Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.

Angiostatin effectively blocks tumor angiogenesis through still poorly understood mechanisms. Given the close association between immune and vascular regulation, we investigated the effects of angiostatin on angiogenesis-associated leukocytes. Angiostatin inhibited the migration of monocytes and, even more markedly, neutrophils. Angiostatin blocked chemotaxis of neutrophils to CXCR2 chemokine receptor agonists (IL-8, MIP-2, and GROalpha), formyl-Met-Leu-Phe (fMLP), and 12-O-tetradecanoylphorbol 13-acetate, and repressed fMLP-induced mitochondrial activity. Two different angiostatin forms (kringles 1-4 and 1-3) were effective, whereas whole plasminogen had no effect. IL-8, MIP-2, and GROalpha induced intense angiogenic reactions in vivo, but no angiogenic response to these factors was observed in neutropenic mice, demonstrating an essential role for neutrophils. Angiostatin potently inhibited chemokine-induced angiogenesis in vivo, and consistent with in vitro observations, both angiostatin forms were active and whole plasminogen had little effect. Angiostatin inhibition of angiogenesis in vivo was accompanied by a striking reduction in the number of recruited leukocytes. In vivo, the inflammatory agent lipopolysaccharide also induced extensive leukocyte infiltration and angiogenesis that were blocked by angiostatin. Neutrophils expressed mRNAs for ATP synthase and angiomotin, two known angiostatin receptors. These data show that angiostatin directly inhibits neutrophil migration and neutrophil-mediated angiogenesis and indicate that angiostatin might inhibit inflammation.

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Tight junctions (TJs) form a barrier to the paracellular diffusion of ions and solutes across epithelia. Although transmembrane proteins of the claudin family have emerged as critical determinants of TJ permeability, little is known about the signaling pathways that control their expression. The aim of this study was to assess the role of three mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH 2-terminal kinases (JNKs), and p38 kinases, in the regulation of epithelial barrier function and claudin expression in mammary epithelial cells. Addition of either PD169316 (a p38 inhibitor) or SP600125 (a JNK inhibitor) induced formation of domes (a phenomenon dependent on TJ barrier function) and enhanced transepithelial electrical resistance, whereas U0126 (an inhibitor of the ERK1/2 activators MEK1/MEK2) had no significant effect. Similar results were obtained using mechanistically unrelated p38 or JNK inhibitors. PD169316 increased the expression of claudin-4 and -8, whereas SP600125 increased claudin-4 and -9 and downregulated claudin-8. Silencing of p38␣ by isoform-specific small interfering RNAs increased claudin-4 and -8 mRNAs, whereas silencing of p38␤ only increased claudin-4 mRNA. Silencing of either JNK1 or JNK2 increased claudin-9 mRNA expression while decreasing claudin-8 mRNA. Moreover, selective silencing of JNK2 increased claudin-4 and -7 mRNAs. Finally, both PD169316 and SP600125 inhibited the paracellular diffusion of Na ϩ and Cl Ϫ across epithelial monolayers. Collectively, these results provide evidence that inhibition of either p38 or JNK enhances epithelial barrier function by selectively modulating claudin expression, implying that the basal activity of these MAPKs exerts a tonic effect on TJ ionic permeability. mitogen-activated protein kinases; domes; transepithelial electrical resistance; tight junction; mammary epithelial cells; c-Jun NH 2-terminal kinase A FUNDAMENTAL FUNCTION of epithelial cells is to maintain homeostasis of the internal milieu by regulating the exchange of substances between compositionally distinct body compartments. Movement of solutes, ions, and water across the epithelial barrier occurs through both the transcellular pathway, owing to the asymmetric cellular distribution of membrane pumps and channels, and the paracellular pathway, via tight junctions (TJs). Whereas the contribution of the transcellular route has been characterized in considerable detail, the molecular mechanisms that regulate TJ permeability are still incompletely understood.

Although loss of cell-cell adhesion and gain of invasive properties play a crucial role in the malignant progression of epithelial tumours, the molecular signals that trigger these processes have not been fully elucidated. In light of the well-established relationship between chronic inflammation and cancer, we hypothesized that pro-inflammatory cytokines disrupt epithelial-cell adhesion and promote cell migration. To test this hypothesis, we used an in vitro model in which 31EG4-2A4 mouse mammary epithelial cells grown in a collagen gel form compact spheroidal colonies. Among the several cytokines examined, tumour necrosis factor α (TNF-α) caused a pronounced 3D scattering of preformed epithelial-cell colonies and induced 31EG4-2A4 cells grown on top of a collagen gel to invade the underlying matrix. In addition, TNF-α abolished contact-mediated inhibition of cell proliferation and stimulated cell growth both in the absence of exogenous mitogens and under anchorage-independent conditions. TNF-α induced the expression of matrix metalloproteinase 9 (MMP-9). Addition of the MMP inhibitor BB-94 abrogated TNF-α-induced 3D scattering. TNF-α also enhanced the attachment of 31EG4-2A4 cells to type-I collagen and markedly increased the expression of the α2 integrin subunit. Addition of a blocking antibody to β1-integrin or of rhodocetin (a specific α2β1 antagonist) to collagen-gel cultures abrogated 3D scattering. Collectively, these results demonstrate an essential role for MMPs and α2β1 integrin in the invasive response of 31EG4-2A4 cells to TNF-α. We propose that the biological activities described in this study contribute to the ability of TNF-α to promote tumour progression and cancer-cell dissemination.