Plasma membrane redox systems in tumor cells

Summary Plasma membrane redox systems in tumor cells are analyzed, their role in proton flux and tumor cell growth is described, and the modulation of their activity by antitumor drugs and growth factors is presented. As an example of the evolution of studies in the characterization of plasma membrane redox systems in tumor cells, we summarized our own results on the model system Ehrlich ascites carcinoma.     

Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression

Cells from human neuroectodermal tumors (retinoblastoma and neuroblastoma) and from neuroblastoma cell lines express a gene, N-myc, which is frequently amplified in these tumors. We report here that N-myc mRNA content is markedly decreased in cells of a neuroblastoma cell line (LA-N-5) following differentiation induced with retinoic acid. Exposure of the cells to retinoic acid induced morphologic changes consistent with neuronal differentiation, and led to a 75% decrease in expression of N-myc mRNA. These results suggest that N-myc expression is intimately related to an undifferentiated phenotype in neuroblastoma cells, and support other studies which relate N-myc expression to the malignant phenotype in neuroblastoma tumors.     

Characterization of N-myc amplification units in human neuroblastoma cells.

A set of DNA clones comprising 48 independent HindIII fragments (215 kilobases of sequence) was derived from the N-myc amplification unit of the neuroblastoma cell line NGP. These clones were used to investigate N-myc amplification units in NGP cells and 12 primary neuroblastoma tumors. Three parameters were evaluated: (i) the number of rearrangements from germ line configuration that had occurred during the amplification process; (ii) the homogeneity of amplification units within individual tumors; and (iii) the conservation of amplified sequences among different tumors. The results indicated that remarkably few rearrangements had occurred during amplification, that the amplification units within any one tumor were quite homogeneous, and that although each tumor contained a unique pattern of amplified DNA fragments, there was considerable similarity between the amplification units of different tumors. In particular, the amplification units were strikingly similar over a contiguous domain of at least 140 kilobases surrounding the N-myc structural gene.     

Excision of N-myc from chromosome 2 in human neuroblastoma cells containing amplified N-myc sequences.

Amplification of one of three growth-stimulating myc genes is a common method by which many tumor types gain a proliferative advantage. In metastatic human neuroblastoma, the amplification of the N-myc locus, located on chromosome 2, is a dominant feature of this usually fatal pediatric cancer. Of the many models proposed to explain this amplification, all incorporate as the initial step either disproportionate overreplication of the chromosomal site or recombination across a loop structure. The original locus is retained within the chromosome in the overreplication models but is excised in the recombination models. To test these models, we have used somatic cell hybrids to separate and analyze the chromosomes 2 from a neuroblastoma cell line containing in vivo amplified N-myc. Our results demonstrate that N-myc is excised from one of the chromosomes, suggesting that deletion is a requisite part of gene amplification in a naturally occurring system.     

N-myc down regulates neural cell adhesion molecule expression in rat neuroblastoma.

In human neuroblastoma, amplification of the N-myc oncogene is correlated with increased metastatic ability. We recently showed that transfection of the rat neuroblastoma cell line B104 with an N-myc expression vector resulted in an increase in metastatic ability and a significant reduction in the expression of major histocompatibility complex class I antigens. We examined whether N-myc causes additional phenotypic changes in these cells. We showed that expression of N-myc leads to a dramatic reduction in the levels of neural cell adhesion molecule (NCAM) polypeptides and mRNAs. Spontaneous revertants of the high N-myc phenotype were found to have regained significant levels of NCAM expression, indicating that the continued expression of N-myc is required to maintain the low NCAM phenotype. NCAM was not reduced in B104 cells transfected with the neomycin resistance vector alone, and other neuronal markers were not specifically reduced in N-myc-transfected B104 cells. As NCAM functions in cell-cell adhesion, decreased NCAM expression could contribute significantly to the increased metastatic potential of N-myc-amplified neuroblastomas.     

Plasma membrane redox system protects cells against oxidative stress

Abstract

Aerobic organisms have developed defensive systems to survive in the presence of oxygen and its highly reactive species (ROS). The cellular mechanisms of protection against oxidative injury include: (i) specific enzymes, such as catalase, glutathione peroxidase and superoxide dismutase; (ii) small hydrophilic molecules, such as ascorbate, glutathione and uric acid; and (iii) hydrophobic agents, such as ubiquinone and α-tocopherol in membranes.¹ Among these, coenzyme Q (CoQ) is the only lipid-soluble antioxidant that can be synthesized in all organisms so far studied.     

Plasma membrane RNase activity in mammalian cells

Plasma membranes isolated from eight different tissues from either man, rat, mouse or rabbit and from tissue culture were shown to inhibit protein synthesis in a cell-free system. From all membranal extracts an RNase endonuclease activity could be isolated which split rRNΔ. In contrast, the polyribosomal structure of rabbit reticulocytes was unaffected, showing that 9S mRNA was not destroyed under these conditions. The Triton X-100 membranal extracts blocked protein synthesis in the elongation stage and all resembled very closely the previously described RNase M activity [2]. A hypothesis is put forward, suggesting that newly formed ribosomes migrate in the cytoplasm while accomplishing protein synthesis. After being engaged in a series of such rounds of protein synthesis they meet with the plasma membrane and are inactivated by the RNase endonucleolytic splitting of their ribosomal RNA (rRNA). It is suggested that this is a mechanism common to all eukaryotic cells.     

Hypoxia promotes apoptosis of human neuroblastoma cell lines with enhanced N-myc expression

We have examined the effect of hypoxia and nutrient depletion on the growth of human neuroblastoma cells with normal or enhanced expression of the N-myc oncogene. The combination of both conditions reduced the growth of neuroblastoma cells with normal N-myc expression. However, this effect was much more pronounced in neuroblastoma cells with enhanced N-myc expression and eventually resulted in apoptosis, presumably by the up-regulation of CD95. Our data suggest that therapeutic induction of tumor hypoxia and nutrient depletion (for example, by anti-angiogenesis) could help to improve the outcome of patients with neuroblastomas carrying the prognostically unfavourable N-myc amplification.     

N-myc expression switched off and class I human leukocyte antigen expression switched on after somatic cell fusion of neuroblastoma cells.

Neuroblastomas often show amplification and high expression of the N-myc oncogene. N-myc expression could be explained as a consequence of gene amplification, but an alternative possibility is that expression primarily results from the inactivation or loss of some factor that normally represses the N-myc gene. To test this idea, we fused N-myc-overexpressing neuroblastoma cell lines with lines that do not express N-myc. In the resulting hybrids, N-myc expression turned out to be switched off, although amplified N-myc copies were still present. This suggests that N-myc overexpression in neuroblastomas results, at least in part, from the inactivation of a suppressor gene that is present in normal cells. In rat neuroblastomas, it has been found that N-myc can switch off class I major histocompatibility complex (MHC) expression. Therefore, we analyzed in our hybrid cells whether suppression of N-myc results in reexpression of human class I MHC genes. Because this was found to be the case, the picture emerges of a hierarchic pathway that connects a putative tumor-suppressor gene with the expression of N-myc and consequently of class I MHC, thus affecting the potential immunogenic properties of neuroblastomas.     

Characterization of plasma membrane redox activity from ehrlich cells

Ferricyanide reductase activity of plasma membranes isolated from Ehrlich ascites tumour cells was very sensitive to trypsin treatment. The decreases of activity observed after treatment with different glycosidases suggests that ferricyanide reductase is a glycoprotein. The opposite effects of phospholipase A₂ and phospholipase C on the redox activity indicate that the phospholipidic environment plays an important role in the function of ferricyanide reductase. Sodium ions at millimolar concentrations, and some divalent cations at micromolar concentrations (Ca²⁺, Mg²⁺, Sr²⁺, and Mn²⁺) behaved as stimulators of ferricyanide reductase activity.     

N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastoma

Amplification of the N-myc gene is correlated with increased metastatic ability of human neuroblastomas. We show here that overexpression of the N-myc gene in a rat neuroblastoma cell line following gene transfer causes down-modulation of class I histocompatibility antigen expression and increases in the in vivo growth rate and metastatic ability of these cells. N-myc-mediated down-modulation of MHC class I antigen expression could be reversed by treatment with interferon without affecting the steady state level of N-myc mRNA. No effect on MHC class I antigen expression was found when the N-myc gene was expressed in rat fibroblasts, indicating that some of the effects caused by N-myc gene amplification are cell-type-specific.     

Plasma membrane redox system activity in tumour cell lines of mammary origins with different proliferation rates

Summary Plasma membrane redox systems seem to play a role in the control of cell growth. In fact, we have found that in mammary tumour cell lines the increase in the proliferation rate is accompanied by a decrease in the plasma membrane redox activity. The oxygen consumption rates, the glycolytic fluxes and other bioenergetic parameters have been studied in two cell strains of Ehrlich ascites tumour with different proliferation rates. In the more proliferative Ehrlich cell strain, the decrease in plasma membrane redox system activity is accompanied by decreased oxygen consumption and glycolytic flux and to a generally less energised status.     

N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma.

Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.