Bombesin antagonist prevents CO2 laser-induced promotion of oral cancer.

We previously reported that CO2 laser incisions in carcinogen-initiated fields promoted cancer development and caused release of growth factors. Here we examined the quantitative and additive properties of this tumor-promoting event and examined whether this promotion could be nullified by treatment with a bombesin antagonist, which down-regulates epidermal growth factor receptors. The model used for cancer promotion was the hamster buccal cheek pouch that had been treated with a carcinogen (9,10-dimethyl-1,2-benzanthracene) for 6 weeks, producing premalignant lesions. These lesions would evolve into a cancer eventually without further treatment. Promotion was measured both by increased fluorescence in response to systemically administered Photofrin, measured noninvasively using an in vivo fluorescence photometer, and by the timing of appearance of clinical tumors. Laser incisions (0-3) were made into the hamster cheek 1 week apart, or three incisions were done 1 day apart. Another group of animals received bombesin antagonist RC-3095 for 4 weeks during the time incisions were made, again measuring promotion. Laser incisions 1 week apart produced additive promotion, whereas three incisions 1 day apart were not statistically different from the group receiving only one incision. RC-3095 treatment completely eliminated the promoting effects of incision and totally stopped promotion for the 4-week period of treatment. After discontinuing treatment with RC-3095, lesion progression resumed at the untreated control rate. This work confirms that the promoting event of a laser incision follows a comparable time course to release of growth factors after such an incision and that it can be eliminated by treatment with bombesin antagonists.

Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.

Proximo–distal specification in the wing disc of Drosophila by the nubbin gene

Mutations in the nubbin (nub) gene have a phenotype consisting of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs since early larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Clones located in the proximal region of the wing blade cause in addition nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior–posterior nor the dorsal–ventral boundary but that the selector genes have been correctly expressed since early larval development. The phenotypes of nub el and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells. We discuss the role of nub in the wing’s proximo–distal axis and in the formation of compartment boundaries.

Pharmacological analysis of cyclooxygenase-1 in inflammation

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50 = 0.009 μM; COX-2 IC50 = 6.3 μM). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2 production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibited in vivo COX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2.

Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFκB

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin’s inhibition of NFκB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, both in vitro and in vivo. To determine in vivo whether clinically relevant levels of salicylate act via adenosine, via NFκB, or via the “inflammatory” cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFκB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also provide in vivo confirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFκB.

The Drosophila LIM-only gene, dLMO, is mutated in Beadex alleles and might represent an evolutionarily conserved function in appendage development

The process of wing patterning involves precise molecular mechanisms to establish an organizing center at the dorsal–ventral boundary, which functions to direct the development of the Drosophila wing. We report that misexpression of dLMO, a Drosophila LIM-only protein, in specific patterns in the developing wing imaginal disc, disrupts the dorsal–ventral (D-V) boundary and causes errors in wing patterning. When dLMO is misexpressed along the anterior–posterior boundary, extra wing outgrowth occurs, similar to the phenotype seen when mutant clones lacking Apterous, a LIM homeodomain protein known to be essential for normal D-V patterning of the wing, are made in the wing disc. When dLMO is misexpressed along the D-V boundary in third instar larvae, loss of the wing margin is observed. This phenotype is very similar to the phenotype of Beadex, a long-studied dominant mutation that we show disrupts the dLMO transcript in the 3′ untranslated region. dLMO normally is expressed in the wing pouch of the third instar wing imaginal disc during patterning. A mammalian homolog of dLMO is expressed in the developing limb bud of the mouse. This indicates that LMO proteins might function in an evolutionarily conserved mechanism involved in patterning the appendages.

Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development.

A novel OTX-related homeodomain transcription factor has been identified on the basis of its ability to interact with the transactivation domain of the pituitary-specific POU domain protein, Pit-1. This factor, referred to as P-OTX (pituitary OTX-related factor), is expressed in primordial Rathke's pouch, oral epithelium, first bronchial arch, duodenum, and hindlimb. In the developing anterior pituitary, it is expressed in all regions from which cells with distinct phenotypes will emerge in the mature gland. P-OTX is able to independently activate and to synergize with Pit-1 on pituitary-specific target gene promoters. Therefore, P-OTX may subserve functions in generating both precursor and specific cell phenotypes in the anterior pituitary gland and in several other organs.