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

BACKGROUND: Many 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). METHODS: We 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. RESULTS: We found no correlation between MYCN expression and the Warburg effect in the cell lines investigated. CONCLUSION: Our 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.

The beta-amyloid protein of Alzheimer's disease increases neuronal CRMP-2 phosphorylation by a Rho-GTP mechanism.

Neuritic abnormalities are a major hallmark of Alzheimer's disease (AD) pathology. Accumulation of beta-amyloid protein (Abeta) in the brain causes changes in neuritic processes in individuals with this disease. In this study, we show that Abeta decreases neurite outgrowth from SH-SY5Y human neuroblastoma cells. To explore molecular pathways by which Abeta alters neurite outgrowth, we examined the activation and localization of RhoA and Rac1 which regulate the level and phosphorylation of the collapsin response mediator protein-2 (CRMP-2). Abeta increased the levels of the GTP-bound (active) form of RhoA in SH-SY5Y cells. This increase in GTP-RhoA correlated with an increase in an alternatively spliced form of CRMP-2 (CRMP-2A) and its threonine phosphorylated form. Both a constitutively active form of Rac1 (CA-Rac1) and the Rho kinase inhibitor, Y27632, decreased levels of the CRMP-2A variant and decreased threonine phosphorylation caused by Abeta stimulation. The amount of tubulin bound to CRMP-2 was decreased in the presence of Abeta but Y27632 increased the levels of tubulin bound to CRMP-2. Increased levels of both RhoA and CRMP-2 were found in neurons surrounding amyloid plaques in the cerebral cortex of the APP(Swe) Tg2576 mice. We found that there was an increase in threonine phosphorylation of CRMP-2 in Tg2576 mice and the increase correlated with a decrease in the ability of CRMP-2 to bind tubulin. The results suggest that Abeta-induced neurite outgrowth inhibition may be initiated through a mechanism in which Abeta causes an increase in Rho GTPase activity which, in turn, phosphorylates CRMP-2 to interfere with tubulin assembly in neurites.

Leukemia inhibitory factor arrests oligodendrocyte death and demyelination in spinal cord injury.

As a consequence of secondary pathophysiological mechanisms elicited after spinal cord injury (SCI), oligodendrocytes die by waves of apoptosis. This ultimately results in demyelination of intact axons leading to a loss of their conducting properties. Preservation of as few as 5% to 10% of myelinated axons in individual tracts can confer locomotor recovery. Thus, strategies aimed at rescuing mature oligodendrocytes ensheathing viable axons are likely to be of therapeutic significance. We report that leukemia inhibitory factor (LIF) can prevent oligodendrocyte apoptosis, notably contralateral to the spinal cord lesion, through the induction of the JAK/STAT and Akt signaling pathways as well as by potentiating the expression of the antiapoptotic molecule, cIAP2. Reduced oligodendrocyte apoptosis after SCI with LIF administration resulted in a substantial decrease in demyelination shown by the preservation of lamellated myelin surrounding viable axons and deposition of the degraded myelin basic protein. The data suggest that LIF signals survival in oligodendrocytes after SCI, prevents the secondary wave of demyelination, and thereby reduces inhibitory myelin deposits.

Enhancing CTL responses to melanoma cell vaccines in vivo: synergistic increases obtained using IFNgamma primed and IFNbeta 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-beta 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 x 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.

Isolation and characterization of a human STAT1 gene regulatory element. Inducibility by interferon (IFN) types I and II and role of IFN regulatory factor-1.

The transcription factor STAT1 plays a pivotal role in signal transduction of type I and II interferons (IFNs). STAT1 activation leads to changes in expression of key regulatory genes encoding caspases and cell cycle inhibitors. Deficient STAT1 expression in human cancer cells and virally mediated inhibition of STAT1 function have been associated with cellular resistance to IFNs and mycobacterial infection in humans. Thus, given the relative importance of STAT1, we isolated and characterized a human STAT1 intronic enhancer region displaying IFN-regulated activity. Functional analyses by transient expression identified a repressor region and type I and II IFN-inducible elements within the STAT1 enhancer sequence. A candidate IRF-E/GAS/IRF-E (IGI) sequence containing GAAANN nucleotide repeats was shown by gel shift assay to bind to IFN regulatory factor-1 (IRF-1), but not to IFN-stimulated gene factor-3 (ISGF-3) or STAT1-3. An additional larger IGI-binding complex containing IRF-1 was identified. Mutation of the GAAANN repeats within the IGI DNA element eliminated IRF-1 binding and the IFN-regulated activity of the STAT1 intronic enhancer region. Transfection of the IFN-resistant MM96 cell line to express increased levels of IRF-1 protein also elevated STAT1, STAT2, and p48/IRF-9 expression and enhanced cellular responsiveness to IFN-beta. Reciprocating regulation between IRF-1 and STAT1 genes and encoded proteins indicates that an intracellular amplifier circuit exists controlling cellular responsiveness to the IFNs.