Ruthenium-Containing Linear Helicates and Mesocates with Tuneable p53-Selective Cytotoxicity in Colorectal Cancer Cells.

The ligands L1 and L2 both form separable dinuclear double-stranded helicate and mesocate complexes with RuII . In contrast to clinically approved platinates, the helicate isomer of [Ru2 (L1 )2 ]4+ was preferentially cytotoxic to isogenic cells (HCT116 p53-/- ), which lack the critical tumour suppressor gene. The mesocate isomer shows the reverse selectivity, with the achiral isomer being preferentially cytotoxic towards HCT116 p53+/+ . Other structurally similar RuII -containing dinuclear complexes showed very little cytotoxic activity. This study demonstrates that alterations in ligand or isomer can have profound effects on cytotoxicity towards cancer cells of different p53 status and suggests that selectivity can be "tuned" to either genotype. In the search for compounds that can target difficult-to-treat tumours that lack the p53 tumour suppressor gene, [Ru2 (L1 )2 ]4+ is a promising compound for further development.

Stress reactivity and cognitive performance in a simulated firefighting emergency.

BACKGROUND: During emergencies maladaptive behavior can reduce survival. This study compared the effects of a basic firefighter training course on 21 volunteers (with no firefighting experience) with age and gender-matched controls. METHODS: Stress reactivity (salivary cortisol and anxiety) were monitored across the course: day 1 (classroom), day 2 (physical equipment training), and day 3 (simulated fire emergency). Cognitive performance (visual attention, declarative and working memory) considered important in surviving a fire emergency were measured immediately post-training or after a 20-min delay. RESULTS: Prior to threat subjects showed an anticipatory cortisol increase but no corresponding increase in self-reported anxiety. On day 3 cortisol was higher in firefighters tested immediately after (10.37 nmol x L(-1) and 20 min after training (7.20 nmol L(-1)) compared to controls (3.13 nmol x L(-1)). Differences in cognitive performance were observed post-threat, with impairments in visual declarative memory in the firefighting subjects tested immediately, and working memory impairments observed in those tested after a 20-min delay. CONCLUSIONS: Cognitive impairments were found following a simulated emergency and could explain maladaptive responses observed during real fires. Moreover, the results suggest the type of cognitive impairments observed may be time dependent, with different cognitive difficulties becoming evident at different times following an emergency.

Cerebrospinal fluid supports viability and proliferation of cortical cells in vitro, mirroring in vivo development.

BACKGROUND: The central nervous system develops around a fluid filled compartment. Recently, attention has turned to the potential role of the fluid (cerebrospinal fluid, CSF) in the developmental process. In particular, the cerebral cortex develops from the germinal epithelium adjacent to the CSF with regulation of cell proliferation and differentiation provided by cells adjacent to the fluid-filled subarachnoid space. METHODS: Histological analysis of fetal rat cortical sections was used to follow the extent of in vivo cortical development. A quantitative analysis of proliferation and migration of cortical cells at E17 - E21 was obtained through immunocytochemical staining of bromodeoxyuridine (BrdU) -labelled cells. In vitro studies were performed on primary cortical cells at days E17-E20, maintained in either Neurobasal media or 100% fetal rat CSF for 72 h before analysis of proliferation. RESULTS: The proliferation potential of primary cortical cells varied depending on the age of extraction. E17 and E20 cells showed little proliferation while E18 and E19 cell showed the maximum. The CSF from fetuses of all ages tested, except E21, was able to maintain primary cortical cells from the developing fetus in vitro and to stimulate and support their proliferation in the absence of any additions. E17 cells showed little proliferation in any media while E19 cells showed maximum proliferation in E19 and E20 CSF. CONCLUSION: CSF composition most probably changes with age, as does the proliferation potential of cells in the developing cerebral cortex. CSF alone supports viability as well as proliferation of cortical cells. CSF must therefore be regarded as an important environmental influence in brain development and can be used in vitro to maintain both the viability of cortical progenitor cells and their age-related proliferative potential.

Bcr-Abl-mediated molecular mechanism for apoptotic suppression in multipotent haemopoietic cells: a role for PKCbetaII.

Bcr-Abl protein tyrosine kinase (PTK) activity is a feature of chronic myeloid leukaemia and confers a survival advantage on haemopoietic progenitor cells. We have expressed conditional mutant of the Bcr-Abl PTK in the FDCP-Mix A4 multipotent haematopoietic cell line in order to examine the molecular mechanisms whereby Bcr-Abl PTK leads to enhanced cell survival under conditions in which normal cells die. Activation of Bcr-Abl PTK does not phosphorylate or activate either ERK-1/2 or JAK-2/STAT-5b, suggesting that these signal transduction pathways are not involved in Abl PTK-mediated suppression of apoptosis in FDCP-Mix cells. However, protein kinase C (PKC) does have a role to play. Inhibition of PKC results in a reversal of Bcr-Abl PTK-mediated survival in the absence of growth factor and Bcr-Abl stimulates translocation of the PKCbetaII isoform to the nucleus. Furthermore, expression of a constitutively activated PKCbetaII in haemopoietic progenitor FDCP-Mix cells stimulates enhanced cell survival when IL-3 is withdrawn. However, expression of this constitutively activated PKC isoform does not suppress cytotoxic drug-induced apoptosis. Thus Bcr-Abl PTK has pleiotropic effects which can suppress cell death induced by a number of stimuli.

Interleukin-3-mediated cell survival signals include phosphatidylinositol 3-kinase-dependent translocation of the glucose transporter GLUT1 to the cell surface.

Maintenance of glucose uptake is a key component in the response of hematopoietic cells to survival factors. To investigate the mechanism of this response we employed the interleukin-3 (IL-3)-dependent murine mast cell line IC2.9. In these cells, hexose uptake decreased markedly upon withdrawal of IL-3, whereas its readdition led to rapid (t(1/2) approximately 10 min) stimulation of transport, associated with an approximately 4-fold increase in Vmax but no change in Km. Immunocytochemistry and photoaffinity labeling revealed that IL-3 caused translocation of intracellular GLUT1 transporters to the cell surface, whereas a second transporter isoform, GLUT3, remained predominantly intracellular. The inhibitory effects of latrunculin B and jasplakinolide, and of nocodazole and colchicine, respectively, revealed a requirement for both the actin and microtubule cytoskeletons in GLUT1 translocation and transport stimulation. Both IL-3 stimulation of transport and GLUT1 translocation were also prevented by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. The time courses for activation of phosphatidylinositol 3-kinase and its downstream target, protein kinase B, by IL-3 were consistent with a role in IL-3-induced transporter translocation and enhanced glucose uptake. We conclude that one component of the survival mechanisms elicited by IL-3 involves the subcellular redistribution of glucose transporters, thus ensuring the supply of a key metabolic substrate.

Defective cell cycle control underlies abnormal cortical development in the hydrocephalic Texas rat.

There is a significant body of evidence to suggest a physiological role for the CSF in both the developing and adult brain. Our recent studies suggest a critical role for this fluid in the developing brain of the hydrocephalic Texas (H-Tx) rat. A key feature of the foetal-onset hydrocephalus in this rat is obstruction in the flow and/or absorption of fluid that is associated with abnormal development of the cerebral cortex resulting in a reduction in the number of neuronal precursors generated. Cells from the affected cerebral cortex do proliferate in vitro and show dose-dependent responses to growth factor stimulation, suggesting that germinal cells are under inhibitory influences in vivo. We tested the hypothesis that the CSF of the affected brains was responsible for the abnormal development. Cells analysed at the time of extraction from affected brains showed an accumulation of cells in the S-phase of the cell cycle, which was reflected in a concentration of cells containing high levels of DNA in the germinal matrix of histological sections of affected brains. CSF from the lateral ventricle of affected foetal brains not only inhibited in vitro proliferation of normal neuronal progenitors, but it also resulted in an accumulation of cells in the S-phase of the cell cycle mimicking the situation in vivo. Fluid from normal foetal brains did not have this effect. From the work detailed here on the mechanistic basis of the deficient cortical development in the foetal hydrocephalic rat brain, we conclude that the content of the CSF is critical in maintaining germinal matrix function and output and, therefore, that the CSF has a vital role in brain development.

Deficient cortical development in the hydrocephalic Texas (H-Tx) rat: a role for CSF.

The objectives of this study were to demonstrate the presence and nature of abnormal cortical development in a rat model of hydrocephalus, the hydrocephalic Texas (H-Tx) rat, and to test the hypothesis that the obstruction of CSF flow in affected animals can be linked to this effect. CSF is secreted continuously by the choroid plexus, located in the lateral, third and fourth ventricles. The fluid flows through the ventricular system, passing over all regions of germinal activity. In the H-Tx rat, obstruction and eventual blockage of CSF flow occurs in the cerebral aqueduct, between the third and fourth ventricles, at embryonic day 18. Prior to obstruction, neurogenesis and migration occur as in normal rats. Here we show that, following obstruction of fluid flow, neurogenesis from the germinal epithelium becomes abnormal. Cell proliferation decreases and proliferating cells are not retained in the germinal layer, as they appear to be in the normal brain. Cell migration is apparently unaffected, although a decrease in the number of migrating cells does occur after CSF obstruction. These data from our study indicate that a rapid primary effect of CSF obstruction, prior to any mechanical effects of fluid accumulation, is to alter the activity of cells in the germinal epithelium of the developing cortex. Further evidence for this is gained from in vitro studies. Once removed from their in vivo environment, cortical cells from the H-Tx rat have the ability to proliferate as normal. CSF extracted from the enlarged ventricles of affected brains is able to inhibit the proliferation of normal cells. Thus, we hypothesize that CSF has a potential role in the developmental process. The damming up and accumulation of CSF, whatever the cause, may result in abnormal cortical development through accumulation of CSF factors that are, or become, inhibitory to normal neuronal proliferation.