In this study, we examined the biological action of IL-17 on human non-small cell lung cancer (NSCLC). Although IL-17 had no direct effect on the in vitro growth rate of NSCLC, IL-17 selectively augmented the secretion of an array of angiogenic CXC chemokines, including CXCL1, CXCL5, CXCL6, and CXCL8 but not angiostatic chemokines, by three different NSCLC lines. Endothelial cell chemotactic activity (as a measure of net angiogenic potential) was increased in response to conditioned medium from NSCLC stimulated with IL-17 compared with those from unstimulated NSCLC. Enhanced chemotactic activity was suppressed by neutralizing mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2 but not to vascular endothelial growth factor-A. Transfection with IL-17 into NSCLC had no effect on the in vitro growth, whereas IL-17 transfectants grew more rapidly compared with controls when transplanted in SCID mice. This IL-17-elicited enhancement of NSCLC growth was associated with increased tumor vascularity. Moreover, treatment with anti-mouse CXCR-2-neutralizing Ab significantly attenuated the growth of both neomycin phosphotransferase gene-transfected and IL-17-transfected NSCLC tumors in SCID mice. A potential role for IL-17 in modulation of the human NSCLC phenotype was supported by the findings that, in primary NSCLC tissues, IL-17 expression was frequently detected in accumulating and infiltrating inflammatory cells and that high levels of IL-17 expression were associated with increased tumor vascularity. These results demonstrate that IL-17 increases the net angiogenic activity and in vivo growth of NSCLC via promoting CXCR-2-dependent angiogenesis and suggest that targeting CXCR-2 signaling may be a novel promising strategy to treat patients with NSCLC.

Digoxin, which is one of the most commonly prescribed drugs for the treatment of heart failure, is mainly eliminated from the circulation by the kidney. P-glycoprotein is well characterized as a digoxin pump at the apical membrane of the nephron. However, little is known about the transport mechanism at the basolateral membrane. We have isolated an organic anion transporter (OATP4C1) from human kidney. Human OATP4C1 is the first member of the organic anion transporting polypeptide (OATP) family expressed in human kidney. The isolated cDNA encodes a polypeptide of 724 aa with 12 transmembrane domains. The genomic organization consists of 13 exons located on chromosome 5q21. Its rat counterpart, Oatp4c1, is also isolated from rat kidney. Human OATP4C1 transports cardiac glycosides (digoxin, K m ‫؍‬ 7.8 M and ouabain, K m ‫؍‬ 0.38 M), thyroid hormone (triiodothyronine, Km ‫؍‬ 5.9 M and thyroxine), cAMP, and methotrexate in a sodiumindependent manner. Rat Oatp4c1 also transports digoxin (K m ‫؍‬ 8.0 M) and triiodothyronine (Km ‫؍‬ 1.9 M). Immunohistochemical analysis reveals that rat Oatp4c1 protein is localized at the basolateral membrane of the proximal tubule cell in the kidney. These data suggest that human OATP4C1͞rat Oatp4c1 might be a first step of the transport pathway of digoxin and various compounds into urine in the kidney.

Immunoassays for haptens depend on competitive hapten-anti-hapten reactions, and consequently their sensitivities are significantly influenced by the affinities of anti-hapten antibodies. Thus, genetically engineered antibodies, which have much higher affinities than native antibodies, should increase assay sensitivities. Here, we created a mutated single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) that allowed immunoassays with a much improved sensitivity. Two steps of affinity maturation were performed on a "wild-type" scFv (scFv#E4-4) composed of V(H) and V(L) domains from a mouse anti-E(2) antibody (Ab#E4-4). First, we conducted complementarity-determining region (CDR)-targeted mutagenesis by "CDR-shuffling". Gene fragments encoding CDRs H2, H3, L1, and L3, each of which contained random point mutations, were combined by "shuffling" into the gene encoding the scFv#E4-4 scaffold. After phage display and repeated panning, we isolated a mutated scFv clone [scFv#m1-e7; Ile(L29)Val] that had 5-fold higher affinity (K(a) = 2.6 x 10(8) M(-1)) compared to the Ab#E4-4 Fab fragment (Fab#E4-4). Next, the entire V(H) and V(L) of this clone were randomly mutated by error-prone polymerase chain reaction (PCR). From this library, we found an improved clone, scFv#m2-c4 (K(a) = 6.3 x 10(8) M(-1); Lys(H19)Arg, Tyr(H56)Phe, Ser(H84)Pro, Glu(H85)Gly, Gln(L27)Arg, Leu(L36)Met, Ser(L63)Gly, and Ser(L77)Gly). ScFv#m2-c4 had more than 10-fold higher sensitivity (the midpoint of its dose-response curve was 0.56 ng) than Fab#E4-4 (midpoint 9.0 ng/assay) in a competitive E(2) radioimmunoassay, and even higher sensitivity [midpoint 21 pg/assay, and a limit of detection of 0.47 pg (1.7 fmol)/assay] in a competitive enzyme-linked immunosorbent assay. Cross-reactivity with selected E(2)-related endogenous steroids strongly suggested that scFv#m2-c4 has improved specificity compared to conventional antibodies.