ALBERT

Notes: Summary Pre-embedding and post-embedding in situ hybridization techniques were compared for the localization of RNAs in the nucleus. 28S rRNA and transcripts of the epidermal growth factor receptor (EGF-receptor) were localized with both hybridization methods. PRE-embedding hybridizations were performed on cells permeabilized with Triton X-100, whereas post-embedding hybridizations were carried out on Lowicryl K4M sections. From these studies it was concluded that, for labelling of 28S rRNA, the post-embedding in situ hybridization is preferred, whereas EGF-receptor transcripts were successfully detected only after pre-embedding in situ hybridization. Furthermore, the detection of the hybrids with ultra-small gold particles was compared to the detection with 6 nm gold particles in both pre- and post-embedding in situ hybridization studies. From our results it is concluded that the use of ultra-small gold particles results in higher label efficiency. Therefore, ultra-small gold particles are preferable to 6 nm gold particles for the detection of hybrids in high-resolution in situ hybridization experiments.

Notes: 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.

Notes: 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/106 cells to 0.36 ± 0.25 nmoles/min/106 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/106 cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 ± 0.25 nmole/min/106 cells in early S phase to 2.10 ± 0.92 nmoles/min/106 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.

Notes: Nerve growth factor (NGF) is required for the growth and development of sensory and sympathetic neurons. Incubation of chick dorsal root ganglionic cells without NGF resulted in a decrease of active (Na+, K+-pump-mediated) K+ influx over a period of several hours. Addition of NGF to NGF-deprived cells caused (1) a return of the active K+ influx to the values occurring in cells continuously exposed to NGF, preceded by (2) a very rapid, but transient overstimulation of the Na+, K+-pump-mediated K+ influx. Restoration of normal Na+, K+-pump activity occurred at NGF concentrations of 1 biological unit/ml or greater, whereas the NGF concentration in the 1-100 biological unit/ml range affected the rapidity with which the pump restoration took place. The transient pump behavior was only observed in NGF-deprived cells and could not be elicited in NGF-supported steady-state cells or in cells having already received delayed NGF once. This transient Na+, K+-pump behavior was exclusively displayed in conjunction with a high intracellular Na+ concentration. Decreasing the external Na+ concentration below 70 mM reduced the hyperstimulation response to NGF, until at 10 mM Na+ the delayed presentation of NGF caused no overshoot at all. The effect of NGF on the Na+, K+-pump was specific for the NGF molecule and could not be mimicked by other proteins.

Notes: We have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25-hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14C-acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol-phospholipid ratio. The experimental modulation of plasma membrane composition by 25-hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistantly under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression.

Notes: Several observations have indicated that clustering of growth factor receptors plays an important role in the action of growth factors. In this investigation, we have used the label fracture method to study the effects of epidermal growth factor (EGF) on the lateral distribution of its receptors in A431 epidermoid carcinoma cells. This method allows a direct visualization of immunogold-labeled plasma membrane receptors on ultrastructural level and in addition permits an quantitative analysis of their lateral distribution. EGF receptors were immunogold-labeled according to standard procedures with the monoclonal anti-EGF receptor antibody 2E9 (lgG1), which binds to the EGF receptor in a 1:1 ratio. In the absence of EGF, EGF receptors located on the surface of A431 cells were found to be clustered, as deduced from Poisson variance analysis (p 〈 0.001). Following treatment of A431 cells with EGF, receptor clustering increased rapidly, reaching the maximum within 10 min. Maximal clustering was maintained for 1 h, after which the lateral distribution of receptors returned to the control situation within another hour.

Notes: Na+ transport properties of neuroblastoma (clone Neuro-2A) cells have been characterized both in exponentially growing cells and during the cell cycle. In exponentially growing cells, Na+ influx followed the kinetics for a one-compartment system with an influx rate of 9.54 ± 0.96 nmoles/minute/106 cells, equilibrium being reached within 2 minutes. The initial rate of influx in the presence of ouabain (5 mM), a concentration completely inhibiting the (Na+-K+)ATPase and thus backflux of tracer, was 11.33 ± 1.50 nmoles/minute/106 cells and, within error, the same as in the absence of ouabain, provided determinations were made within 3 minutes of ouabain addition.Na+ influx rate was determined at intervals during the cell cycle of synchronized Neuro-2A cells using a 3-minute pulse of 22Na+ in the presence of ouabain. On passing from mitosis to G1-phase Na+ influx decreases from 12.13 ± 1.93 to 7.40 ± 0.90 nmoles/minute/106 cells but increases rapidly and transiently approximately twofold upon entry into S-phase. This transient increase coincides with a transient stimulation of the (Na+-K+) pump activity. It then returns to a steady level of ˜ 12 nmoles/minute/106 cells for most of the remainder S-phase.Intracellular Na+ concentration during the cell cycle was determined from the equilibrium content of 24Na+ and data on the intracellular H2O volume, published previously (Boonstra et al., 1981b). Na+ concentration is maximal in mitosis at 56.96 ± 6.05 mM and decreases rapidly tourfold as cells enter G1-phase. With progression through S-phase a steady increase from 13.80 ± 1.25 mM to 37.35 ± 2.91 mM is observed. Combining the Na+ concentration with the K+ concentration obtained previously (Boonstra et al., 1981b), the K+:Na+ ratio was obtained during the cell cycle. The ratio had a value of between 3 and 5 during most of the cell cycle, but was significantly higher in G1-phase, where the loss of Na+ is considerably greater than the loss of K+. The values for Na+ concentration were combined with membrane potential measurements reported previously (Boonstra et al., 1980b) to obtain the Na+ electrochemical gradient across the cell membrane in the cell cycle. This had a value of 63.2 ± 2.6 mV in mitosis and increased rapidly to reach a maximum value of 84.0 ± 5.5 mV during G1-phase, thereafter maintaining a value of between 73 and 80 mV. The transient increase in Na+ influx and in (Na+-K+)ATPase-mediated K+ influx at the G1/S-phase transition are specifically inhibitable by the diuretic amiloride (0.2 mM) but amiloride has little effect on Na+ or K+ influx in other phases of the cell cycle. This indicated activation of an amiloride-sensitive transport system specically at the G1/S-phase transition and a causal relationship between increased Na+ entry and transient stimulation of the pump at this point in the cell cycle. Further, amiloride (0.1, 0.2 mM) addition was shown to give rise to a significant lengthening of the cell cycle and to cause a partial blockage of the cells specifically in G1-phase, suggesting an important role for the transient ion flux changes in controlling entry into and progression through S-phase.

Notes: Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.

Notes: Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20-30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high-calcium cells were deprived of extracellular sources of cholesterol but increased (8-10 fold) in deprived low-calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low-calcium cells but had no effect on high-calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I-LDL was 20-30 times higher than in keratinocytes cultured under high-calcium conditions. Switching of the cells from the low-calcium to the high-calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I-LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high-calcium level, whereas both were observed at the low-calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.