1 Exposure of human mammary carcinoma cell line MCF-7 to TNF-a leads to apoptotic cell death within 24 h. In search for apoptosis-preventing signals, we identi®ed glucocorticoids as potent deathpreventing compounds. Ten nM dexamethasone provided a signi®cant protective e ect whereas 100 nM dexamethasone roughly blocked 80 ± 90% of TNF-a-induced apoptosis.2 Surprisingly, dexamethasone exerted a protective e ect even when supplied several hours after TNF-a. This points to a powerful inhibition of even advanced apoptotic processes by dexamethasone. 3 To further pinpoint the anti-apoptotic glucocorticoid action, we investigated the expression levels of several members of the inhibitors of apoptosis (IAPs) family of proteins in response to TNF-a and dexamethasone. IAP proteins directly block caspase protease activities including caspase-3, caspase-7, and caspase-9. Exposure of MCF-7 cells to TNF caused an extensive downregulation of cIAP1, cIAP2, and XIAP protein levels. The decline of the IAP protein levels temporally paralleled the appearance of apoptotic DNA fragments which started 12 ± 14 h following TNF-a addition and maximal e ects were seen within 24 h. 4 Coincubation of cells with TNF-a and dexamethasone potently blocked cIAP1, cIAP2, and XIAP downregulation. 5 TNF-a-mediated IAP protein downregulation was not a ected by proteasome inhibitors like lactacystin, ALLN or ALLM, whereas it was blocked by the broad-spectrum caspase inhibitor Z-VAD-fmk which also prevented TNF-a-induced apoptotic cell death. These data suggest that inhibition of IAP downregulation mediated by a caspase proteolytic activity constitutes the antiapoptotic action of glucocorticoids in MCF-7 carcinoma cells.

The observed injury-mediated increase in HO-1 mRNA and protein at the wound site was due to infiltrating HO-1 expressing monocytic cells. Macrophage-derived HO-1 expression was not under regulatory control by NO in skin repair. We provide evidence that HO-1 might exert a regulatory role in macrophage-derived cytokine release.

In this paper, we investigate the potential of a high-performance liquid chromatography technique to determine pKa values of drug candidates that show poor solubility in water. The determination of pKa values by this method is in principle not new, but it exhibits simplicity, requires lower quantities of drugs and solvents, and minimal analysis time. The method is an alternative to existing methodology, in which this determination is not readily feasible.

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INTRODUCTION.Mesangial cells and RAW 264.7 macrophages respond to different NO donors within 16-24h or 6-8h, respectively, with apoptotic cell death (1). RAW 264.7 macrophages also die in response to endogenous NO production. In contrast, endogenous NO production failed to significantly induce cell death in mesangial cells (2,3). We hypothesized that differences in the expression of antiapoptotic proteins in particular the inhibitor of apoptosis (IAP) protein family might be responsible for this cell type-specific behaviour. METHOD.To this end, by performing RNase Protection Assay and Western Blot Analysis, we compared IAP expression in relation to apoptosis induction in response to NO and cytokines in both cell types. RESULTS.In mesangial cells IL-1β and TNF-α induced cIAP1 mRNA expression within 3 h. In contrast, XIAP mRNA levels remained unaffected by cytokines. Although coincubation of cells with IL-1β and TNF-α or IL-1β and bFGF resulted in a synergistic induction of iNOS, comparable potentiating effects were absent regarding cIAP1 induction. Also exogenously released NO from NO donors promoted cIAP1 mRNA upregulation in mesangial cells whereas XIAP mRNA was downregulated. However, these changes seen on the mRNA level were not adequately translated onto the protein level and corresponding values for cIAP1 and XIAP were only slightly affected. In contrast, in LPS/interferon-γ-stimulated RAW 264.7 macrophages, we found a massive NO-dependent downregulation of cIAP1 and XIAP protein level that correlated temporally with induction of apoptosis. This effect was at least partially reversed by L-NMMA, an inhibitor of NOS activity.In summary, in macrophages we found a direct correlation between the downregulation of IAP protein levels and the induction of apoptosis by endogenous NO. By contrast, a stable level of IAP protein in mesangial cells might represent a mechanism constituting resistance of the cells against endogenously produced NO.ACKNOWLEDGEMENT.