Brunella Del Re

LINE-1 retrotransposition in human neuroblastoma cells is affected by oxidative stress

Long interspersed element-1s (LINE-1 or L1s) are abundant retrotransposons that occur in mammalian genomes and that can cause insertional mutagenesis and genomic instability. L1 activity is generally repressed in most cells and tissues but has been found in some embryonic cells and, in particular, in neural progenitors. Moreover, L1 retrotransposition can be induced by several DNA-damaging agents. We have carried out experiments to verify whether L1 retrotransposition is affected by oxidative DNA damage, which plays a role in a range of human diseases, including cancer and inflammatory and neurodegenerative disease. To this purpose, BE(2)C neuroblastoma cells, which are thought to represent embryonic precursors of sympathetic neurons, have been treated with hydrogen peroxide and subjected to an in vitro retrotransposition assay involving an episomal L1RP element tagged with enhanced green fluorescent protein. Our results indicate that hydrogen peroxide treatment induces an increase in the retrotransposition of transiently transfected L1RP and an increase in the expression of endogenous L1 transcripts. An increase of γ-H2AX foci and changes in the mRNA levels of MRE11, RAD50, NBN and ERCC1 (all involved in DNA repair) have also been found. Thus, oxidative stress can cause L1 dysregulation.

Interferon-mediated enhancement of metastasis. Are MHC antigens involved?

The relationship between major histocompatibility complex (MHC) antigens and metastasis was investigated on B16 melanoma variants. B16 cell lines express low amounts of murine MHC (H-2) antigens. A high expression can be induced in line B16-A by in vitro treatment with immune interferon (IFN-gamma) or by in vivo transplant in allogeneic mice. The increase of H-2 antigens correlated with an enhancement of lung colonization in young syngeneic mice. The higher metastatic capacity of B16-A cells with induced high levels of H-2 antigens was observed also in adult mice and in young mice pretreated with cyclophosphamide. These results were confirmed investigating the behaviour of a mutant B16 clone (B78H1) which was selectively resistant to the H-2-inducing action of IFN-gamma: lung colonization ability was not increased by IFN pretreatment. The study of variants derived from individual B16-A lung colonies revealed a wide range of H-2 levels. Variants with a low expression had a low colonization ability; one out of two variants with a high H-2 expression also was poorly colonizing. IFN-gamma-mediated H-2 expression appeared to act as an enhancer, rather than a determinant of B16 metastatic capacity.

Synergic effect of retinoic acid and extremely low frequency magnetic field exposure on human neuroblastoma cell line BE(2)C

The aim of the present study was to assess whether exposure to a sinusoidal extremely low frequency magnetic field (ELF-MF; 50 Hz, 1 mT) can affect proliferation and differentiation in the human neuroblastoma cell line BE(2)C, which is representative of high risk neuroblastomas. Cells were subjected to ELF-MF exposure in the presence or absence of a neuronal differentiating agent (all-trans-retinoic acid, ATRA) for 24-72 h. In each experiment, ELF-MF-exposed samples were compared to sham-exposed samples. Cells exposed to ELF-MF combined with retinoic treatment showed a decreased cellular proliferation and an increased proportion of G(0)/G(1) phase cells compared to cells exposed to either treatment alone. Moreover, ELF-MF- and ATRA-treated cells showed more differentiated morphological traits (a higher neurite number/cell, an increased neurite length), together with a significant increase of mRNA levels of p21(WAF1/CIP1) and cdk5 genes, both involved in neuronal differentiation. In addition, the expression of cyp19 gene, which is involved both in neuronal differentiation and stress response, was evaluated; cyp19 gene expression was enhanced by ATRA treatment and significantly enhanced further by ELF-MF exposure combined with ATRA. In conclusion, our data suggest that ELF-MF exposure can strengthen ATRA effects on neuroblastoma cells.

In vivo and in vitro production of haemopoietic colony-stimulating activity by murine cell lines of different origin: a frequent finding.

The production of colony-stimulating factor (CSF) by murine transformed cells was investigated in 10 cell lines derived from spontaneous or chemically induced tumours and from cells transformed by SV40 or Moloney-MSV; histologic types included carcinomas, sarcomas and melanoma. Nine of 10 supernatants contained CSF activity as judged by in vitro proliferation and differentiation of normal murine monocytic and granulocytic progenitors in agar cultures. Tumours induced with CSF-producing cells caused alterations of haemopoiesis which can include leukocytosis, granulocytosis and splenomegaly. Haemopoietic alterations were also evident in the absence of a local tumour in mice bearing large experimental lung metastases. Production of CSF seems to be a frequent finding among murine cell lines, and its biological and immunological consequences on host-tumour relationships should be taken into account.

Effect of extremely low frequency magnetic field exposure on DNA transposition in relation to frequency, wave shape and exposure time

Purpose: To examine the effect of extremely low frequency magnetic field (ELF-MF) exposure on transposon (Tn) mobility in relation to the exposure time, the frequency and the wave shape of the field applied. Materials and methods: Two Escherichia coli model systems were used: (1) Cells unable to express β-galactosidase (LacZ-), containing a mini-transposon Tn10 element able to give ability to express β-galactosidase (LacZ+) upon its transposition; therefore in these cells transposition activity can be evaluated by analysing LacZ+ clones; (2) cells carrying Fertility plasmid (F+), and a Tn5 element located on the chromosome; therefore in these cells transposition activity can be estimated by a bacterial conjugation assay. Cells were exposed to sinusoidal (SiMF) or pulsed-square wave (PMF) magnetic fields of various frequencies (20, 50, 75 Hz) and for different exposure times (15 and 90 min). Results: Both mini-Tn10 and Tn5 transposition decreased under SiMF and increased under PMF, as compared to sham exposure control. No significant difference was found between frequencies and between exposure times. Conclusions: ELF-MF exposure affects transposition activity and the effects critically depend on the wave shape of the field, but not on the frequency and the exposure time, at least in the range observed.

Evaluation of LINE-1 mobility in neuroblastoma cells by in vitro retrotransposition reporter assay: FACS analysis can detect only the tip of the iceberg of the inserted L1 elements

Long Interspersed Nuclear Elements (L1) are retroelements generally repressed in most differentiated somatic cells. Their activity has been observed in some undifferentiated and tumour cells and could be involved in tumour onset and progression. Growing evidences show that the L1 activation can occur in neuronal precursor cells during differentiation process. Neuroblastoma is a tumour originating from neuronal precursor cells, and, although the molecular basis of its progression is still poorly understood, the implication of L1 activation has not yet been investigated. In this study L1 mobility in neuroblastoma BE(2)C cells was assessed using the in vitro retrotransposition assay consisting in an episomal EGFP-tagged L1(RP) element, whose mobility can be evaluated by cytofluorimetric analysis (FACS) of EGFP expression. FACS results have shown a low retrotransposition activity. To detect L1(RP) integrated in transcriptionally repressed genomic sites, both a cell treatment with a stimulator of reporter gene promoter, and a quantitative Real-Time PCR analysis were performed. A retrotransposition activity ten and one thousand times that of FACS was found, respectively. These results point out that the real rate of L1 retrotransposition events in tumour cells might be considerably higher than that reported so far by evaluating only the reporter gene expression.

Assessing LINE-1 retrotransposition activity in neuroblastoma cells exposed to extremely low-frequency pulsed magnetic fields.

Mobile genetic elements represent an important source of mutation and genomic instability, and their activity can be influenced by several chemical and physical agents. In this research we address the question whether exposure to extremely low-frequency pulsed magnetic fields (EMF-PMF) could affect the mobility of the human LINE-1(RP) retrotransposon. To this purpose, an in vitro retrotransposition assay was used on human neuroblastoma BE(2) cells exposed for 48h to 1mT, 50Hz PMF, or sham-exposed. Moreover, since it is well known that retrotransposition causes DNA double-strand breaks (DSB), an estimation of γ-H2AX foci, which is a marker of DNA DSB, was carried out on PMF- and sham-exposed samples. The results show that PMF-exposed cells had a lower number of both retrotransposition events and DNA DSB compared with sham-exposed samples. These results suggest that exposure to PMF can interfere with retrotransposition activity by inducing a decrease of retrotransposition events.

Cell-host, LINE and environment: Three players in search of a balance.

Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship.

An evaluation of genotoxicity in human neuronal-type cells subjected to oxidative stress under an extremely low frequency pulsed magnetic field

The possible genotoxicity of extremely low frequency magnetic field (ELF-MF) exposure is still a controversial topic. The most of the reported data suggests that it alone does not affect DNA integrity, but several recent reports have suggested that sinusoidal ELF-MF may increase the effect of known genotoxic agents. Only a few studies deal with non sinusoidal ELF-MF, including pulsed magnetic field (PMF), which are produced by several devices. The aim of this study is to investigate whether PMF exposure can interfere with DNA damage and repair in the presence of a genotoxic oxidative agent in neuronal type cells. To this purpose gamma-H2AX foci formation, which is a sensitive marker of DNA double strand breaks (DSB), was investigated at different points of time (1, 24, 48, 72h) after the H2O2 treatment (300μM for 1h) under PMF exposure (1mT, 50Hz) in human neuroblastoma BE(2)C cells. Moreover, cytotoxicity evaluation, by MTT assay and cell cycle analysis, was performed at various points of time after the treatment. Taken together, results suggest that PMF exposure does not interfere with genotoxicity and cytotoxicity induced by oxidative stress.