so that proliferation was optimal within a cell volume window. In contrast, external acidification decreased proliferation independently of cell size. Using electrophysiological techniques, we showed that changes in cell size were dependent on both the uptake of nutrients and the passive influx of ions. Furthermore, an increase in cell size was associated with an increase in total proteins/cell. Another way to influence cell growth and proliferation is to alter the activity of the PI‐3 kinase and target of rapamycin (TOR) signaling pathway. In NG108‐15 cells, pharmacological inhibition of these proteins with LY 294002 and rapamycin respectively decreased proliferation but did not modify cell size. In contrast, aphidicolin treated cells did not proliferate, but they continued to increase in size. Altogether these results indicate that the proliferation of NG108‐15 cells is controlled by both cell size‐dependent and independent mechanisms that include extracellular pH and PI‐3 kinase activity.

The Boltzmann equation in molecular biology

In the 1870s, Ludwig Boltzmann proposed a simple equation that was based on the notion of atoms and molecules and that defined the probability of finding a molecule in a given state. Several years later, the Boltzmann equation was developed and used to calculate the equilibrium potential of an ion species that is permeant through membrane channels and to describe conformational changes of biological molecules involved in different mechanisms including: open probability of ion channels, effect of molecular crowding on protein conformation, biochemical reactions and cell proliferation. The aim of this review is to trace the history of the developments of the Boltzmann equation that account for the behaviour of proteins involved in molecular biology and physiology.

Roles of cell volume in molecular and cellular biology

Extracellular tonicity and volume regulation control a great number of molecular and cellular functions including: cell proliferation, apoptosis, migration, hormone and neuromediator release, gene expression, ion channel and transporter activity and metabolism. The aim of this review is to describe these effects and to determine if they are direct or are secondarily the result of the activity of second messengers.

Sodium-dependent activity of aquaporin-1 in rat glioma cells: a new mechanism of cell volume regulation

Cell volume controls many functions and is itself regulated. To study cell volume regulations, the mean volume of C6-BU-1 rat glioma cells was electronically measured under isotonic and anisotonic conditions. Two isotonic solutions were used containing either normal (solution 1) or low (solution 2) NaCl. Anisotonicity was induced by changing NaCl or sucrose concentrations in solutions 1 and 2, respectively. The cells behaved like perfect osmometers when the tonicity was increased. In contrast, just after hypotonic challenges, the cell volume was smaller than that predicted by a perfect osmometer. This deviation reveals a new mechanism, which we call the volume increase limitation (VIL). When hypotonicity was induced by decreasing NaCl, a classical slow regulatory volume decrease (RVD) was also observed in addition to VIL. The cells expressed aquaporin-1 sensitive to HgCl2 and decreased by siRNA, which both reduced fast volume changes. In this study, we show that: (1) RVD is proportional to the change in external Cl− concentration and is inhibited by Cl− channel and K+–Cl− cotransporter blockers; (2) cell swelling due to the influx of H2O through aquaporins shows rectification with decreasing osmolarity and is sensitive to the internal Na+ concentration; (3) VIL is linearly related with hypotonicity and is abolished in solutions 1 and 2 by the Na+ ionophore monensin and in solution 1 by the Na+–K+ ATPase inhibitor ouabain. These results suggest that VIL is triggered by the decrease in internal Na+ caused by hyponatrema and cell swelling. In addition to RVD, VIL should protect cells during hyposmotic stress.

Calcium and voltage-dependent alterations of cell volume in neuroblastoma×glioma hybrid NG108-15 cells

Abstract. Intracellular calcium ([Ca2+]i), cell volume, membrane potential and currents were measured in neuroblastoma×glioma hybrid cells to gain insight into how [Ca2+]i controls cell volume. [Ca2+]i was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin. The increase in [Ca2+]i induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution. Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution. In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling. Both cell volume changes were inhibited by the Cl– channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid. The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA. Under all these conditions, the membrane input resistance was decreased. Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current. Taken together, these results suggest that, if cell volume is closely dependent on [Ca2+]i and the activity of Cl– channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential.

A proton pump contributes to neuroblastoma x glioma cell membrane potentials

Abstract The contribution of an electrogenic proton pump to the membrane potential of neuroblastoma × glioma hybrid NG 108–15 cells was determined with whole-cell voltage and current recordings and cell volume measurements with the preparation bathed in symmetrical 140 mM KCl solutions. The effects of the K+ channel blockers tetraethylammonium and 4-aminopyridine and of the H+-ATPase inhibitor bafilomycin A1 on the membrane potential and input resistance revealed that the membrane potential is generated by an outward H+ pump current of 5–15 pA in equilibrium with an inward passive current. This conclusion is supported by both current- and voltage-clamp results obtained when the preparation was bathed in a Na+-containing external solution after K+ channel blockade with Cs+ in the pipette. Additional support was obtained by measurement of the volume of cells incubated in solutions containing 140 mM KCl. Tetraethylammonium induced a bafilomycin-sensitive increase in inward K+ current and an increase in cell volume of 76% which we believe to be a consequence of the K+ influx. Finally, comparison of membrane potentials obtained in experiments using Na+-containing external, and K+-containing pipette solutions and after K+ channel blockade with Cs+ in the pipette also showed that, under normal physiological conditions, the resting membrane potential is essentially determined by an electrogenic H+ pump.