Insulin stimulation of glucose entry in chick fibroblasts and HeLa cells

3 to 5-fold stimulation by insulin of the rate of D-glucose entry into chick fibroblasts in culture is observed in cells transferred for several hours to medium containing little (50 to 100 μg/ml) or no D-glucose. No stimulation is obtained in cells trans-ferred for the same period to “high” glucose (1 mg/ml) medium. The latter finding probably accounts for the consistent failure of earlier studies to demonstrate stimulation. Pyruvate alone is as stimulatory as insulin and reverses the D-glucose “repression”. Hela cells are as sensitive to insulin as chick cells.     

Salicylic acid has a dual role in the activation of defence-related genes in parsley

Systemic acquired resistance is an inducible plant defence state, the activation of which depends mostly on the accumulation of salicylic acid (SA). During the past several years, it has been demonstrated that pretreatment of cultured parsley cells with SA potentiates the elicitation of several defence responses that are local in whole plants, including the accumulation of phenylpropanoid products. Here it is reported that while anionic peroxidase and mannitol dehydrogenase encoding genes are directly responsive to SA, pretreating parsley cells with SA not only enhances elicitation of the phenylpropanoid genes phenylalanine ammonia-lyase and 4-coumarate:CoA ligase but also of genes for PR-10 and a hydroxyproline-rich glycoprotein. Enhanced induction of these genes was seen at low levels of endogenous free SA. Enhancement of phenylalanine ammonia-lyase gene activation was proportional to the length of SA pretreatment. Furthermore, the ability of SA analogues to both potentiate elicited and directly induce defence gene activation correlated with their biological activity to promote plant disease resistance. In summary, these results emphasize that SA has at least a dual role in plant defence gene activation.     

Transient deactivation of ERK signalling is sufficient for stable entry into G0 in primary avian fibroblasts

Re-entry into the cell cycle from quiescence requires the activation of mitogen-activated protein (MAP) kinases of the extracellular-signal-regulated kinase (ERK) family [1,2]. The relationship between ERK and cell-cycle control is, however, complex, as ERK activation can also lead to terminal differentiation [3] or a senescence-like growth arrest [4]. Here, we report that reversible cell-cycle exit induced by serum withdrawal in primary avian fibroblasts is associated with rapid deactivation of ERK, but ERK activity is subsequently regenerated and sustained at high levels in fully quiescent (G0) cells. As in proliferating cells, ERK activation during G0 required the MAPkinase kinase MEK and was partially dependent on cell adhesion. Active, phosphorylated ERK was concentrated in the nucleus in cycling cells, but was largely confined to the cytoplasm during G0. This was unexpected, as activatory phosphorylation mediated by MEK is thought to play an important role in promoting nuclear translocation [5,6]. These results indicate that transient deactivation of ERK signalling can be sufficient for stable cell-cycle exit, and that MEK-mediated phosphorylation is not sufficient for nuclear translocation of active ERK in G0. Cytoplasmic sequestration may prevent active ERK from accessing critical nuclear cell-cycle targets, thus allowing quiescent or post-mitotic cells to retain ERK activity for other physiological functions.     

Thermolabile analogue proteins and the entry of HeLa and CHO cells into division

Entry of HeLa and CHO-10 cells into mitosis can be inhibited by incorporation of p-fluorophenylalanine at certain temperatures, 37 °C for the former cell type and 39.5 °C for the latter. At lower temperatures, 32 °C in the former and 37 °C in the latter, the analogue does not inhibit entry of cells into mitosis. The possibility that the analogue is not incorporated at the permissive temperatures has been ruled out; indeed incorporation is relatively greater at the permissive temperatures. The results suggest that the physiological properties of analogue protein molecules differ depending on the temperature at which they are synthesized; the higher the temperature the more likely they are to malfunction.     

Albumin has no role in the uptake of copper by human fibroblasts

The mechanism of copper uptake by cells has been the subject of controversy for some time. This paper examines the possibility of a role for albumin in the uptake of copper by fibroblasts. Although the cells could accumulate copper from a copper-albumin complex, there was no evidence for either copper-albumin or albumin receptors on the cell surface. The possibility of a surface exchange mechanism for copper was examined. While copper uptake showed saturation with increasing concentrations of labelled copper-albumin, adding unlabelled copper to the incubation medium did not inhibit uptake. Adding albumin or histidine to the copper-albumin complex resulted in an inhibition of copper uptake. The results can only be explained by the cell taking up free copper from the incubation medium, with the albumin then releasing its copper to maintain the equilibrium between free and bound metal. Since, in vivo there is essentially no free copper in serum, it is concluded that albumin is most unlikely to play a role in the uptake of copper by fibroblasts.     

Cytochalasin B-induced depolymerization of F-actin in chick embryo fibroblasts is dependent on cell density and anchorage to substratum

The effect of cytochalasin B on F-actin amount and organization was measured in chick embryo fibroblasts (CEF) grown on solid substratum at low density, at high density, and suspended in a fluid medium. It was found that: 1) Cytochalasin B induced decrease in F-actin content only in cells growing at low density, in density-inhibited or suspended cells cytochalasin B had no effect on F-actin amount. 2) In cells grown at low density F-actin filaments organized in stress fibers are more resistant to cytochalasin B than F-actin which is not organized in fibrils. In cell density-inhibited or suspended in a fluid medium F-actin filaments are insensitive to the action of cytochalasin B, although they are not organized in stress fibers. These results are interpreted to reflect the influence of contact reactions on treadmilling in F-actin filaments.     

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

Quantification of entry into and exit from the cell cycle in human lymphocyte cultures

A mathematical model is presented for the analysis of transition between cycling and non-cycling compartments by cells responding to a growth stimulus. The cellular age distribution as a function of time is derived from sequential [3H]thymidine pulse labeling indices. Rates of entry into and exit from the cycling compartment are determined on the basis of labeling indices obtained after instantaneous and long duration [3H]thymidine pulses. Analysis of an experiment involving sequential measurements over the whole lifespan of a human lymphocyte culture stimulated by phytohemagglutinin is presented as an example of the application of this method.     

Lipid phosphate phosphatase-2 activity regulates S-phase entry of the cell cycle in Rat2 fibroblasts

Lipid phosphates are potent mediators of cell signaling and control processes including development, cell migration and division, blood vessel formation, wound repair, and tumor progression. Lipid phosphate phosphatases (LPPs) regulate the dephosphorylation of lipid phosphates, thus modulating their signals and producing new bioactive compounds both at the cell surface and in intracellular compartments. Knock-down of endogenous LPP2 in fibroblasts delayed cyclin A accumulation and entry into S-phase of the cell cycle. Conversely, overexpression of LPP2, but not a catalytically inactive mutant, caused premature S-phase entry, accompanied by premature cyclin A accumulation. At high passage, many LPP2 overexpressing cells arrested in G(2)/M and the rate of proliferation declined severely. This was accompanied by changes in proteins and lipids characteristic of senescence. Additionally, arrested LPP2 cells contained decreased lysophosphatidate concentrations and increased ceramide. These effects of LPP2 activity were not reproduced by overexpression or knock-down of LPP1 or LPP3. This work identifies a novel and specific role for LPP2 activity and bioactive lipids in regulating cell cycle progression.     

Regulation of mitotic activity and the cell cycle in primary chick muscle cells by neurotransferrin

We previously demonstrated that neurotransferrin (NTF), a transferrin extracted from adult chicken peripheral nerves, promotes growth of primary chick muscle cells in the absence of embryo extract. NTF was shown to stimulate DNA synthesis and cell proliferation. In the present study, we demonstrate that NTF is a mitogen using two independent methods; counts of orcein-stained mitotic figures and analysis of cell cycle kinetics with a fluorescence-activated cell sorter. In low-density cultures mitotic activity increases with increasing doses of NTF followed by a plateau at concentrations greater than 6 μg/ml. Residual, embryonic mitotic activity progressively declines with time after plating muscle cells in the absence of NTF. Absence of NTF for 2 days causes cells to lose irreversibly their myogenic potential. In the presence of NTF, mitotic activity increases for 2 days followed by a decline concurrent with myoblast fusion and formation of myotubes. Cell cycle analysis showed that NTF addition causes cell populations to shift from G_t to S and G₂ + M within 18.5 hr. Muscle cells, plated at high densities in the absence of NTF, show mitotic activities similar to those plated at low densities in the presence of NTF. Addition of NTF to high-density cultures is ineffective in stimulating mitosis. These studies show that at typical cell plating densities, NTF is a required mitogen for primary chick muscle cell cultures.     

Aging of human fibroblasts is a succession of subtle changes in the cell cycle and has a final short stage with abrupt events

The proliferation of diploid human embryonic lung fibroblasts was analysed at different population doubling levels with growth curves, autoradiography after tritiated thymidine ([³H]TdR) labelling and staining of mitoses after incorporation of bromodeoxyuridine (BrdU). The parameters analysed allow for the first time the distinction between different changes in cell growth that occur during in vitro aging. The percent of slowly and rapidly dividing cells is steady during most of the lifespan; the number of cells capable of synthesizing DNA during a 24-h period is always high with a subtle decline during the second half of the lifespan up to the last 4–5 population doublings when the fall becomes pronounced. There is a constant decline of the rate of entrance into DNA synthesis and a stepwise increase in the sensitivity to cell cycle inhibition during cell crowding. Towards the middle of the lifespan, the population becomes more sensitive to low inocula which fail to accelerate the rate of entrance into the cycle. These changes lead to a final, abrupt stage of profound disorganization of proliferation taking place during the last 4–5 doublings.