Analysis of mechanisms contributing to AraC-mediated chemoresistance and re-establishment of drug sensitivity by the novel heterodinucleoside phosphate 5-FdUrd-araC

Chemoresistance is a biological response of cells to survive toxic stress. During cancer treatment the development of chemoresistance is a major problem. The mechanisms how cells become insensitive, and which downstream pathways are affected are not completely understood. Since it has not been well analysed which and how many regulative disorders are subsummised under the term “chemoresistance”, we examined and measured arabinosylcytosine (AraC)-mediated desensitation of two mechanisms relevant for tissue homeostasis, cell cycle inhibition and apoptosis induction. MCF-7 cells harbouring ectopic mutated p53 were suitable for this investigation because they activated these mechanisms subsequently and became insensitive to AraC with regard to cell cycle inhibition and apoptosis induction. The major causal mechanism of acquired resistance against AraC was most likely through the inhibition of the first step of AraC phosphorylation within the cell, which is rate limiting for its activation. With regard to cell cycle inhibition AraC-resistant cells were also resistant against 5-fluorodeoxyuridine (5-FdUrd), but fully responsive to 5-FdUrd-induced apoptosis, evidencing that cell cycle and apoptosis are independent of each other. Apoptosis correlated with AIF-activation and was independent of Caspase 7, whereas cell cycle inhibition correlated with cyclinD1 expression but not with induction of p21 or p27. The phosphate conjugated 5-FdUrd-araC heterodimer (5-Fluoro-2′-desoxyuridylyl-(3′→5′)-Arabinocytidine), which is a prodrug of AraC-monophosphate, reactivated AIF and down-regulated cyclin D1 in AraC-resistant cells and circumvented resistance to apoptosis and to cell cycle inhibition. Also, cells which were resistant to 5-FdUrd or doxorubicin were sensitive to 5-FdUrd-araC. This investigation demonstrates that chemoresistance affects apoptosis induction and cell cycle inhibition independently and that detailed knowledge about the affected downstream pathways would enable the design of targeted intervention with small molecules to restore chemosensitivity. The project was funded by the Jubilaeumsfonds of the Austrian Nationalbank (No.: 10843) to M. F.-S.     

Effects of Feed Contaminant Deoxynivalenol on Plasma Cytokines and mRNA Expression of Immune Genes in the Intestine of Broiler Chickens

An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1β, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κβ1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1β, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1β, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κβ1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes.     

Living roots magnify the response of soil organic carbon decomposition to temperature in temperate grassland

Increasing atmospheric carbon dioxide (CO₂) concentration is both a strong driver of primary productivity and widely believed to be the principal cause of recent increases in global temperature. Soils are the largest store of the world's terrestrial C. Consequently, many investigations have attempted to mechanistically understand how microbial mineralisation of soil organic carbon (SOC) to CO₂ will be affected by projected increases in temperature. Most have attempted this in the absence of plants as the flux of CO₂ from root and rhizomicrobial respiration in intact plant‐soil systems confounds interpretation of measurements. We compared the effect of a small increase in temperature on respiration from soils without recent plant C with the effect on intact grass swards. We found that for 48 weeks, before acclimation occurred, an experimental 3 °C increase in sward temperature gave rise to a 50% increase in below ground respiration (ca. 0.4 kg C m^?2; Q₁₀ = 3.5), whereas mineralisation of older SOC without plants increased with a Q₁₀ of only 1.7 when subject to increases in ambient soil temperature. Subsequent ¹⁴C dating of respired CO₂ indicated that the presence of plants in swards more than doubled the effect of warming on the rate of mineralisation of SOC with an estimated mean C age of ca. 8 years or older relative to incubated soils without recent plant inputs. These results not only illustrate the formidable complexity of mechanisms controlling C fluxes in soils but also suggest that the dual biological and physical effects of CO₂ on primary productivity and global temperature have the potential to synergistically increase the mineralisation of existing soil C.