Thrombopoietin stimulates migration and activates multiple signaling pathways in hepatoblastoma cells.

Thrombopoietin (TPO), a cytokine that participates in the differentiation and maturation of megakaryocytes, is produced in the liver, but only limited information is available on the biological response of liver-derived cells to TPO. In this study, we investigated whether HepG2 cells express c-Mpl, the receptor for TPO, and whether TPO elicits biological responses and intracellular signaling in this cell type. Specific transcripts for c-Mpl were detected in HepG2 cells by RT-PCR, and expression of the protein was demonstrated by Western blot analysis and immunofluorescence. Exposure of HepG2 cells to TPO was associated with a dose-dependent increase in cell migration and chemoinvasion through Matrigel-coated filters. A checkerboard analysis showed that the effects of TPO on cell migration were dependent on both chemotaxis and chemokinesis. Exposure of HepG2 cells to TPO resulted in the activation of different members of the MAPK family, including ERK and JNK, as assessed using phosphorylation-specific antibodies and immune complex kinase assays. TPO also activated phosphatidylinositol 3-kinase (PI3K) and the downstream kinase Akt in a time-dependent manner. Finally, activation of c-Mpl was associated with increased activation of nuclear factor-kappaB. With the use of specific inhibitors, tyrosine phosphorylation and activation of PI3K were found to be required for the induction of migration in response to TPO. We conclude that TPO exerts biological actions on cultured hepatoblastoma cells via activation of c-Mpl and its downstream signaling.

In vitro antileukemic effect of a new anthracycline analogue, MEN 11079.

The biological activity of MEN 11079, a new daunorubucin analogue with a fluorine atom in C(8) of ring A, was investigated in the human leukemic cell lines K-562 and in mononuclear cells (MNCs) of 40 patients with acute myeloid leukemia (AML) and the activity compared to two well-characterized anthracyclines, idarubicin (IDA) and doxorubicin (DOXO). IDA and MEN 11079 were more active than DOXO in cytotoxicity tests (WST-1 assay). IDA and MEN 11079 ID(50) values were also significantly different from each other (K-562: P=0.038; MNCs: P=0.003). Moreover, the range was 0.002-4.300 microM for IDA and 0.002-0.670 microM for MEN 11079, in the MNCs. Therefore, the latter appeared to assure a smaller variability of response in the AML cells. Apoptosis assays (performed using Annexin-V assay and propidium iodide) and cell cycle studies demonstrated that the MEN 11079 effective concentration was 10-fold lower than the DOXO and IDA ones. MDR (Pgp and MRP1 proteins), as measured by semiquantitative RT-PCR, cytofluorimetric and functional analysis of proteins, was similarly elicited by IDA and MEN 11079. In conclusion, the response of the cells to the new anthracycline indicates that there is greater cytotoxic activity of this molecule than IDA and DOXO. Its narrower ID(50) range may allow for a more predictable response in the clinical setting.

Antioxidant protection in cultured corneal cells and whole corneas submitted to UV-B exposure.

Several corneal pathologies are characterized by the presence of reactive oxygen species (ROS); therefore, we evaluated the protection afforded by pirenoxine and melatonin to corneal cell culture and whole rabbit cornea from ultraviolet exposure and other oxidant systems. Rabbit cornea cell (SIRC) plates and whole corneas were exposed to UV-B (80 or 800 mJ/cm2) or incubated with fMLP-stimulated autologous macrophages, in the presence or absence of pirenoxine or melatonin (10(-5) M). The protective activity of compounds was assessed by measuring superoxide anion formation, inhibition of oxidation and mitochondrial viability. Moreover the ex vivo protective effect of pirenoxine and melatonin was verified in the whole cornea submitted to UV-B exposure in vitro. Our experimental data demonstrate that pirenoxine and melatonin were able to inhibit the superoxide formation and oxidative effect in cell culture and whole rabbit corneas submitted to UV-B exposure or to incubation with fMLP-stimulated autologous macrophages. Mitochondrial viability was restored in epithelial cells of rabbit cornea but not in SIRCs. Moreover, both compounds are also able to increase ex vivo epithelial corneal cell defences against the in vitro UV-B induced lipid peroxidation.

Lyn kinase is activated following thrombopoietin stimulation of the megakaryocytic cell line B1647.

BACKGROUND AND OBJECTIVES: B1647 is a cell line derived from bone marrow cells of a patient with acute myeloid leukemia (M2) with a complete erythro-megakaryocytic phenotype and bears both k and p isoforms of c-mpl. Interestingly, spontaneous B1647 cell proliferation is significantly potentiated by thrombopoietin (TPO). DESIGN AND METHODS: We aimed to evaluate the proliferative signal transduction events following the activation of c-mpl and we stimulated B1647 cells with TPO 40 ng/mL for 3, 7, 15 and 30 minutes; cells were then lysed and whole lysates were immunoprecipitated with anti-phosphotyrosine antibodies. RESULTS: In our hands, TPO stimulation induced phosphorylation of several substrate proteins in B1647 cells. The increase in tyrosine phosphorylation from background spontaneous activation was transient, maximal after 10 minutes and declined to reach constitutive levels after 30 minutes. In particular, protein substrates between 50 and 140 kDa appeared to be selectively phosphorylated by TPO. We demonstrated that Jak2, Stat3 and Shc were activated in B1647 cells after TPO, as already shown for different cell lines by other authors. Moreover, Lyn kinase activation was detected. Grb2 co-immunoprecipitated with phosphorylated proteins. The phosphorylation of Syk kinase was not demonstrated, whereas Vav was activated by TPO. INTERPRETATION AND CONCLUSIONS: The pattern of protein phosphorylation determined in B1647 cells by TPO testifies the role of this cytokine in sustaining cell growth and indicates Lyn tyrosine kinase as a possible target protein in transduction of the TPO proliferative signal.

Up-regulated expression of fractalkine and its receptor CX3CR1 during liver injury in humans.

BACKGROUND/AIMS: Little is known about the role of fractalkine (CX3CL1) in the liver. The aim of this study was to investigate the expression patterns of fractalkine and its receptor CX3CR1 in normal human liver and in conditions of injury. METHODS: Distribution and expression of fractalkine and its receptor were investigated using immunohistochemistry, in situ hybridization, flow cytometry and reverse transcriptase-polymerase chain reaction. In vitro experiments were conducted in HepG2 cells. RESULTS: Both fractalkine and CX3CR1 were up-regulated during chronic injury, in areas of portal and lobular inflammation. In severe acute hepatitis, fractalkine and CX3CR1 were expressed at high levels not only in areas of inflammation but also in regenerating epithelial cells within bile duct-like structures, which showed co-expression of fractalkine and cytokeratin-7 or CX3CR1. The human hepatocarcinoma cell line HepG2 expressed fractalkine at the gene and protein level, and HepG2-conditioned medium was chemotactic for cells overexpressing CX3CR1. Transcripts for CX3CR1 were detected in HepG2, and exposure of these cells to recombinant fractalkine induced cell migration. CONCLUSIONS: This study shows that the fractalkine system is up-regulated during liver damage, and suggests that fractalkine may play a role in the recruitment and adhesion of inflammatory cells and in the biology of liver epithelial cells.