Incidence of Abcd1 level on the induction of cell death and organelle dysfunctions triggered by very long chain fatty acids and TNF-α on oligodendrocytes and astrocytes.

X-linked adrenoleukodystrophy (X-ALD) is characterized by ABCD1 deficiency. This disease is associated with elevated concentrations of very long chain fatty acids (C24:0 and C26:0) in the plasma and tissues of patients. Under its severe form, brain demyelination and inflammation are observed. Therefore, we determined the effects of C24:0 and C26:0 on glial cells:oligodendrocytes, which synthesize myelin, and astrocytes, which participate in immune response. So, 158N murine oligodendrocytes, rat C6 glioma cells, rat primary cultures of neuronal-glial cells, and of oligodendrocytes were treated for various periods of time in the absence or presence of C24:0 and C26:0 used at plasmatic concentrations found in X-ALD patients (1-5 µM) and higher (10, 20, 40 µM). To evaluate the importance of extrinsic and intrinsic factors, the part taken by TNF-α and reduced Abcd1 level was studied. Whatever the cells considered, no effects on cell growth and/or viability were detected at 1-5 µM, more or less pronounced effects were identified at 10 µM, and an induction of cell death with increased permeability to propidium iodide and loss of transmembrane mitochondrial potential was observed at 20-40 µM. On 158N, cell death was characterized by (i) an increased superoxide anion production at the mitochondrial level; (ii) the presence of vacuoles of different sizes and shapes; a destabilization of lysosomal membrane and a cytoplasmic redistribution of lysosomes; (iii) a modulation of Abcd3/PMP70 and Acox-1 protein expression, and a decrease in catalase activity at the peroxisomal level. When TNF-α was combined with C24:0 or C26:0 and used on 158N cells, C6 cells, and on 158N cells after siRNA mediated knockdown of Abcd1, no or slight potentiation was revealed. Thus, on the different cell models used, an induction of cell death with marked cellular dysfunctions at the mitochondrial, lysosomal, and peroxisomal levels were found with C24:0 and C26:0 at 20 µM and higher. However, in our experimental conditions, plasmatic concentrations of these fatty acids were unable to induce cell death, and organelle dysfunctions on oligodendrocytes and astrocytes, and additional intrinsic and environmental factors, such as reduced Abcd1 level and/or TNF-α, were ineffective to potentiate their side effects.

Impact of 7-ketocholesterol and very long chain fatty acids on oligodendrocyte lipid membrane organization: evaluation via LAURDAN and FAMIS spectral image analysis.

In the context of multiple sclerosis and X-linked adrenoleukodystrophy, 7-ketocholesterol (7KC) and very long chain fatty acids (C24:0, C26:0) are supposed to induce side effects respectively on oligodendrocytes which are myelin (which is a lipoproteic complex) synthesizing cells. The effects of 7KC (25, 50 µM), C24:0 and C26:0 (10, 20 µM) on cell viability and lipid membrane organization were investigated on 158N murine oligodendrocytes. Concerning 7KC and fatty acids (at 20 µM only): 1) cell growth was strongly inhibited; 2) marked induction of cell death was revealed with propidium iodide (PI); 3) no apoptotic cells were found with C24:0 and C26:0 (absence of cells with condensed and/or fragmented nuclei, of FLICA positive cells and of PI negative/SYTO16 negative cells); 4) some apoptotic cells were detected with 7KC. Fatty acids (at 20 µM only) and 7KC also induced a disorganization of lipid membranes revealed with Merocyanine 540. So, to point out the effects of 7KC (25 µM), C24:0 and C26:0 (20 µM) on the lateral organization of lipid membranes, we used LAURDAN, which gives simultaneous information about morphology and phase state of lipid domains: its emission is blue in the ordered lipid phase, green in the disordered lipid phase. To overcome the qualitative filtering settings of blue and green emission colors, data obtained by mono- and bi-photon confocal microscopy were analyzed by spectral analysis. Sequences of emission images were obtained on both mono- and bi-photon confocal microscopes and processed by means of Factor Analysis of Medical Image Sequences (FAMIS), which is a relevant tool to unmix emission spectra and provide pure color images. Only 7KC was capable to induce a green emission with LAURDAN. Thus, at concentrations inducing oligodendrocyte cell death, 7KC (25 µM) is more efficient than C24:0 and C26:0 (20 µM), to trigger lateral lipid membrane disorganization.

Identification of DDB2 protein as a transcriptional regulator of constitutive SOD2 gene expression in human breast cancer cells.

Manganese superoxide dismutase plays a role in breast tumor cell growth, which depends on its constitutive expression. However, the mechanisms responsible for the regulation of constitutive SOD2 gene expression at different malignant phenotype in breast cancers remain to be determined. The present study reports the identification and characterization of a DNA sequence located in the proximal promoter of the SOD2 gene, which forms a complex with a nuclear protein from breast tumor MCF-7 cells. Purification of this complex showed that it contained DDB2 (damaged DNA binding 2), a well known protein involved in nucleotide excision DNA repair and cell cycle regulation. Functional analysis of the proximal promoter of the SOD2 gene or modulation of DDB2 expression allowed us to demonstrate that DDB2 regulates negatively the constitutive expression of the SOD2 gene in breast cancer cells. We demonstrate that the binding of DDB2 was associated with the loss of acetylated H3 histones and the decrease in the binding of Sp1 but not AP-2alpha transcription factors to the SOD2 proximal promoter. In addition, we show that DDB2 exerts, at least in part, a control of breast cancer cell growth through its negative regulation of constitutive expression of the SOD2 gene. For the first time, these data give supporting evidence that DDB2 is a new transcriptional regulator, and they provide insight into the molecular function of breast cancer cell growth, which will have an important clinical interest.

Damaged DNA binding protein 2 plays a role in breast cancer cell growth.

The Damaged DNA binding protein 2 (DDB2), is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER)-positive (MCF-7 and T47D), but not in ER-negative breast cancer (MDA-MB231 and SKBR3) or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold) higher in ER-positive than in ER-negative tumor samples (P = 0.0208) from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase). These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.

Role of manganese superoxide dismutase on growth and invasive properties of human estrogen-independent breast cancer cells.

Manganese superoxide dismutase (MnSOD) is known to play a role in cancer. MnSOD exerts a tumor suppressive effect in estrogen-dependent human breast cancer cells. In the present study we investigated the in vitro role of MnSOD in the growth of some aggressive and highly metastatic estrogen-independent breast cancer cells, i.e., MDA-MB231 and SKBR3 cells. We show that estrogen-independent cells expressed a significantly higher basal MnSOD level compared to estrogen-dependent human breast cancer cell lines (MCF-7 and T47D). For MDA-MB231 cells, the high-MnSOD level was accompanied by an overproduction of intracellular hydrogen peroxide (H2O2) and by a low expression of the major H2O2-detoxifying enzymes, catalase, and peroxiredoxin 3, compared to MCF-7 cells. Suppression of MnSOD expression by antisense RNA was associated with a decrease of H2O2 content and caused a stimulation of growth with a reduced cell doubling time but induced a decrease of colony formation. Furthermore, treatment of MDA-MB231 cells with H2O2 scavengers markedly reduced tumor cell growth and colony formation. In addition, MnSOD suppression or treatment with H2O2 scavengers reduced the invasive properties of MDA-MB231 cells up to 43%, with a concomitant decrease of metalloproteinase-9 activity. We conclude that MnSOD plays a role in regulating tumor cell growth and invasive properties of estrogen-independent metastatic breast cancer cells. These action are mediated by MnSOD-dependent H2O2 production. In addition, these results suggest that MnSOD up-regulation may be one mechanism that contributes to the development of metastatic breast cancers.