Intradermal injection of phytohemagglutinin (PHA) in the wattles of chickens elicited marked swellings often accompanied by induration. Histologically, this reaction was characterized by a perivascular accumulation of lymphocytes and macrophage migration in the central layer of the wattles. Heterophilic infiltration was observed mostly at early hours and waned thereafter. Sometime basophils were prominently located around the vessels. These responses were significantly decreased in cases of neonatal thymectomy. Therefore, the PHA skin test was considered to be a thymus-dependent response. The use of the PHA skin test in chickens may provide useful information for the evaluation of thymus-dependent function.

DNA topoisomerase I was purified to near homogeneity from a clonal line of human lymphoblastic leukemia cells, RPMI 8402, that is resistant to camptothecin, a cytotoxic alkaloid from Camptotheca acuminata, and compared with that of the parent wild-type cells. As assayed by relaxation of the supercoiled plasmid DNA and by formation of enzymelinked DNA breaks, the purified enzyme from the resistant cells was shown to be >125-fold as resistant to camptothecin as the wild-type enzyme, comparable to a cellular resistance index of about 300. Therefore, the cellular resistance appears to be due to the resistance of the enzyme. The amount of the immunoreactive enzyme protein in whole extract appeared to be reduced to less than half that of the wild-type enzyme. These results establish that DNA topoisomerase I is the cellular target of camptothecin and that DNA topoisomerase I is essential for the survival of mammalian cells.The DNA topoisomerases are enzymes that catalyze the concerted breakage and rejoining of the DNA backbone and thereby are presumed to participate in various genetic processes (1-5). The type I topoisomerases transiently cut and reseal one DNA strand so that the linking number changes by steps of one. Genetic and functional studies of the enzymes have been largely limited to prokaryotes and the lower eukaryote yeast. Viable mutants ofEscherichia coli defective in the topA gene encoding topoisomerase I were isolated (6, 7), but these proved to have mutations in DNA gyrase genes that compensated for the mutation in topA (8-10). This finding suggested an essential role for the enzyme in regulating the degree of supercoiling of DNA by counteracting the activity of the type II enzyme. In contrast, however, topoisomerase I-deficient mutants of yeast were isolated and shown to be viable, although they possessed the wild-type allele of topoisomerase II (11,12). The effect of the topoisomerase I mutation, however, was manifested by an additional mutation in topoisomerase II, implicating the complementary role of the latter (12).Topoisomerase I was previously found associated with transcriptionally active chromatin in mammalian cells (13,14). It also appears to be catalytically active on transcriptionally active genes in Drosophila polytene chromosomes (15) as well as on nucleolus-associated ribosomal genes (16-19). These experiments suggest a functional role for the enzyme in transcriptional events involving either RNA polymerase I or II.The availability of mutants and specific inhibitors of this enzyme as was the case in prokaryotes might help dissect and establish the role of this enzyme in DNA metabolism. We previously reported that heparin is a potent inhibitor of a mammalian DNA topoisomerase I (20, 21), but its broad specificity limits its usefulness for this purpose. Camptothecin (CPT) is a cytotoxic alkaloid isolated from Camptotheca acuminata (22, 23), which has a strong antitumor activity against a wide range of experimental tumors. CPT inhibits RNA and DNA synthesis and causes rapid and rev...

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Oxidative stress is a critical mediator in liver injury of steatohepatitis. The transcription factor Nrf2 serves as a cellular stress sensor and is a key regulator for induction of hepatic detoxification and antioxidative stress systems. The involvement of Nrf2 in defense against the development of steatohepatitis remains unknown. We aimed to investigate the protective roles of Nrf2 in nutritional steatohepatitis using wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. WT and Nrf2-null mice were fed a methionine- and choline-deficient (MCD) diet for 3 and 6 wk, and the liver tissues were analyzed for pathology and for expression levels of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. In WT mice fed an MCD diet, Nrf2 was potently activated in the livers, and steatohepatitis did not develop over the observation periods. However, in Nrf2-null mice fed an MCD diet, the pathological state of the steatohepatitis was aggravated in terms of fatty changes, inflammation, fibrosis, and iron accumulation. In the livers of the Nrf2-null mice, oxidative stress was significantly increased compared with that of WT mice based on the increased levels of 4-hydroxy-2-nonenal and malondialdehyde. This change was associated with the decreased levels of glutathione, detoxifying enzymes, catalase, and superoxide dismutase activity. Correlating well with the liver pathology, the mRNA levels of factors involved in fatty acid metabolism, inflammatory cytokines, and fibrogenesis-related genes were significantly increased in the livers of the Nrf2-null mice. These findings demonstrate that Nrf2 deletion in mice leads to rapid onset and progression of nutritional steatohepatitis induced by an MCD diet. Activation of Nrf2 could be a promising target toward developing new options for prevention and treatment of steatohepatitis.

The protective action of ursodeoxycholic acid (UDCA) in cholestatic liver diseases may be mediated by choleresis, detoxification, and cytoprotection against oxidative stress. Nrf2, one transcription factor, serves as a cellular stress sensor and is a key regulator for hepatic induction of detoxifying enzymes, antioxidative stress genes, and numerous Mrp family members. We aimed to investigate whether UDCA induces hepatic Mrp expression along with that of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. The protein level, subcellular localization, and mRNA level of Mrp family members were assessed in livers of Keap1 gene-knockdown (Keap1-kd) mice and those of UDCA-fed wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. Nuclear levels of Nrf2 in livers of Keap1-kd mice markedly increased, resulting in constitutive activation of Nrf2. Keap1-kd mice have high-level expression of hepatic Mrp2, Mrp3, and Mrp4 relative to WT mice. UDCA potently increased nuclear Nrf2 expression level in livers of WT mice, and the treatment showed maximal hepatic induction of Mrp2, Mrp3, and Mrp4 in association with enhanced membranous localizations in an Nrf2-dependent manner. UDCA similarly increased nuclear Nrf2 expression level in rat hepatocytes. Chromatin immunoprecipitation assays using mouse hepatocytes revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of Mrp2, Mrp3, and Mrp4. These findings demonstrate an important role of Nrf2 in the induction of Mrp family members in livers and suggest that a therapeutic mechanism of UDCA action is, via Nrf2 activation, a stimulation of detoxification and antioxidative stress systems, along with Mrp-mediated efflux transport.

Inchinkoto, a herbal medicine, and its ingredients dually exert Mrp2/MRP2-mediated choleresis and Nrf2-mediated antioxidative action in rat livers. Am J Physiol Gastrointest Liver Physiol 292: G1450 -G1463, 2007. First published October 12, 2006; doi:10.1152/ajpgi.00302.2006, a herbal medicine, has been recognized in Japan and China as a "magic bullet" for jaundice. To explore potent therapeutic agents for cholestasis, the effects of ICKT or its ingredients on multidrug resistance-associated protein 2 (Mrp2/ MRP2)-mediated choleretic activity, as well as on antioxidative action, were investigated using rats and chimeric mice with livers that were almost completely repopulated with human hepatocytes. Biliary excretion of Mrp2 substrates and the protein mass, subcellular localization, and mRNA level of Mrp2 were assessed in rats after 1-wk oral administration of ICKT or genipin, a major ingredient of ICKT. Administration of ICKT or genipin to rats for 7 days increased bile flow and biliary excretion of bilirubin conjugates. Mrp2 protein and mRNA levels and Mrp2 membrane densities in the bile canaliculi and renal proximal tubules were significantly increased in ICKT-or genipin-treated rat livers and kidneys. ICKT and genipin, thereby, accelerated the disposal of intravenously infused bilirubin. The treatment also increased hepatic levels of heme oxygenase-1 and GSH by a nuclear factor-E2-related factor (Nrf2)-dependent mechanism. Similar effects of ICKT on MRP2 expression levels were observed in humanized livers of chimeric mice. In conclusion, these findings provide the rationale for therapeutic options of ICKT and its ingredients that should potentiate bilirubin disposal in vivo by enhancing Mrp2/MRP2-mediated secretory capacities in both livers and kidneys as well as Nrf2-mediated antioxidative actions in the treatment of cholestatic liver diseases associated with jaundice.

Topoisomerase (topo) I and II are nuclear enzymes which are novel targets of cancer chemotherapy. A new camptothecin (CPT) analog, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyl-oxy-CPT (CPT-11), is a topo-I inhibitor with a higher activity and less toxicity than CPT. To investigate topo-I and -II-targeting chemotherapy in an in vivo model, we studied the effect of sequential or co-treatment using CPT-11 and adriamycin (ADR) a topo-II inhibitor, in 6 human tumor xenografts (2 esophageal, 2 gastric and 2 colon tumor lines). In sequential treatment, adriamycin was administered i.v. 24 hr after CPT-11 treatment, and no antagonistic effect of this treatment schedule was observed. ADR cytotoxicity was potentiated significantly by CPT-11 pretreatment in the case of 2 esophageal and 2 gastric tumor lines and 1 colon tumor line. On the other hand, co-treatment abolished the sensitivity to CPT-11 and ADR in all 6 tumor lines. Moreover, CPT-11 did not significantly enhance the cytotoxicity of other agents tested, including mitomycin C (MMC) and cisplatin (CDDP). Flow cytometry and dot-blot analyses showed that CPT-11 pretreatment induced an increase in the S-phase cell population with an increase of topo-II mRNA expression after 24 and 48 hr, respectively, in the esophageal and colon tumor lines. These results suggest that CPT-11 can modulate topo-11 levels to enhance the effect of topo-II inhibitors in some human tumors, and this suggests a new clinical method of topo-I and -II targeting chemotherapy for human solid tumors.

An early phase II study of a new camptothecin analog and an inhibitor of topoisomerase I, CPT-11, was conducted in 62 patients with refractory leukemia and lymphoma by four different treatment schedules in a multiinstitutional cooperative study. CPT-11 therapy resulted in four complete remissions (CRs) and three partial remissions (PRs) in 29 assessable non-Hodgkin's lymphoma (NHL) patients, one PR in three Hodgkin's disease (HD), one CR and one PR in 11 acute lymphoblastic leukemia (ALL), and one PR in 15 acute myelogenous leukemia (AML) patients. Single infusion of 200 mg/m2 every 3 to 4 weeks produced no response in both leukemia and lymphoma patients. Sixty-minute infusions of 40 mg/m2/d for 5 days every 3 to 4 weeks or for 3 days weekly produced four CRs (17%) and four PRs (17%) in 24 patients with malignant lymphoma. Sixty-minute infusions of 20 mg/m2 twice a day for 7 days every 3 to 4 weeks resulted in one CR and two PRs in 12 patients with acute leukemia. No response was seen in an acute leukemia patient by another treatment schedule. CPT-11 was effective in two (15%) of 13 primarily refractory leukemia and lymphoma cases, in two of four relapsed cases, and in seven (17%) of 41 relapsed and refractory cases. Major side effects were leukopenia (91%) and gastrointestinal (GI) (76%). CPT-11 was shown to be effective against refractory leukemia and lymphoma, and thus deserves further clinical study; the novel antitumor activity mode of this drug predicts no cross-resistance to presently available antitumor drugs.