Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus

Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated beta-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between T0 and T7 for Mad-1 and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-1 and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.     

Imatinib inhibits various types of activating mutant kit found in gastrointestinal stromal tumors

Mutations of proto-oncogene c-KIT in gastrointestinal stromal tumors (GISTs) are considered to cause a constitutive activation of KIT responsible for their oncogenesis. Imatinib has therapeutic potential for GISTs because of its inhibitory effect on KIT kinase activity. To investigate the effect of Imatinib on various c-KIT mutations found in GISTs, we examined kinase activity of KIT, cell proliferation and tumorigenicity of transfectants with various c-KIT mutations. Murine lymphoid Ba/F3 cells transfected with one of the three types of mutants (KIT(del559-560), KIT(642Glu), and KIT(820Tyr)) or wild-type KIT were used for the experiments. Phosphorylation of KIT, mitogen-activated protein (MAP) and Akt was studied by immunoblotting with or without immunoprecipitation. In vitro studies on cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylcetrazolium bromide colorimetric assay and in vivo tumorigenicity assay using nude mice were also carried out. Imatinib could inhibit the KIT, MAP and Akt phosphorylation of all the transfectants but had a weaker effect on KIT(820Tyr). Imatinib potently inhibited the proliferation of cells transfected with KIT(820Tyr) at the concentration of 10 microM whereas it inhibited the other 3 types at 1 microM. Moreover, Imatinib could inhibit the tumor formation in nude mice transplanted with transfectants. In various types of activating mutant KIT, Imatinib could inhibit the constitutive activation of KIT signal transduction and cell proliferation both in vitro and in vivo although the effect of Imatinib on KIT(820Tyr) was weaker than that on KIT(del559-560) or KIT(642Glu).     

Dietary factors and the risk of gastric cancer among Japanese women: a comparison between the differentiated and non-differentiated subtypes

PURPOSE: Using data from the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), a case-referent study was performed to clarify whether dietary habits differentially impact on the risk of female gastric cancers of different histological subtypes. METHODS: The study subjects comprised 508 histologically confirmed female gastric cancer cases (156 differentiated, 352 non-differentiated), identified via hospital cancer registry and surgical records between 1988 to 1998. The referents were 36,490 cancer-free first-visit female outpatients over 30 years old presenting at the center within the same period of time. The odds ratios were estimated by logistic regression analyses and adjusted for potential confounding factors. RESULTS: Frequent vegetable and fruit conferred reduced risk of both differentiated and non-differentiated female gastric cancers in similar patterns. In addition, reduced risk was observed for intake of fish and soybean products, particularly for the non-differentiated type. CONCLUSIONS: These results suggest that both differentiated and non-differentiated female gastric cancers are preventable by frequent intake of vegetable and fruit, fish and soybean products, suggested to be common protective factors, although the possibility of some variation in the impact of the diet on different histopathological entities needs to be further addressed.     

Comprehensive evaluation of canine renal papillary necrosis induced by nefiracetam,a neurotransmission enhancer

The effects of nefiracetam, a neurotransmission enhancer, on renal biochemistry and morphology with toxicokinetic disposition were investigated in both in vivo and in vitro systems. In the in vivo studies with rats, dogs, and monkeys, only the dog exhibited renal papillary necrosis. Namely, when beagle dogs were orally administered with 300 mg/kg/day of nefiracetam over 11 weeks, decreased urinary osmotic pressure was noted from week 5, followed by increases in urine volume and urinary lactate dehydrogenase from week 8. The first morphological change was necrosis of ductal epithelia in the papilla in week 8. In toxicokinetics after 3 weeks of repeated oral administration to dogs, nefiracetam showed somewhat high concentrations in serum and the renal papilla as compared with rats and monkeys. As for metabolites, although metabolite-18 (M-18) concentration in the renal papilla of dogs was between that in rats and monkeys, the concentration ratios of M-18 in the papilla to cortex and papilla to medulla were remarkably high. In the in vitro studies, while nefiracetam itself showed no effects on the synthesis of prostaglandin E2 and 6-keto-prostaglandin F1alpha, a stable metabolite of prostaglandin I2, in canine renal papillary slices, only M-18 among the metabolites clearly decreased both prostaglandin syntheses. The basal prostaglandin synthesis in canine renal papillary slices was extremely low relative to those in rats and monkeys. Taken together, certain factors such as basal prostaglandin synthesis, M-18 penetration into the renal papilla leading to an intrarenal gradient, and inhibitory potential of M-18 on prostaglandin synthesis were considered to be crucial for the occurrence of renal papillary necrosis in dogs.     

A relationship between the mevalonate pathway and isoprenoid production in actinomycetes

Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate. In addition to this pathway, some Strepromyces strains have the mevalonate pathway to produce terpenoid antibiotics. We have previously shown that a gene cluster for biosynthesis of terpentecin, a diterpene antibiotic, was located in adjacent the mevalonate pathway gene cluster. In this study, a mevalonate pathway gene cluster was cloned from Actinoplanes sp. strain A40644, an isoprenoid antibiotic BE-40644 producer, to examine whether the mevalonate pathway genes and isoprenoid biosynthetic genes are clustered in genomic DNA. By sequencing flanking regions a probable BE-40644 biosynthetic gene cluster was found in the downstream region of the mevalonate pathway gene cluster. Heterologous expression of a 9-kb fragment confirmed that a set of the BE-40644 biosynthetic genes was involved in the fragment. This result suggested that the presence of the mevalonate pathway might be a good landmark to detect the production of isoprenoid compounds by actinomycetes.     

Novel benzylidene-9(10H)-anthracenones as highly active antimicrotubule agents. Synthesis,antiproliferative activity,and inhibition of tubulin polymerization

A novel series of 10-benzylidene-9(10H)-anthracenones and 10-(phenylmethyl)-9(10H)-anthracenones were synthesized and evaluated for antiproliferative activity in an assay based on K562 leukemia cells. The 3-hydroxy-4-methoxybenzylidene analogue 9h was found to be the most active compound (IC(50) K562: 20 nM). Structure-activity relationships are also considered. The highly active compound 9h and the 2,4-dimethoxy-3-hydroxybenzylidene analogue 9l were tested against five tumor cell lines using the XTT assay, including multidrug resistant phenotypes. Induction of cell death in a variety of tumor cell lines was determined in a monolayer assay using propidium iodide. Noteworthy, all compounds within the series induced elongations in K562 cells similar to vinblastine-treated cells. The effect of the lead compound 9h on K562 cell growth was associated with cell cycle arrest in G2/M. Concentrations for 50% KB/HeLa cells arrested in G2/M after treatment with 9h and 9l were determined and found to be in the range of 0.2 microM. Additionally, we monitored the dose dependent caspase-3-like protease activity in K562 cells and MCF-7/Casp-3 cells treated with 9h, indicating induction of apoptosis. Western blotting analysis demonstrated that 9h caused a shift in tubulin concentration from the polymerized state found in the cell pellet to the unpolymerized state found in the cell supernatant. Seven compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds such as colchicine, podophyllotoxin, and nocodazole. In general, the antiproliferative activity correlated with inhibition of tubulin polymerization. The most active compounds strongly displaced [(3)H]colchicine from its binding site in the tubulin, yielding IC(50) values 3- to 4-fold lower than that of colchicine. The novel benzylidene-9(10H)-anthracenones described in the present study constitute an interesting group of highly active and easily accessible antimitotic agents that inhibit tubulin polymerization.     

Anti-angiogenic and anti-tumor activities of isoflavonoids from the rhizomes of Belamcanda chinensis

The present study was carried out to clarify whether tectorigenin and tectoridin isolated from the rhizomes of Belamcanda chinensis (Iridaceae) inhibit angiogenesis by the experimental methods in vitro and in vivo. Tectorigenin and tectoridin decreased angiogenesis of both chick embryos in the chorioallantoic membrane assay and basic fibroblast growth factor-induced vessel formation in the mouse Matrigel plug assay. Both compounds also reduced the proliferation of calf pulmonary arterial endothelial (CPAE) cells and found to possess relatively weak gelatinase/collagenase inhibitory activity in vitro. Tectorigenin exhibited a much stronger anti-proliferative activity than its glycoside, tectoridin and was almost equipotent to that of genistein, a reference drug. Tectorigenin, when administered subcutaneously at the dose of 30 mg/kg for 20 days to mice implanted with murine Lewis lung carcinoma (LLC), caused a significant inhibition of tumor volume by 30.8 %. Tectorigenin and tectoridin, when treated i. p. at the same dosage for 10 days to ICR mice bearing sarcoma 180, caused a significant suppression in tumor weight by 44.2 and 24.8 %, respectively.     

Protective effects of glutathione on 5-fluorouracil-induced myelosuppression in mice

The protective effects of glutathione (GSH) administration on myelosuppression induced by 5-fluorouracil (5-FU) were investigated in female BALB/c mice. Animals were allocated to four groups (16 mice/group). GSH was given orally at a dose of 800 mg/kg to groups 3 and 4 for 21 consecutive days (day 0 to day 20). 5-FU was repeatedly administered at a dose of 40 mg/kg to groups 2 and 3 for 1 week (day 7 to day 13) by gavage. Group 3 served as a combined treatment group and group 1 as a non-treated control group. The total observation period was 3 weeks. Body weight was measured once a week. A decrease in body weight due to 5-FU treatment was observed in groups 2 and 3 on day 14. Although the body weight in group 2 had not increased by 1-week after cessation of 5-FU treatment, the value in group 3 markedly recovered. Hematology, total nucleated myelocyte count and histopathology of bone marrow were carried out on day 14 and day 21. In groups 2 and 3, these examinations showed thrombocytopenia, leukopenia, reticulocytopenia and myelosuppression on day 14. However, platelets and bone marrow were less affected in group 3 than in group 2. On day 21, the thrombocytopenia in groups 2 and 3 was resolved. The myelosuppression, leukopenia and reticulocytopenia resolved in group 3, but not in group 2. Although simple microcytic anemia occurred delayed on day 21, it was less severe in group 3 than in group 2. Therefore, GSH may have preventive effects against 5-FU-induced hematopoietic toxicity, and accelerate recovery after cessation of 5-FU treatment.     

Attenuation of arbekacin-induced nephrotoxicity in rats by pazufloxacin mesilate

The protective effect of pazufloxacin (PZFX) mesilate, a parenteral quinolone antimicrobial agent, on arbekacin (ABK)-induced nephrotoxicity was evaluated with 8-week-old male Sprague-Dawley rats. Animals were injected with ABK at a dose of 32 mg/kg intramuscularly, or a combination of ABK in the same manner with PZFX mesilate at a dose of 208 mg/kg (160 mg/kg convert to PZFX, active principle of PZFX mesilate) intravenously once a day for 4 days. In consequent, ABK induced increases in protein, beta 2-microglobulin and N-acetyl-beta-(D)-glucosaminidase in urine, and histopathological phospholipidosis in kidneys. The extent of these changes was reduced when ABK was given in a combination with PZFX mesilate. Renal cortex level of ABK increased after an administration of ABK 1 hour to 4 hours; however, the increase was suppressed by coadministration of PZFX mesilate. Taken together, these results suggest that PZFX mesilate has the protective effect on ABK-induced nephrotoxicity, and that this was attributable to a suppression of uptake of ABK in cortical renal tubules.     

Comparative studies on activities of antimicrobial agents against causative organisms isolated from patients with urinary tract infections (2001). III. Secular changes in susceptibility

The bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa) isolated from patients diagnosed as urinary tract infections (UTIs) in 10 institutions in Japan were supplied between September and December, 2001. Then, the susceptibilities of these bacteria to various antimicrobial agents were examined, and the results were compared with those obtained between 1992 and 2000. Comparison was made by classifying strains isolated from patients into those in uncomplicated UTIs and those in complicated UTIs (including with or without indwelling catheter). The drug sensitivity of S. aureus in this year was comparable to those in up to the previous year, and S. aureus showed the best susceptibility to vancomycin (VCM). E. faecalis showed good susceptibility to ampicillin and imipenem, and the MIC90s were 2 micrograms/mL. The susceptibility of E. faecalis to VCM was also good. E. coli showed good susceptibility to the drugs except penicillins. Among cephems, the susceptibility to cefozopran (CZOP) was better (MIC90: < or = 0.125 microgram/mL). Just as the last report, the decreases in susceptibility of E. coli to quinolones were also observed in the patients with complicated UTIs. The susceptibility of Klebsiella spp. to all the test drugs did not significantly change in 2001 and was generally good but not to penicillins. Among cephems, Klebsiella spp. showed good susceptibility to flomoxef, cefpirome, cefixime, and CZOP with < or = 0.125 microgram/mL of MIC90s either in uncomplicated or complicated UTIs. Although the drug sensitivity of P. aeruginosa was generally low, the detection of the strains that showed good susceptibility to quinolones and carbapenems (MIC: < or = 0.125-2 micrograms/mL) were relatively frequent.