Destruction of existing tumor blood vessels may be achieved by targeting vascular endothelial growth factor (VEGF) signaling, which mediates not only endothelial cell proliferation but also endothelial cell survival. In this study, however, intravital microscopy failed to demonstrate that targeting of VEGFR-2 (by the tyrosine kinase inhibitor SU5416) induces significant regression of experimental tumor blood vessels. Immunohistochemistry, electron microscopy, expression analyses, and in situ hybridization provide evidence that this resistance of tumor blood vessels to VEGFR-2 targeting is conferred by pericytes that stabilize blood vessels and provide endothelial cell survival signals via the Ang-1/Tie2 pathway. In contrast, targeting VEGFR-2 plus the platelet-derived growth factor receptor (PDGFR)-beta system (PDGFR-beta) signaling (by SU6668) rapidly forced 40% of tumor blood vessels into regression, rendering these tumors hypoxic as shown by phosphorescence quenching. TUNEL staining, electron microscopy, and apoptosis blocking experiments suggest that VEGFR-2 plus PDGFR-beta targeting enforced tumor blood vessel regression by inducing endothelial cell apoptosis. We further show that this is achieved by an interference with pericyte-endothelial cell interaction. This study provides novel insights into the mechanisms of how 1) pericytes may provide escape strategies to anti-angiogenic therapies and 2) novel concepts that target not only endothelial cells but also pericyte-associated pathways involved in vascular stabilization and maturation exert potent anti-vascular effects.

Cadmium-mediated toxicity of cultured proximal tubule (PT) cells is associated with increased production of reactive oxygen species (ROS) and apoptosis. We found that cadmium-dependent apoptosis (Hoechst 33342 and annexin V assays) decreased with prolonged CdCl 2 (10 M) application (controls: 2.4 ؎ 1.6%; 5 h: ؉5.1 ؎ 2.3%, 20 h: ؉5.7 ؎ 2.5%, 48 h: ؉3.3 ؎ 1.0% and 72 h: ؉2.1 ؎ 0.4% above controls), while cell proliferation was not affected. Reduction of apoptosis correlated with a time-dependent up-regulation of the drug efflux pump multidrug resistance P-glycoprotein (

Resveratrol, a polyphenol present in wine and grapes, can inhibit tumor cell growth in vitro and tumorigenesis in vivo. Some of its effects have been linked to activation of the p53 tumor suppressor; however, p53 is frequently mutated in tumors, particularly in the common and often therapy-resistant colon cancers. Using the human wild-type p53-expressing HCT116 colon carcinoma cell line and HCT116 cells with both p53 alleles inactivated by homologous recombination, we show in the current study that resveratrol at concentrations comparable to those found in some foods can induce apoptosis independently of p53. The cell death is primarily mitochondria-mediated and not receptor-mediated. No cells survived in cultures continuously exposed to 100 M resveratrol for 120 hr. When compared with 5-FU, resveratrol stimulated p53 accumulation and activity only weakly and with delayed kinetics and neither the increased levels nor the activity affected apoptosis detectably. The apoptosis agonist Bax was overproduced in response to resveratrol regardless of p53 status, yet the kinetics of Bax expression were influenced by p53. Remarkably, apoptosis was preceded by mitochondrial proliferation and signs of epithelial differentiation. Thus, resveratrol triggers a p53-independent apoptotic pathway in HCT116 cells that may be linked to differentiation. The antifungal phytoalexin resveratrol (3,5,4Ј-trihydroxy-transstilbene) is a constituent of many plant species and present at particularly high levels in grapes and wine. 1 As a polyphenol, it is not only a potent inhibitor of radical formation (ED 50 27 M) but acts as a pleiotropic effector molecule to inhibit initiation, promotion and progression of malignant transformation. Resveratrol inhibits the cyclooxygenase (ED 50 15 M) and hydroperoxidase (ED 50 3.7 M) activities of COX-1 and the hydroperoxidase activity of COX-2 (ED 50 85 M). 2 Furthermore, it functions as an inhibitor of platelet aggregation, a modulator of lipid and lipoprotein metabolism 3 and an effective blocker of ribonucleotide reductase (ED 50 100 M) that provides deoxyribonucleotides for DNA synthesis. 4 It also inhibits DNA polymerase 5 and mimics estradiol. 6 Several of these activities constitute the basis for the chemopreventive action of resveratrol, exemplified by its antimutagenic activity in the Ames assay, inhibition of tumorigenesis in a 2-stage murine skin-cancer model 2 and inhibition of cell proliferation in vitro. 7,8 The presence of the functional wild-type form of the p53 tumor suppressor correlates with the sensitivity of mouse tumors and at least some human tumors to therapeutic agents. 9 -11 p53 is stabilized and activated as a transcription factor in response to a variety of cellular stresses, 12 the result being either cell-cycle arrest, mostly through transcriptional activation of the p21 WAF/Cip1 inhibitor of cyclin-dependent kinases, or apoptosis, depending on the cell context. Apoptosis often involves changes to mitochondria. 13 One of the major predictive parameters indicating commitment...

Tumour necrosis factor (TNF)-alpha-induced apoptosis is associated with several nuclear and cell surface alterations, in particular with the condensation of chromatin and the fragmentation of the cell nucleus, formation of blebs on the cell surface and breakdown of the plasma membrane. However, there is little information about the relationship between the cell surface alterations and the nuclear changes during apoptosis. To study this, cultured WEHI cells were exposed to TNF-alpha over different time periods. The cytological changes were studied using a correlative approach, which allowed observation of the same cell consecutively under light, scanning and transmission electron microscopy. The earliest sign of cell alteration was a reduction of the number of microvilli after 15 min of TNF-alpha exposure. This reaction was reversible (reappearance of microvilli) and took place during the first hour, in which neither nuclear alterations nor plasma membrane breakdown were observed. The changes in the nucleus began with condensation of chromatin after approximately 1 h of TNF-alpha-exposure. After 4-5 h the microvilli disappeared again, particularly in areas where the formation of blebs (blebbing) was observed. Strikingly, cell surface alterations (bleb formation) were detected only in those cells that presented with condensed chromatin, and not in cells with a normal chromatin pattern, proving at least a close correlation between nuclear and cell surface changes during the process of apoptosis.

Cells were cultivated on transparent conductive substrates. glass slides coated with indium oxide: individual cells were marked with a diamond indentor. Cell cultures were frozen ( -15"C), thawed, and then stained with fluorescent dyes to determine cell damage. The marked cells were examined by phase contrast. fluorescence, and Nomarski DIC microscopy. After aldehyde and osmium tetroxide fixation, the cell preparations were sequentially treated with tannic acid, uranyl acetate, and lead citrate. The same marked cell could be sequentially studied by light microscopy (LM; in water immersion conditions), scanning electron microscopy (SEM; after dehydration and critical point drying), and transmission electron microscopy (TEM: after embedding of cell samples in epoxy resin and laser marking of the cell previously marked with a diamond indentor). The method used ensures good preservation of cell morphology, cell surface relief, and intracellular structures. The treatment used renders the cells conductive and permitted SEM of uncoated culture cells on conductive substrates.

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