In-vivo visualization of tumor microvessel density and response to anti-angiogenic treatment by high resolution MRI in mice

Purpose

Inhibition of angiogenesis has shown clinical success in patients with cancer. Thus, imaging approaches that allow for the identification of angiogenic tumors and the detection of response to anti-angiogenic treatment are of high clinical relevance.

Experimental Design

We established an in vivo magnetic resonance imaging (MRI) approach that allows us to simultaneously image tumor microvessel density and tumor vessel size in a NSCLC model in mice.

Results

Using microvessel density imaging we demonstrated an increase in microvessel density within 8 days after tumor implantation, while tumor vessel size decreased indicating a switch from macro- to microvessels during tumor growth. Moreover, we could monitor in vivo inhibition of angiogenesis induced by the angiogenesis inhibitor PTK787, resulting in a decrease of microvessel density and a slight increase in tumor vessel size.

Conclusions

We present an in vivo imaging approach that allows us to monitor both tumor microvessel density and tumor vessel size in the tumor. Moreover, this approach enables us to assess, early-on, treatment effects on tumor microvessel density as well as on tumor vessel size. Thus, this imaging-based strategy of validating anti-angiogenic treatment effects has high potential in applications to preclinical and clinical trials.     

IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A(1), A(2a), A(2b) and A(3) adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA (N6-(R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A(2b) receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A(2b) dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.     

TICO: a tool for improving predictions of prokaryotic translation initiation sites

SUMMARY: We provide the tool 'TICO' (Translation Initiation site COrrection) for improving the results of conventional gene finders for prokaryotic genomes with regard to exact localization of the translation initiation site (TIS). At the current state TICO provides an interface for direct post processing of the predictions obtained from the widely used program GLIMMER. Our program is based on a clustering algorithm for completely unsupervised scoring of potential TIS locations. AVAILABILITY: Our tool can be freely accessed through a web interface at http://tico.gobics.de/ CONTACT: maike@gobics.de     

Immediate early gene X1 (IEX-1) is organized in subnuclear structures and partially co-localizes with promyelocytic leukemia protein in HeLa cells

Immediate early gene X1 (IEX-1) represents a stress response gene involved in growth control and modulation of apoptosis. Here, we report a detailed analysis of IEX-1 with respect to its intracellular localization. By means of confocal laser scanning microscopy, a green fluorescent protein-IEX-1 fusion protein transfected into HeLa cells, as well as endogenous IEX-1, could be detected in distinct subnuclear structures. This particular subnuclear localization of IEX-1 was not observed with a green fluorescent protein-IEX-1 fusion protein lacking a putative nuclear localization sequence, along with a decreased effect on apoptosis. Double immunofluorescence staining revealed a partial co-localization of endogenous promyelocytic leukemia protein (PML) and IEX-1 in these subnuclear structures. Nuclear localization of IEX-1 is also enhanced upon treatment of cells with leptomycin B, an inhibitor of the nuclear exporter CRM1. These observations indicate that IEX-1 is specifically shuttled to and from the nucleus. Overexpression experiments using PML isoforms III and IV revealed distinct intranuclear interaction of IEX-1 and PML. Coprecipitation experiments showed physical interaction between IEX-1 and PML. The close structural relation of IEX-1-containing nuclear subdomains and PML nuclear bodies suggests a function of IEX-1 related to the multiple functions of these unique subnuclear regions, particularly during stress response and growth control.     

Hypotonic stress induces E-cadherin expression in cultured human keratinocytes

Human epidermis marks the interface between internal and external environments with the major task being to maintain body hydration. Alternating exposure of skin to a dry or humid environment is likely to cause changes in the epidermal water gradient resulting in osmotic alterations of epidermal keratinocytes. The present in vitro approach studied the effect of hypotonicity on cell-cell contact. It was demonstrated that hypotonic stress applied to human epithelial cells (HaCaT, A-431) induced upregulation of E-cadherin at both, the protein and mRNA level. 5'-deletional mutants of the E-cadherin promoter identified an element ranging from -53 to +31 that conveyed strong transactivation under hypotonic stress. In order to define relevant upstream regulators members of the MAP kinase family, the epidermal growth factor receptor (EGFR) and protein kinase B/Akt (PKB/Akt) were investigated. Hypotonic conditions led to a fast activation of ERK1/2, SAPK/JNK, p38, EGFR and PKB/Akt with distinct activation patterns. Experiments using specific inhibitors showed that p38 contributes to the E-cadherin transactivation under hypotonic conditions. Further upstream, adhesion was found to be a prerequisite for E-cadherin transactivation in this model. In summary, the present study provides evidence that E-cadherin is an osmo-sensitive gene that responds to hypotonic stress. The function of this regulation may be found in morphological changes induced by cell swelling. It is likely that induction of E-cadherin contributes to the stabilization between adjacent cells in order to withstand the physical forces induced by hypotonicity.     

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as well as the indoles indole-3-carbinol and its condensation product 3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce apoptosis. In this study, we compared the cell growth inhibitory potential of the four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+)) and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116). Using sulforhodamin B staining to assess cell proliferation, we found that the isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of treatment. Apoptosis induction was analyzed by immunoblotting of poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC (10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM) induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines, suggestive of a p53-independent mechanism of apoptosis induction. In cultured 40-16 cells, activation of caspase-9 and -7 detected by Western blotting indicated involvement of the mitochondrial pathway. We detected time- and concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L) as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly correlated with apoptosis induction measured by PARP cleavage. Taken together, we demonstrated that the glucosinolate breakdown products investigated in this study have distinct profiles of cell growth inhibition, potential to induce p53-independent apoptosis and to modulate Bcl-2 family protein expression in human colon cancer cell lines.     

An unsupervised classification scheme for improving predictions of prokaryotic TIS

Background  

Although it is not difficult for state-of-the-art gene finders to identify coding regions in prokaryotic genomes, exact prediction of the corresponding translation initiation sites (TIS) is still a challenging problem. Recently a number of post-processing tools have been proposed for improving the annotation of prokaryotic TIS. However, inherent difficulties of these approaches arise from the considerable variation of TIS characteristics across different species. Therefore prior assumptions about the properties of prokaryotic gene starts may cause suboptimal predictions for newly sequenced genomes with TIS signals differing from those of well-investigated genomes.