Survivin is an inhibitor of apoptosis protein (IAP) that is markedly overexpressed in most cancers. We identified two novel functionally divergent splice variants, i.e. non-antiapoptotic survivin-2B and antiapoptotic survivin-deltaEx3. Because survivin-2B might be a naturally occurring antagonist of antiapoptotic survivin variants, we analyzed the subcellular distribution of these proteins. PSORT II analysis predicted a preferential cytoplasmic localization of survivin and survivin-2B, but a preferential nuclear localization of survivin-deltaEx3. GFP-tagged survivin variants confirmed the predicted subcellular localization and additionally revealed a cell cycle-dependent nuclear accumulation of survivin-deltaEx3. Moreover, a bipartite nuclear localization signal found exclusively in survivin-deltaEx3 may support cytoplasmic clearance of survivin-deltaEx3. In contrast to the known association between survivin and microtubules or centromeres during mitosis, no corresponding co-localization became evident for survivin-deltaEx3 or survivin-2B. In conclusion, our study provided data on a differential subcellular localization of functionally divergent survivin variants, suggesting that survivin isoforms may perform different functions in distinct subcellular compartments and distinct phases of the cell cycle.

Rationale: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. Objective: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. Methods and Results: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm 2 ) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled 15 N-nitrite, whole body UVA irradiation (20 J/cm 2 ) induced significant levels of 15 N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11؎2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R

Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colonyforming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications - these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled.

Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects in various cells and tissues. Although NO formation in human skin has been convincingly demonstrated, little is known about the NO-mediated effects in skin physiology and pathology. Here, we investigate the influence of NO on proliferation, differentiation, and apoptosis of primary cultures of normal human keratinocytes and fibroblasts. Four different NO donors at concentrations ranging from 0.01 to 5 mM were added every 12 h or 24 h to primary cultures of human keratinocytes and fibroblasts, and cells cultured for up to 3 d in the presence of these compounds. Cultures were examined for necrosis or apoptosis using trypan blue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increase in cell numbers using neutral red staining. In addition, keratinocytes were stained for cytokeratin 6 expression to assess differentiation. We find that both keratinocytes and fibroblasts are highly resistant towards necrosis- or apoptosis-inducing effects of NO. In both cell types NO modulates cell growth, albeit in a cell-type specific pattern: cytostasis becomes significant in fibroblasts at concentrations of > or = 0.25 mM of the NO donor. In keratinocytes a biphasic effect is found with increased proliferation at low concentrations ranging from 0.01 to 0.25 mM and cytostasis at concentrations of > or = 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at higher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate that NO modulates proliferation and differentiation in human skin cells, a finding that will help to explain the pathophysiology of human skin diseases. Moreover, these findings suggest that NO generation in human skin diseases is not directly associated with local cell destruction, in contrast to findings in several other human diseases.