Session 2: The U.S. Perspective

This panel provides an overview of the current state of protection of moral rights in the United States, including discussion of the “patchwork” approach of federal and state laws, as well as judicial opinions

Non-Coding Keratin Variants Associate with Liver Fibrosis Progression in Patients with Hemochromatosis

Background: Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with endstage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods: The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCRamplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previouslygenerated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results: We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. I

Origin of myofibroblasts in liver fibrosis

Most chronic liver diseases of all etiologies result in progressive liver fibrosis. Myofibroblasts produce the extracellular matrix, including type I collagen, which constitutes the fibrous scar in liver fibrosis. Normal liver has little type I collagen and no detectable myofibroblasts, but myofibroblasts appear early in experimental and clinical liver injury. The origin of the myofibroblast in liver fibrosis is still unresolved. The possibilities include activation of endogenous mesenchymal cells including fibroblasts and hepatic stellate cells, recruitment from the bone marrow, and transformation of epithelial or endothelial cells to myofibroblasts. In fact, the origin of myofibroblasts may be different for different types of chronic liver diseases, such as cholestatic liver disease or hepatotoxic liver disease. This review will examine our current understanding of the liver myofibroblast

ROS release by PPARβ/δ-null fibroblasts reduces tumor load through epithelial antioxidant response.

Tumor stroma has an active role in the initiation, growth, and propagation of many tumor types by secreting growth factors and modulating redox status of the microenvironment. Although PPARβ/δ in fibroblasts was shown to modulate oxidative stress in the wound microenvironment, there has been no evidence of a similar effect in the tumor stroma. Here, we present evidence of oxidative stress modulation by intestinal stromal PPARβ/δ, using a FSPCre-Pparb/d <sup>-/-</sup> mouse model and validated it with immortalized cell lines. The FSPCre-Pparb/d <sup>-/-</sup> mice developed fewer intestinal polyps and survived longer when compared with Pparb/d <sup>fl/fl</sup> mice. The pre-treatment of FSPCre-Pparb/d <sup>-/-</sup> and Pparb/d <sup>fl/fl</sup> with antioxidant N-acetyl-cysteine prior DSS-induced tumorigenesis resulted in lower tumor load. Gene expression analyses implicated an altered oxidative stress processes. Indeed, the FSPCre-Pparb/d <sup>-/-</sup> intestinal tumors have reduced oxidative stress than Pparb/d <sup>fl/fl</sup> tumors. Similarly, the colorectal cancer cells and human colon epithelial cells also experienced lower oxidative stress when co-cultured with fibroblasts depleted of PPARβ/δ expression. Therefore, our results establish a role for fibroblast PPARβ/δ in epithelial-mesenchymal communication for ROS homeostasis

Molecular mechanisms of response of human peripheral blood mononuclear cells to ionizing radiation

Introduction: The aim of this study was to compare reaction of quiescent and proliferating PHA (mitogenic lectin phytohemagglutinin)-stimulated human peripheral blood mononuclear cells (PBMCs) to γ-radiation (IR) and analyze changes of proteins related to repair of DNA damage and apoptosis, such as γH2A.X, p53 and its phosphorylations on serine 15 and 392, and p21. Materials and methods: PBMC were acquired from venous blood of normal donors by centrifugation over Histopague. The lymphocytes were cultured in IMDM medium suplemented with 20% fetal calf serum and antibiotics. For mitogenic stimulation of lymphocytes was added 10 μg/ml PHA. The viability of the PBMC has been identified by flow cytometry. Western blotting has been used for p53 and its phosphorylation form, p21 and H2AX detection. T-lymphocytes and PBMC were irradiated and lysed. To enhance detection sensitivity the Total p53 and PathScan p53ser15 Sandwich ELISA Kits were used. Results: PBMCs were stimulated to enter the cell cycle by treatment with the PHA. After 72 h long stimulation by PHA 96 % of CD3+cells were CD25+ and 12 % entered cell cycle (S+ and G2 phase). PHA-stimulation itself causes increase in γH2A.X, p53, and p21, but not phosphorylation of p53. After the irradiation of these stimulated PBMCs we detected increase in p53 and its phosphorylations on serine 15 and 392, and further increase in p21 from 4 h after the irradiation. Also level of γH2A.X increased significantly. Increase of p53 phosphorylation on serine 15 is dose-dependent 4 h after the irradiation in the whole studied dose range (0.5-7.5 Gy) in stimulated PBMCs. We compared p53, p53ser15 and ser392, p21 and H2AX between grouply of phytohemaglutinin-stimulated and non-stimulated PBMC 4 hours after 0,5-7,5Gy as well as 1-72 hours after 4Gy irradiation.. We were unable to detect p53 accumulation or phosphorylation in response unstimulated PBMC to gamma radiation induced DNA damage. We induced cell cycling by phytohemaglutinin in the T-cell fraction of PMBC preparations. Althougly total p53 protein expression was unchanged after 4 Gy irradiation, we found p53ser392 time-dependent expression (1-72 hours, 4 Gy) and p53ser15 dose-dependent expression in range of 0,5-7,5 Gy 4 hours after irradiation in phytohemaglutinin- stimulated PBMC. Neither total p53 protein nor p53ser392 was not altered by irradiation in non-stimulated PBMC. Conclusions: We suggested that p53 protein accumulation is a common mechanism for induction apoptosis of irradiated cells. We suggest that this pathway is activated in proliferating lymphocytes, unlike quiescent lymphocytes

Fractionated dose of 60 Gy changes molecular response of HL-60 cells to irradiation

Objective: The cells of human promyelocyte leukaemia HL-60 lack functional protein p53 and react to ionizing radiation in doses up to 10 Gy by long cell cycle arrest in G2 phase, during which they repair radiation-induced DNA damage. Abrogation of G2 cell cycle arrest has radiosensitizing effect. In this work we evaluated changes in molecular response of HL-60-R cells (HL-60 irradiated by 10 cycles of radiation with total dose of 60 Gy, given over a period of 3 months) to irradiation by the dose of 2 and 8 Gy. Results: Both types of cells (HL-60 and HL-60-R) have high basal level of ERK1/2 phosphorylated on serine 202/204. This corresponds with their quick proliferation. Irradiation by the doses of 2 and 8 Gy induces decrease of ERK1/2 phosphorylation after 4 h in both cell types. However, after irradiation by the dose of 2 Gy in HL-60-R cells ERK1/2 phosphorylation is restored after 24 h, while in HL-60 cells the decrease is longer and ERK1/2 phosphorylation is restored only after 72 h. This may be related to different repair capacity, as the dose of 2 Gy is not lethal. This dose also induces in HL-60-R cells upregulation of cdk inhibitor p21, which is not detectable in HL-60 cells. Increased p21 is responsible for cell cycle arrest. On the other hand, in HL-60 cells the phosphorylation of check-point kinase 2 (chk-2) on threonine 68 occurs, while it is not observed in HL-60-R cells. Apoptosis induction by the dose of 8 Gy was lower in HL-60-R then in HL-60 cells. No difference in expression of anitiapoptotic mitochondrial protein Mcl-1 was found. Conclusion: In contrary to HL-60 cells, the HL-60 irradiated by 10 cycles of radiation with total dose of 60 Gy, given over a period of 3 months (HL-60-R) react to irradiation by p53 independent increase in p21, and not by activation of chk-2. Also kinetics of Erk1/2 phosphorylation is different in these cell types. HL-60-R are more resistant to radiation-induced apoptosis, but significant difference in D0 was not found (HL-60 2.5 Gy, HL-60-R 2.6 Gy). P21 might prevent apoptosis induction and trigger permanent cell-cycle arrest, which can also contribute to regression of this leukemia after therapy