Reduction of glyceraldehyde-3-phosphate dehydrogenase activity in Alzheimer's disease and in Huntington's disease fibroblasts

New functions have been identified for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) including its role in neurodegenerative disease and in apoptosis. GAPDH binds specifically to proteins implicated in the pathogenesis of a variety of neurodegenerative disorders including the beta-amyloid precursor protein and the huntingtin protein. However, the pathophysiological significance of such interactions is unknown. In accordance with published data, our initial results indicated there was no measurable difference in GAPDH glycolytic activity in crude whole-cell sonicates of Alzheimer's and Huntington's disease fibroblasts. However, subcellular-specific GAPDH-protein interactions resulting in diminution of GAPDH glycolytic activity may be disrupted or masked in whole-cell preparations. For that reason, we examined GAPDH glycolytic activity as well as GAPDH-protein distribution as a function of its subcellular localization in 12 separate cell strains. We now report evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite unchanged gene expression. In the postnuclear fraction, GAPDH was 27% less glycolytically active in Alzheimer's cells as compared with age-matched controls. In the nuclear fraction, deficits of 27% and 33% in GAPDH function were observed in Alzheimer's and Huntington's disease, respectively. This evidence supports a functional role for GAPDH in neurodegenerative diseases. The possibility is considered that GAPDH:neuronal protein interaction may affect its functional diversity including energy production and as well as its role in apoptosis.     

Signalling networks that cause cancer

Cancer is caused by the stepwise accumulation of mutations that affect growth control, differentiation and survival. The view that mutations affect discrete signalling pathways, each contributing to a specific aspect of the full malignant phenotype, has proved to be too simplistic. We now know that oncogenes and tumour suppressors depend on one another for their selective advantage, and that they affect multiple pathways that intersect and overlap. The interactive nature of each genetic change has important implications for cancer therapy and for the stepwise model of carcinogenesis.     

Genetic instability and darwinian selection in tumours

Genetic instability has long been hypothesized to be a cardinal feature of cancer. Recent work has strengthened the proposal that mutational alterations conferring instability occur early during tumour formation. The ensuing genetic instability drives tumour progression by generating mutations in oncogenes and tumour-suppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumour. Here, we discuss the role of genetic instability in tumour formation and outline future work necessary to substantiate the genetic instability hypothesis.     

Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone

The aim of this study was to examine the effect of three structurally different anticancer drugs-the pro-oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA-DPH and 12-AS) and spin labels (5-DS and methyl-12-DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2-5 microM). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20 microM) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure.     

Effect of toluene diisocyanate and its corresponding amines on viability and growth of human lung fibroblasts in culture

Toluene diisocyanate (TDI) is a highly volatile chemical known to cause occupational asthma in exposed workers. TDI-induced asthma is associated with airway epithelium injury and repair, and subepithelial fibrosis. We investigated the effect of TDI and its hydrolysis products, the 2,4- and 2,6-toluenediamines (TDA), on viability and growth of human lung fibroblasts (HLFs) in culture, using a tetrazolium-based cell viability assay. The effects of increasing concentrations of each of these chemicals were evaluated on quiescent cells seeded at two densities (2500 and 5000 cells/well) and treated for 24 or 48 h. TDI (10^–4–10^–5 mol/L, as a mixture of 80% 2,4-TDI and 20% 2,6-TDI) exhibited a partial but significant cytotoxic effect (10–24%, p<0.05) on HLFs. This effect was observed at both cell densities, and was time- and concentration-dependent. 2,4-TDA, at lower concentrations (10^–8–10^–6 mol/L) applied for 48 h, also partially reduced HLF viability (10–15%, p<0.05), whereas it tended to trigger cell growth at concentrations higher than 10^–5 mol/L. 2,6-TDA exhibited both a cytotoxic and a proliferative effect on HLFs that depended on concentration, time of exposure and cell culture density. Significant cytotoxicity was only observed after 24 h of treatment with 10^–7–10^–6 mol/L 2,6-TDA, and reached greater intensity in cells cultured at the highest density. In contrast, 2,6-TDA stimulated HLF growth only after 48 h of incubation at 10^–4 mol/L on cells cultured at the lowest density. Taken together, our results showed that TDI and 2,4-TDA somewhat decreased HLF viability, whereas 2,6-TDA appeared to exhibit both a cytotoxic and a growth stimulatory effect on these cells. TDI and 2,4-TDA are thus suggested to contribute to airway epithelium damage associated with TDI-induced asthma, whereas 2,6-TDA might either trigger epithelial damage or induce cell proliferation that could contribute to epithelium repair or subepithelial fibrosis.     

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.2. Characterization with selective ligands, dithiothreitol, and GTPS, indicated that only the AT₂ subtype was expressed.3. AT₂ receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT₂ receptors being expressed earlier than ACE. In contrast, high expression of AT₂ receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.4. Our results imply a possible relation of AT₂ receptors to cell growth and cell–cell contact.     

Lack of evidence for an immunosuppressive role for MUC1

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1- and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells. Received: 20 August 1998 / Accepted: 3 December 1998     

Tumour escape: antitumour effectors too much of a good thing?

Although even spontaneous tumours are immunogenic and are commonly infiltrated by tumour antigen-specific T cells (at least in melanoma), most tumours are not completely rejected by the host, and cancer progresses. There is a growing realisation that many responses defined as antitumour effector mechanisms act as double-edged swords and under different conditions either become ineffective or even protumorigenic. Examples are interleukin 2 (also proapoptotic for activated T cells), interferon (by induction of ligands for T and NK cell inhibitory receptors), angiogenesis inhibition (by hypoxia-mediated induction of growth factors promoting metastasis), and macrophage free radical-mediated cytotoxicity (by inhibiting T cells). Immune selection pressure itself, resulting in outgrowth of resistant tumour variants could also be viewed in this light. On the other hand, knowledge of the many tumour escape pathways offers the theoretical possibility of reconstituting antitumour immunity. Tumour escape from immunosurveillance represents the last series of hurdles to be overcome in formulating truly effective cancer immunotherapy, but given the immense plasticity of the tumour cell, and the complex balance between pro- and antitumour activity of the very same effector pathways, this remains a major challenge.This article is based on presentations made at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

A cellular automaton model for tumour growth in inhomogeneous environment

Most of the existing mathematical models for tumour growth and tumour-induced angiogenesis neglect blood flow. This is an important factor on which both nutrient and metabolite supply depend. In this paper we aim to address this shortcoming by developing a mathematical model which shows how blood flow and red blood cell heterogeneity influence the growth of systems of normal and cancerous cells. The model is developed in two stages. First we determine the distribution of oxygen in a native vascular network, incorporating into our model features of blood flow and vascular dynamics such as structural adaptation, complex rheology and red blood cell circulation. Once we have calculated the oxygen distribution, we then study the dynamics of a colony of normal and cancerous cells, placed in such a heterogeneous environment. During this second stage, we assume that the vascular network does not evolve and is independent of the dynamics of the surrounding tissue. The cells are considered as elements of a cellular automaton, whose evolution rules are inspired by the different behaviour of normal and cancer cells. Our aim is to show that blood flow and red blood cell heterogeneity play major roles in the development of such colonies, even when the red blood cells are flowing through the vasculature of normal, healthy tissue.