Stress-induced senescence in human and rodent astrocytes

There is an increasing awareness that astrocytes, the most abundant cell type in the central nervous system, are critical mediators of brain homeostasis, playing multifunctional roles including buffering potassium ions, maintaining the blood-brain barrier, releasing growth factors, and regulating neurotransmitter levels. Defects in astrocyte function have been implicated in a variety of diseases including age-related diseases such Alzheimer's disease and Parkinson's disease. However, little is known about the age-related changes that occur in astrocytes and if these cells are able to generate a senescent phenotype in response to stress. In this report we have examined whether astrocytes can initiate a senescence program similar to that described in other cell types in response to a variety of stresses. Our results indicate that after oxidative stress, proteasome inhibition, or exhausted replication, human and mouse astrocytes show changes in several established markers of cellular senescence. Astrocytes appear to be more sensitive to oxidative stress than fibroblasts, suggesting that stress-induced senescence may be more pronounced in the brain than in other tissues.     

Polyphosphoinositides in myelin

1. On fractionation of guinea-pig forebrain homogenates by differential and gradient-density centrifugation most of the polyphosphoinositides were recovered in the myelin-rich particles. 2. The phospholipids of pure preparations of myelin contained di- and tri-phosphoinositide in proportions 2-3 times greater than in the whole-brain phospholipids. 3. Di- and tri-phosphoinositide appeared in young rat brain during the period of myelination. 4. After the administration of [(32)P]phosphate to guinea pigs the labelling of the polyphosphoinositides in isolated pure myelin was as great as in the whole brain, whereas little synthesis of the other myelin phospholipids had occurred. 5. When brain subcellular fractions were incubated with [gamma-(32)P]ATP, some triphosphoinositide labelling occurred in the myelin-rich fraction whereas the active labelling of diphosphoinositide was localized mainly in the mitochondrial fraction. 6. The Na(+), K(+) and Mg(2+) plus Ca(2+) concentrations in purified myelin have been determined. The Mg(2+) plus Ca(2+) content present showed close acid-base equivalence to the polyphosphoinositides. 7. It is concluded that di- and tri-phosphoinositide are rapidly-metabolizing components of the myelin sheath or intimately associated structures.     

Proteoglycans in normal and neoplastic monocytes

35S proteoglycans produced by normal and neoplastic (U-937) monocytes after a 20-h pulse with [35S]sulfate in vitro have been isolated and compared. Both cell types produce exclusively chondroitin sulfate proteoglycan (CSPG), which are released into the medium and are not contained within the cells. The neoplastic cell-derived molecules were much larger in molecular size, due to the substitution of galactosaminoglycan chains, with an approximate Mr of 60,000. The corresponding chains in monocyte CSPG had an Mr of approx. 20,000. The latter chains were also found to be more sulfated than their neoplastic counterparts.     

Retinoid-induced apoptosis in normal and neoplastic tissues

Vitamin A and its derivatives (collectively referred to as retinoids) are required for many fundamental life processes, including vision, reproduction, metabolism, cellular differentiation, hematopoesis, bone development, and pattern formation during embryogenesis. There is also considerable evidence to suggest that natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects in human diseases such as cancer. Therefore it is not surprising that a significant amount of research was dedicated to probe the molecular and cellular mechanisms of retinoid action during the past decade. One of the cellular mechanisms retinoids have been implicated in is the initiation and modulation of apoptosis in normal development and disease. This review provides a brief overview of the molecular basis of retinoid signaling, and focuses on the retinoid-regulation of apoptotic cell death and gene expression during normal development and in pathological conditions in vivo and in various tumor cell lines in vitro.     

Circadian concepts in normal and neoplastic breast

Normal breast exhibits rhythmic properties linked to the hormonal environment of the gland in animals and humans. Breast tumors also display rhythmic properties; however, they differ from those found normally in animals and humans. Breast cancer in humans is characterized by disruption or modification of normal circadian patterns, which may be of prognostic value. The relationships between melatonin biology and breast cancer require exploration. The present work summarizes the data concerning circadian concepts in breast cancer and explores future directions in the breast cancer treatment by chronomodulation of medications during the 24h, taking advantage of the circadian time structure of breast tissue to improve the treatment outcome.     

Circadian concepts in normal and neoplastic breast

Normal breast exhibits rhythmic properties linked to the hormonal environment of the gland in animals and humans. Breast tumors also display rhythmic properties; however, they differ from those found normally in animals and humans. Breast cancer in humans is characterized by disruption or modification of normal circadian patterns, which may be of prognostic value. The relationships between melatonin biology and breast cancer require exploration. The present work summarizes the data concerning circadian concepts in breast cancer and explores future directions in the breast cancer treatment by chronomodulation of medications during the 24h, taking advantage of the circadian time structure of breast tissue to improve the treatment outcome.     

Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro . In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo , but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.     

Gene expression patterns in in vivo normal adult astrocytes compared with cultured neonatal and normal adult astrocytes

This paper presents data on the basal gene expression patterns, determined by microarray analysis, for cultured neonatal and normal adult striatal astrocytes, and for comparison, for astrocytes isolated directly from adult rat striatum (in vivo adult astrocytes). Of the 1176 genes on the Clontech array, 1101 were expressed in one of the three types of astrocyte samples. Nineteen of the genes were expressed only in the astrocytes taken directly from adult rats (in vivo adult). The cultured neonatal astrocytes expressed many genes at a two-fold or greater level than their expression in cultured adult astrocytes, including genes in the adhesion, cytoskeleton, and extracellular matrix (ECM) family, signal transduction genes, and genes related to apoptosis, DNA-binding, and cell cycle regulation. Overall the results support the concept that cultured neonatal astrocytes are more "activated" than cultured adult cells, although the adult cells expressed higher levels of many metabolic enzyme and protease/protease inhibitor genes.     

Calcium release from intracellular stores in rodent astrocytes and neurons in situ

Endoplasmic reticular Ca(2+) stores, instrumental for intra- and intercellular calcium signalling, can be depleted by different receptor agonists. In the present study, the functional status of ER Ca(2+) stores was probed by cyclopiazonic acid (CPA, 10-30 microM, inhibitor of SERCA-dependent ER Ca(2+) uptake) and/or caffeine (20 mM, ryanodine receptor activator) in astrocytes and neurons of rat and mouse acute hippocampal brain slices (Stratum radiatum, Stratum moleculare), and in cultured astrocytes, using confocal microscopy and conventional Ca(2+) imaging. Astrocytes and neurons in situ, identified by their Ca(2+) response in K(+)-free saline (Dallwig and Deitmer [J. Neurosci. Methods 116 (2002) 77]), had a resting cytosolic Ca(2+) level of 105 and 157 nM, respectively (P<0.05). CPA evoked a Ca(2+) transient, which was faster and larger in neurons than in astrocytes, indicating larger Ca(2+) leak of neuronal Ca(2+) stores. Caffeine evoked a Ca(2+) rise in most neurons (>80%), but only in less than 40% of astrocytes. The glial Ca(2+) transients in the presence of caffeine had a large and variable delay (>50 s), as compared to those in neurons (< or =10 s), and appeared to be spontaneous and/or secondary to the neuronal Ca(2+) response, leading to release of neuronal transmitters. Astrocytes in culture responded to CPA, but never to caffeine with a Ca(2+) rise. Our results indicate that astrocytes, in contrast to neurons, lack caffeine-sensitive Ca(2+) stores, and have a relatively smaller leak from CPA-sensitive Ca(2+) stores than neurons.     

Protein phosphorylation in normal and neoplastic hepatocytes]

Partially purified preparations of protein kinase were isolated from the cytoplasm and nuclei of rat liver and hepatoma 27 and characterized in terms of their substrate specificity. The protein kinases from normal liver and hepatoma revealed some differences in phosphorylating protein substrates. Hepatoma protein kinases were found to phosphorylate arginine-rich histones (H3, H4); differences in phosphorylation of histone H1 were revealed. Hepatoma protein kinases phosphorylated the C-terminal fragment of histone H1, whereas normal liver protein kinases produced no such effect. It was assumed that the phosphorylation of histone H1 in rapidly dividing and resting cells is operated through different channels.     

Polyamine acetylations in normal and neoplastic growth processes

Summary The expression patterns of cytosolic and nuclear polyamine acetyltransferases were studied in normal and neoplastic growth processesin vivo andin vitro to evidentiate the roles played by these enzymes in cell proliferation. In regenerating liver, cytosolic spermidine/spermine N¹-acetyltransferase showed similar augments of mRNA level and enzymatic activity during the prereplicative period (4–8 h), whereas spermidine N⁸-acetyltransferase activity increased later (24 h) when DNA synthesis was maximally enhanced. In fibroblasts continuously dividing, the messenger for spermidine/spermine N¹-acetyltransferase rapidly accumulated after serum-stimulation. In cultured Morris hepatoma cells stimulated to logarithmic growth, spermidine N⁸-acetyltransferase activity remained at plateau for 1 day declining thereafter, while spermidine/spermine N¹-acetyltransferase activity immediately decreased. In Yoshida AH-130 hepatoma cells transplanted in rat peritoneum, spermidine N⁸-acetyltransferase and spermidine/spermine N¹-acetyltransferase activities rose, respectively, in concomitance with elevated proliferation-rate and quasi-stationary phase of growth. Since the expression of cytosolic and nuclear acetyltransferases underwent different temporal activation, an involvement of these enzymes in separate metabolic processes controlling normal and neoplastic growth may be suggested.     

Glucocorticoid receptors and lymphocytolysis in normal and neoplastic lymphocytes

When cells of the thymus or mouse leukemias P288 and L1210 are exposed in vitro to the potent synthetic glucocorticoid, 3H-Triamcinolone acetonide, the steroid enters the cells passively and binds to macromolecules in the cytoplasm. At 37 degrees C this hormone-receptor complex enters the nucleus and associates with the chromatin. The association with chromatin occurs not only in the corticosteroid-sensitive rat thymocytes and mouse tumors P288 and P1798S but also in the corticosteroid-resistant mouse tumors L1210 and P1798R. An apparent correlation, although not absolute, exists between the content of glucocorticoid-binding macromolecule and the sensitivity of the lymphocytes studied to the lytic effect of glucocorticoids; the sensitive cells having more receptor than the resistant cells. The process of lysis is attributed to the release from the much larger stores of triglyceride in thymus and sensitive lymphoma cells, of a large pool of FFA which causes focal damage to the nuclear membrane resulting in karyorrhexis and, subsequently, to cytolysis. Resistance is attributed to the capacity for preventing the accumulation of greater than about 0.5 fmole FFA/cell. Resistant cells induced to accumulate greater amounts, even for a few minutes, ultimately undergo lysis. Most effective in accomplishing this are branched chain fatty acids of C-8 and higher, which block FFA metabolism, causing accumulation which results in cytolysis.     

Orotate uptake and metabolism in normal and neoplastic tissues

The uptake of [3H]orotate was greater in mouse liver than in hepatoma but the difference was less marked than in the rat. Of the tissues examined, a high uptake of [3H]orotate was restricted to the liver and kidney in rat, mouse and guinea-pig. We confirmed that a high orotate diet greatly increases the ratio of UTP to ATP concentration in rat liver but we observed that there is little change of this nucleotide ratio in kidney. Evidence was obtained for a different pattern of orotate metabolism in rat liver and kidney.     

Accumulation of noradrenaline and its oxidation products by cultured rodent astrocytes

The accumulation of [³H]noradrenaline ([³H]NA) and its oxidation products was studied in primary cultures of cerebral astrocytes. Astroglial accumulation of radiolabeled catecholamine ([³H] NA and oxidation products) was enhanced by manganese or iron, but it was inhibited by unlabeled NA, dopamine or ascorbate. Tissue:medium ratios of radioactivity increased as extracellular [³H]NA was oxidized. When extracellular oxidation was prevented by ascorbate, as confirmed by high performance liquid chromatography with electrochemical detection, either ouabain pretreatment or nominally Na⁺-free incubation medium inhibited approximately one-half of specific [³H]NA accumulation by rat (but not mouse) astrocytes. These observations suggest that neurological responses to trace metals and ascorbate may arise from the effects of these agents on the clearance of extracellular catecholamines. Astrocytes can accumulate oxidation products of NA more rapidly than they take up NA itself, but ascorbate at physiological concentrations prevents the oxidation process in extracellular fluid. Furthermore, in the presence of ascorbate, Na⁺-dependent transport mediates a significant component of NA accumulation in rat astrocytes.     

Stability analysis of normal and neoplastic growth

A model for normal and abnormal tissue growth is defined by a system of differential equations. The model consists of a proliferating compartment, which is characterized by a decreasing growth rate, and a nonproliferating one. The compartments are also parametrized by their death and transfer rates. A stability analysis of the system shows in a precise way how abnormal growth may result from a perturbation of the parameters leading to a change in an equilibrium of the tissue size.     

Gangliosides of normal and neoplastic human melanocytes

The major ganglioside component isolated from diploid human melanocytes is sialosyllactosylceramide (GM3 86-91% of total sialic acid). The corresponding disialo derivative (GD3) is found as a minor component (2-6% of total sialic acid) in the membranes of these cells. In human melanoma cells, grown in tissue culture, GD3 is the predominant ganglioside component (48-63% of total sialic acid). Withdrawal of TPA from the culture medium of normal melanocytes or addition of TPA to the medium of melanoma cells had no significant effect on GM3/GD3 ratios. We conclude that the difference between the composition of gangliosides is related to the normal vs transformed phenotypes of melanocytes.