The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases

Mitogen-induced activation of p70(s6k) is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.     

The in-vitro generation of dendritic cells from blast cells in acute leukaemia

Recent clinical observations indicate that ibuprofen may alleviate the radiation-induced dysuria that almost invariably occurs during radiation therapy for prostate cancer. Because the use of ibuprofen could consequently become common during radiation therapy for prostate cancer, we have been interested in the potential interactions between ibuprofen and ionizing radiation on prostate tumor cells. The effects of gamma-irradiation and/or ibuprofen on PC3 and DU-145 human prostate carcinoma cells were evaluated in vitro using three model systems. Clonogenic survival was determined by plating cells 24 h after treatment of nearly confluent monolayers. Analysis of cell growth, cell detachment, and apoptotic cell death was carried out over a period of up to 9 days after treatment of PC3 and DU-145 monolayers. The effect of ibuprofen and/or radiation was also probed by observing the inhibition of growth of established PC3 and DU-145 colonies that were treated on the 14th day of colony growth. Ibuprofen enhanced the radiation response of prostate cancer cells in all three in vitro models. Both the cytotoxic and radiosensitizing effects of ibuprofen seem to require concentrations that are higher than those reported to inhibit prostaglandin synthesis, suggesting that other molecular mechanisms may be responsible for ibuprofen cytotoxicity.     

A review of nonparametric tests for changepoint problems, with application to a recombinant drug therapy clinical trial

We review general classes of nonparametric tests for the changepoint problem. We extend these tests to analyze the results of a clinical trial examining whether pretreatment of surgical patients with erythropoietin reduces their subsequent need for blood transfusions. We find a changepoint in baseline hemoglobin levels, below which pretreatment with erythropoietin appears particularly beneficial relative to placebo.     

Determination of background serum density for lipoprotein ultracentrifugation

It is necessary to know the density of serum exclusive of its macromolecules (background density) prior to density adjustment with solid potassium bromide for ultra-centrifugal separation of lipoprotein fractions. To evaluate this, we compared the densities of the corresponding ultrafiltrates or dialysates of both human and equine sera produced by ultrafiltration and equilibrium dialysis method for macromolecule removal. Excellent correlation is found between background densities determined following ultrafiltration or equilibrium dialysis. These data validate the use of ultrafiltration as a simple, direct method for determination of background serum densities but reveal equilibrium dialysis to be more time consuming and less precise. Using ultrafiltration, we find the background density for equine serum to be 1.004 g/ml, and initial investigation suggests this value may be altered by freezing, prolonged refrigeration (3 months), or heating to inactivate lecithin: cholesterol acyltransferase.     

Does age of the host affect growth rates of skeletal malignancies?

Statistical analysis of bone tumor growth rates as a function of age at initiation of radiation-induced skeletal malignancies in our animal colony indicated that the p value for an association between these parameters was <0.05, suggesting a correlation in beagle dogs. The youngest animals appeared to exhibit the most slowly growing tumors, and the trend was toward more rapidly growing tumors with increasing age. Less effective immune systems in older animals were invoked as a possible explanation of this relationship.     

NSAIDS inhibit the IL-1 beta-induced IL-6 release from human post-mortem astrocytes: the involvement of prostaglandin E2

Epidemiological studies have shown that steroidal as well as non-steroidal anti-inflammatory drugs lower the risk of developing Alzheimer's Disease (AD). A suppressive effect of these anti-inflammatory drugs on local inflammatory events in AD brains has been suggested, however the mechanisms responsible are still unknown. In this study we investigated at cellular level the influence of two anti-inflammatory drugs-dexamethasone and indomethacin--and an experimental specific cyclooxygenase-2 inhibitor, BF389, on the production of the pro-inflammatory cytokine IL-6 and the inflammatory mediator PGE2 by human astrocytes. Two human post-mortem astrocyte cultures (A157 and A295) and astroglioma cell lines (U251 and U373 MG) were found to secrete considerable amounts of IL-6 upon stimulation with IL-1beta. The glucocorticoid dexamethasone inhibited the IL-1beta-activated release of IL-6 from the postmortem astrocyte cultures A157 and A295 and from the astroglioma cell lines. The non-specific cyclooxygenase inhibitor indomethacin and BF389 only suppressed the IL-6 release by post-mortem astrocyte culture A157. This post-mortem astrocyte culture was found to produce large amounts of PGE2 upon stimulation with IL-1beta, whereas in the supernatants of the postmortem astrocyte culture A295 and the astroglioma cell lines, low PGE2 concentrations were detected. Addition of exogenous PGE2 prevented the inhibitory effect of indomethacin and BF389 on the IL-1beta-activated IL-6 release from A157 astrocytes and largely potentiated the IL-1-induced release of IL-6 from all astrocytes/astroglioma cells tested. Dexamethasone also inhibited the PGE2 release from the astrocytes and astroglioma cells, however the inhibitory effect of dexamethasone on the IL-1beta-activated IL-6 release could not be prevented by the addition of PGE2. The observed reduction of IL-6 and/or PGE2 from astrocytes may be involved in the mechanism underlying the beneficial effects of these drugs in AD.