Irene Hatzinisiriou

Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast.

We have developed a method for monitoring autophagy using Rosella, a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Its mode of action relies upon differences in pH between different cellular compartments and the vacuole. Here we demonstrate its utility in yeast (Saccharomyces cerevisiae) by expression in the cytosol, and targeting to mitochondria or to the nucleus. When cells were cultured in nitrogen depleted medium, uptake of the compartment labelled with the biosensor (i.e. cytosol, mitochondria, or nucleus) into the vacuole was observed. We showed that this vacuolar uptake was, for cytosol and mitochondria, an ATG8-dependent process while the uptake of the nucleus was significantly reduced in the absence of Atg8p and can be said to be partially ATG8-dependent. We further demonstrated the value of the biosensor as a reporter of autophagy by employing fluorescence-activated cell sorting of discrete populations of cells undergoing autophagy.

Enhancing CTL responses to melanoma cell vaccines in vivo : synergistic increases obtained using IFNγ primed and IFNβ treated B7-1 + B16-F10 melanoma cells

Sequentially treating human melanoma cell lines by priming with interferon‐gamma before adding interferon‐beta was previously found to be the most efficient protocol for producing concurrently increased expression of the three surface antigens B7‐1, intercellular adhesion molecule‐1 and human histocompatibility leucocyte antigens Class I. The present study describes similar outcomes when the same sequential intercellular adhesion molecule‐based protocol is applied to murine B16‐F10 melanoma cells as well as preclinical studies using the B16‐F10 model as a poorly immunogenic melanoma. Thus, treating B16‐F10 cells or a highly expressing B7‐1 transfected subline (B16‐F10/B7−1 hi) by priming with interferon‐gamma for 24 h before adding interferon‐β for a further 48 h (interferon‐gamma 72/beta 48) increased expression of all three surface antigens, particularly major histocompatibility complex class I whose increased expression was sustained for several days. As a whole tumour cell vaccine, interferon‐gamma 72/beta 48 treated B16‐F10 cells produced greater levels of cytoxic T lymphocyte response compared to vaccines prepared from cells treated with a single type of interferon. Furthermore, B16‐F10 cells expressing high levels of B7‐1 and treated using the interferon‐gamma 72/beta 48 protocol (interferon‐gamma 72/beta 48‐treated B16‐F10/B7−1 hi) produced substantially increased cytoxic T lymphocyte responses with a fivefold greater synergy than the combined results of either interferon treated or B7‐1 expressing cells tested individually. The resulting CD8+ cytoxic T lymphocyte showed greater specificity for B16‐F10 cells with tenfold higher killing than for syngeneic EL‐4 lymphoma cells. Killing proceeded via the perforin‐mediated pathway. CTL responses were induced independent of CD4+ T helper cells. The majority of mice receiving interferon‐gamma 72/beta 48‐treated B16‐F10/B7−1 hi vaccine in vivo remained tumour free after challenge with 5 × 105 live B16‐F10 cells expressing intermediate B7‐1 levels. The novel strategy described will help enhance vaccine potency when applied clinically to prepare whole cell based cancer vaccine therapies.

SLIMMER (FHL1B/KyoT3) Interacts with the Proapoptotic Protein Siva-1 (CD27BP) and Delays Skeletal Myoblast Apoptosis

The fhl1 gene encoding four-and-a-half LIM protein-1 (FHL1) and its spliced isoform, SLIMMER, is mutated in reducing body myopathy, X-linked myopathy with postural muscle atrophy, scapuloperoneal myopathy, and rigid spine syndrome. In this study we have identified a novel function for SLIMMER in delaying skeletal muscle apoptosis via an interaction with the proapoptotic protein Siva-1. Siva-1 was identified as a SLIMMER-specific-interacting protein using yeast two-hybrid screening, direct-binding studies, and glutathione S-transferase pulldown analysis of murine skeletal muscle lysates. In C2C12 skeletal myoblasts, SLIMMER and Siva co-localized in the nucleus; however, both proteins exhibited redistribution to the cytoplasm following the differentiation of mononucleated myoblasts to multinucleated myotubes. In sections of mature skeletal muscle from wild type mice, SLIMMER and Siva-1 co-localized at the Z-line. SLIMMER and Siva-1 were also enriched in Pax-7-positive satellite cells, muscle stem cells that facilitate repair and regeneration. Significantly, SLIMMER delayed Siva-1-dependent apoptosis in C2C12 myoblasts. In skeletal muscle sections from the mdx mouse model of Duchenne muscular dystrophy, SLIMMER and Siva-1 co-localized in the nucleus of apoptotic myofibers. Therefore, SLIMMER may protect skeletal muscle from apoptosis.

Lack of correlation between MYCN expression and the Warburg effect in neuroblastoma cell lines

BackgroundMany cancers preferentially meet their energy requirements through the glycolytic pathway rather than via the more efficient oxidative phosphorylation pathway. It is thought that this is an important adaptation in cancer malignancy. We investigated whether use of glycolysis for energy production even in the presence of oxygen (known as the Warburg effect) varied between neuroblastoma cell lines with or without MYCN amplification (a key indicator of poor disease outcome in neuroblastoma).MethodsWe examined ATP and lactate production, oxygen consumption and mitochondrial energisation status for three neuroblastoma cell lines with varying degrees of MYCN amplification and MYCN expression.ResultsWe found no correlation between MYCN expression and the Warburg effect in the cell lines investigated.ConclusionOur results suggest preferential use of glycolysis for energy production and MYCN expression may be independent markers of neuroblastoma malignancy in vitro if not in vivo.

Use of cytokines in cancer vaccines/immunotherapy: recent developments improve survival rates for patients with metastatic malignancy.

Historically, patients with disseminated cancer have had poor prognoses and chemotherapy has been of little benefit. However, several different avenues of clinical research are providing reasons for hope. The advent of cytokine immunotherapy, particularly in combination with chemotherapy (biochemotherapy) has seen significantly improved outcomes for metastatic disease. Early biochemotherapy trials often revealed more than 20% complete responses. Unfortunately, Phase III trials have not confirmed earlier expectations for reasons that are not clear, but may reflect the inclusion of patients with refractory brain or other metastases in later trials. More recently, cancer vaccine therapies have provided significantly improved patient survival rates. It is not uncommon for 5-year survival rates of post-surgical patients recovering from metastatic malignancy who receive cancer vaccine therapy to reach more than 50%. Cytokines have become an integral part of cancer therapy and are also under trial together with cancer vaccines as post-surgical adjuvant therapies providing significant gains in long term survival rates. New insights from several different areas of research into the properties of tumour cells and their significance for immunosurveillance point to the importance of the tumour cells themselves as antigen presenting cells. Recent developments with genetically deficient animals and cancer cells have provided greater understanding at the molecular level of the importance of a functioning antigen presenting system operating inside tumour cells. This new knowledge offers support for further enhancing patient survival by combining previous therapies such as use of cytokines in biochemotherapy together with immunization using cytokine activated whole cell cancer vaccines in the future.