Silver nanospheres are cytotoxic and genotoxic to fish cells.

Nanoparticles are being widely investigated for a range of applications due to their unique physical properties. For example, silver nanoparticles are used in commercial products for their antibacterial and antifungal properties. Some of these products are likely to result in silver nanoparticles reaching the aquatic environment. As such, nanoparticles pose a health concern for humans and aquatic species. We used a medaka (Oryzias latipes) cell line to investigate the cytotoxicity and genotoxicity of 30nm diameter silver nanospheres. Treatments of 0.05, 0.3, 0.5, 3 and 5microg/cm(2) induced 80, 45.7, 24.3, 1 and 0.1% survival, respectively, in a colony forming assay. Silver nanoparticles also induced chromosomal aberrations and aneuploidy. Treatments of 0, 0.05, 0.1 and 0.3microg/cm(2) induced damage in 8, 10.8, 16 and 15.8% of metaphases and 10.8, 15.6, 24 and 24 total aberrations in 100 metaphases, respectively. These data show that silver nanoparticles are cytotoxic and genotoxic to fish cells.

Development of fragment-specific osteopontin antibodies and ELISA for quantification in human metastatic breast cancer.

BACKGROUND: Osteopontin (OPN) is associated with human cancers, and circulating blood OPN may have diagnostic or prognostic value in clinical oncology. METHODS: To evaluate OPN as a cancer biomarker, we generated and characterized five novel mouse monoclonal antibodies against the human full-length OPN (fl-OPN). Epitopes recognized by four antibodies (2C5, 2F10, 2H9, and 2E11) map to N-terminal OPN (aa1-166); one (1F11) maps to C-terminal OPN (aa167-314). These antibodies recognize recombinant and native OPN by ELISA and immunoblot, cross reacting with human and mouse OPN. Two of these novel antibodies (2F10 and 1F11) were used to develop a quantitative enzyme linked immunosorbent assay (ELISA) for fl-OPN. RESULTS: In comparison with commercially available ELISAs, our assay had high accuracy in measuring fl-OPN standards, and high sensitivity. Specifically, our ELISA has a linear dose response between 0.078 ng/ml-10 ng/ml, with a sensitivity of 13.9 pg/ml. We utilized this assay to quantify fl-OPN in the plasma of healthy volunteers in comparison with patients with metastatic breast cancer. The average circulating plasma fl-OPN in healthy volunteers was 1.2 ng/ml, compared to 4.76 ng/ml in patients with metastatic breast cancer (p = 0.0042). Although the increase in fl-OPN in cancer patients is consistent with previous studies, the measured quantity varied greatly between all existing fl-OPN ELISAs. CONCLUSION: Because OPN is a complex molecule with diversity from alternative splicing, post-translational modification, extracellular proteolytic modification, and participation in protein complexes, we suggest that further understanding of specific isoform recognition of multiple OPN species is essential for future studies of OPN biomarker utility.

Widespread delta-like-1 expression in normal adult mouse tissue and injured endothelium is reflected by expression of the Dll1LacZ locus.

BACKGROUND: Our study characterizes Delta-like 1 (Dll1) in the adult mouse, particularly in normal versus injured vasculature, with the aid of the transgenic Dll1(LacZ) line. METHODS: Normal mouse adult tissues or those from the Dll1(LacZ) reporter line were analyzed for Dll1 expression and promoter activity. Vascular tissue was analyzed before and after carotid artery ligation. RESULTS: In wild-type mice, Dll1 transcript expression was widespread. Similarly, the Dll1(LacZ) reporter had beta-galactosidase activity detectable in the cerebellum, cerebrum, spinal cord, liver, lung and cornea, although the normal adult vasculature had no reporter expression. Following arterial ligation, there was acute induction of Dll1(LacZ) reporter expression, both in the ligated left carotid artery, and the uninjured right contralateral artery. Expression returned to low/undetectable levels 4-10 days after arterial ligation. CONCLUSION: The expression of Dll1 in the adult mouse is more widespread than previously realized, although not in resting large arteries in the adult mouse. Following arterial injury, Dll1 promoter activity is induced selectively in the endothelial cells of both the injured artery and the contralateral uninjured artery. Our results show that while overall expression in the adult mouse is widespread, Dll1 may be selectively expressed in the endothelium of injured vasculature, similar to the endothelial-restricted expression of Dll4.

Prevention of TGF-beta-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways.

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-beta (TGF-beta). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-beta-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-beta-induced apoptosis.

Activation of EDTA-resistant gelatinases in malignant human tumors.

Among the many proteases associated with human cancer, seprase or fibroblast activation protein alpha, a type II transmembrane glycoprotein, has two types of EDTA-resistant protease activities: dipeptidyl peptidase and a 170-kDa gelatinase activity. To test if activation of gelatinases associated with seprase could be involved in malignant tumors, we used a mammalian expression system to generate a soluble recombinant seprase (r-seprase). In the presence of putative EDTA-sensitive activators, r-seprase was converted into 70- to 50-kDa shortened forms of seprase (s-seprase), which exhibited a 7-fold increase in gelatinase activity, whereas levels of dipeptidyl peptidase activity remained unchanged. In malignant human tumors, seprase is expressed predominantly in tumor cells as shown by in situ hybridization and immunohistochemistry. Proteins purified from experimental xenografts and malignant tumors using antibody- or lectin-affinity columns in the presence of 5 mmol/L EDTA were assayed for seprase activation in vivo. Seprase expression and activation occur most prevalently in ovarian carcinoma but were also detected in four other malignant tumor types, including adenocarcinoma of the colon and stomach, invasive ductal carcinoma of the breast, and malignant melanoma. Together, these data show that, in malignant tumors, seprase is proteolytically activated to confer its substrate specificity in collagen proteolysis and tumor invasion.

IL-4 suppresses the expression and the replication of hepatitis B virus in the hepatocellular carcinoma cell line Hep3B.

IL-4 has been known as a Th2 cytokine and can act on B cells, T cells, and monocytes. In this study we demonstrate that IL-4Rs are expressed on human hepatocellular carcinoma (HCC) cells. We found that IL-4 suppresses hepatitis B surface Ag (HBsAg) mRNA and HBsAg production in the Hep3B cell line, which contains an integrated hepatitis B virus (HBV) genome and constitutively secretes HBsAg. When Hep3B cells are further transfected with the plasmid pHBV3.6 that contains >1 U of HBV genome, IL-4 could suppress the production of all HBV RNA and secreted HBsAg and hepatitis B virus e Ag. Furthermore, an endogenous DNA polymerase activity assay shows a decrease in HBV DNA after IL-4 treatment. Using luciferase reporter assays we have demonstrated that IL-4 could suppress the activity of the surface promoter II and the core promotor (CP). To delineate how IL-4 suppressed the transcription of HBV genes, we have examined the effect of IL-4 on the expression of transcription factors that are known to bind to the core upstream regulatory sequence, which colocalizes with enhancer II of the HBV genome. Our results demonstrate that IL-4 suppresses the expression of C/EBPalpha. Furthermore, overexpression of C/EBPalpha blocked 43 and 30% of the IL-4-mediated suppression of CP activity and IL-4-induced suppression of pregenomic RNA, respectively. Finally, we have demonstrated that mutations affecting the C/EBPalpha-binding sites on core upstream regulatory sequence/enhancer II completely abolish the IL-4-mediated suppression of CP activity. Thus, down-regulation of C/EBPalpha may be involved in the anti-HBV effect of IL-4 in Hep3B cells.