-
[show abstract]
[hide abstract]
ABSTRACT: The impact of oxidative stress on mobilization of endogenous retroviruses and their effects on cell fate is unknown. We investigated the action of H2O2 on retrotransposition of an EGFP-tagged mouse LTR-retrotransposon, VL30, in an NIH3T3 cell-retrotransposition assay. H2O2 treatment of assay cells caused specific retrotranspositions documented by UV microscopy and PCR analysis. Flow cytometric analysis revealed an unusually high dose- and time-dependent retrotransposition frequency induced, ∼420,000-fold at 40 μM H2O2 compared to the natural frequency, which was reduced by ectopic expression of catalase. Remarkably, H2O2 moderately induced the RNA expression of retrotransposon B2 without affecting the basal expression of VL30s and L1 and significantly induced the expression of various endogenous reverse transcriptase genes. Further, whereas treatment with 50 μM FeCl2 alone was ineffective, cotreatment with 10 μM H2O2 and 50 μM FeCl2 caused a 6-fold higher retrotransposition induction than H2O2 alone, which was associated with cytotoxicity. H2O2- or H2O2/FeCl2-induced retrotransposition was significantly reduced by the iron chelator DFO or the antioxidant NAC, respectively. Furthermore, both H2O2-induced retrotransposition and associated cytotoxicity were inhibited after pretreatment of cells with DFO or the reverse transcriptase inhibitors efavirenz and etravirine. Our data show for the first time that H2O2, acting via iron, is a potent stimulus of retrotransposition contributing to oxidative stress-induced cell damage.
Free radical biology & medicine 04/2012; 52(10):2072-81. · 5.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The impact of long terminal repeat (LTR) retrotransposition on cell fate is unknown. Here, we investigated the effect of VL30 retrotransposition on cell death in SV40-transformed mouse SVTT1 cells. Transfection of a VL30 retrotransposon decreased the clonogenicity of SVTT1 by 17-fold, as compared to parental NIH3T3 cells. Correlated levels of retrotransposition frequency and cell death rates were found in retrotransposition-positive SVTT1 cloned cells, exhibiting DNA fragmentation, nuclear condensation, multinucleation and cytoplasmic vacuolization. Analysis of activation of effector caspases revealed a caspase-independent cell death mechanism. However, cell death was associated with p53 induction and concomitant upregulation of PUMAalpha and Bax and downregulation of Bcl-2 and Hsp70 protein expression. Moreover, we found partial loss of colocalization of large T-antigen (LT)/p53 and p53 translocation to mitochondria, leading to mitochondrial outer membrane permeabilization (MOMP) accompanied by lysosomal membrane permeabilization (LMP). Interestingly, treatment with the antioxidant N-acetylcysteine abolished cell death, suggesting the involvement of mitochondrial-derived reactive oxygen species, and resulted in an increase of retrotransposition frequency. Importantly, the induction of cell death was VL30 retrotransposon-specific as VL30 mobilization was induced; in contrast, mobilization of the non-LTR L1 (LINE-1, long interspersed nuclear element-1), B2 and LTR MusD retrotransposons decreased. Our results provide, for the first time, strong evidence that VL30 retrotransposition mediates cell death via mitochondrial and lysosomal damage, uncovering the role of retrotransposition as a nuclear signal activating a mitochondrial-lysosomal crosstalk in triggering cell death.
Cell Research 04/2010; 20(5):553-62. · 8.19 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vanadium exerts a variety of biological effects, including antiproliferative responses through activation of the respective signaling pathways and the generation of reactive oxygen species. As epidermal cells are exposed to environmental insults, human keratinocytes (HaCaT) were used to investigate the mechanism of the antiproliferative effects of vanadyl(IV) sulfate (VOSO(4)). Treatment of HaCaT cells with VOSO(4) inhibited proliferation and induced apoptosis in a dose-dependent manner. Inhibition of proliferation was associated with downregulation of cyclins D1 and E, E2F1, and the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1). Induction of apoptosis correlated with upregulation of the c-fos oncoprotein, changes in the expression of clusterin (CLU), an altered ratio of antiapoptotic to proapoptotic Bcl-2 protein family members, and poly(ADP-ribose) polymerase-1 cleavage. Forced overexpression of c-fos induced apoptosis in HaCaT cells that correlated with secretory CLU downregulation and upregulation of nuclear CLU (nCLU), a pro-death protein. Overexpression of Bcl-2 protected HaCaT cells from vanadium-induced apoptosis, whereas secretory CLU overexpression offered no cytoprotection. In contrast, nCLU sensitized HaCaT cells to apoptosis. Our data suggest that vanadium-mediated apoptosis was promoted by c-fos, leading to alterations in CLU isoform processing and induction of the pro-death nCLU protein.
FEBS Journal 07/2009; 276(14):3784-99. · 3.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: For many years, there has been uncertainty concerning the reason for Hsp70 translocation to the nucleus and nucleolus. Herein, we propose that Hsp70 translocates to the nucleus and nucleoli in order to participate in pathways related to the protection of the nucleoplasmic DNA or ribosomal DNA from single-strand breaks. The absence of Hsp70 in HeLa cells, via Hsp70 gene silencing (knockdown), indicated the essential role of Hsp70 in DNA integrity. Therefore, HeLa Hsp70 depleted cells were very sensitive in heat treatment and their DNA breaks were multiple compared to that of control HeLa cells. The molecular mechanism with which Hsp70 performs its role at the level of nucleus and nucleolus during stress was examined. Hsp70 co-localizes with PARP1 in the nucleus/nucleoli as was observed in confocal studies and binds to the BCRT domain of PARP1 as was revealed with protein-protein interaction assays. It was also found that Hsp70 binds simultaneously to XRCC1 and PARP-1, indicating that Hsp70 function takes place at the level of DNA repair and possibly at the base excision repair system. Making a hypothetical model, we have suggested that Hsp70 is the molecule that binds and interrelates with PARP1 creating the repair proteins simultaneously, such as XRCC1, at the single-strand DNA breaks. Our data partially clarify a previously unrecognized cellular response to heat stress. Finally, we can speculate that Hsp70 plays a role in the quality and integrity of DNA.
Cell Stress and Chaperones 01/2009; 14(4):391-406. · 3.01 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Although human diseases of retrotransposition-derived etiology have been documented, retrotransposon RNA expression and the occurrence of retrotransposition events in the human oocyte are not studied. We investigated the RNA expression of L1 and HERV-K10 retrotransposons in human oocytes by RT-PCR analysis with designed primers. Using denucleated germinal vesicles (GVs), we detected RT-PCR products of expressed L1, HERV-K10 and, unexpectedly, SINE-R, VNTR and Alu (SVA) retrotransposons. Their transcript specificities were identified as such following RNA-FISH and their origin by cloning and sequence alignment analyses. Assessing the expression level in comparison with somatic cells by densitometry analysis, we found that although in normal lymphocytes and transformed HeLa cells their profile was in an order of L1 > HERV-K10 > SVA, remarkably this was reversed in oocytes. To investigate whether de novo retrotransposition events occur and reverse transcriptases are expressed in the human oocyte, we introduced in GVs either a retrotransposition active human L1 or mouse reverse transcriptase deficient-VL30 retrotransposon tagged with an EGFP-based retrotransposition cassette. Interestingly, in both the cases, we observed EGFP-positive oocytes, associated with an abnormal morphology for L1 and granulation for VL30, and the retrotransposition events were confirmed by PCR. Our results: (i) show that L1, HERV-K10 and SVA retrotransposons are transcriptionally expressed and (ii) provide evidence, for the first time, for retrotransposition events occurring in the human oocyte. These findings suggest that both, network of retrotransposon transcripts and controlled retrotranspositions, might serve important functions required for oocyte development and fertilization while the uncontrolled ones might explain the onset of genetic disorders.
Human Molecular Genetics 01/2009; 18(7):1221-8. · 7.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Carcinogenesis by vanadium is thought to occur through induction of DNA-double-strand breaks (DSBs) but its mechanism is not fully understood. We investigated the effect of vanadium on induction of viral-like 30 element (VL30) retrotransposition using a NIH3T3 cell-retrotransposition assay based on a recombinant VL30/EGFP element. Incubation of assay cells with vanadyl sulphate (VOSO(4)) induced retrotransposition frequency in a dose and time-dependent manner, measured by fluorescence-activated cell scanning (FACS) and retrotransposition events were confirmed by UV microscopy and PCR analysis. Among vanadium salts with different valence tested, vanadyl (4+) ions were the most potent retrotransposition inducers. VOSO(4), at 50 muM induced retrotranspositions at an unusually high frequency of up to 0.185 events per cell per generation. VOSO(4), acting at the transcription level, strongly induced VL30 and endogenous reverse transcriptase (enRT) transcripts with maxima at 50 muM and 100 muM of 22 and 18-fold, respectively. VOSO(4)-induced retrotransposition frequency was inhibited by 42% with efavirenz, an inhibitor of enRTs, while paraquat, a DNA-DSBs inducer, had no effect. Furthermore, it was completely abolished with deferoxamine, a metal chelator, while reduced by 75% with N-acetyl-cysteine, a general antioxidant. Remarkably, H(2)O(2) reproduced inducible retrotransposition linking for the first time oxidative stress to induction of retrotransposition. We propose that VOSO(4)-induced VL30 retrotransposition through H(2)O(2) generation may be an alternative mutagenic, DNA-DSBs independent, mechanism leading to carcinogenesis.
Journal of Molecular Biology 12/2007; 374(1):80-90. · 4.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Heat shock protein-70 (Hsp70) is the main heat-inducible member of the 70-kDa family of chaperones that assist cells in maintaining proteins functional under stressful conditions. In the present investigation, the role of Hsp70 in the molecular mechanism of hydrogen peroxide-induced DNA damage to HeLa cells in culture was examined. Stably transfected HeLa cell lines, overexpressing or lacking Hsp70, were created by utilizing constitutive expression of plasmids containing the functional hsp70 gene or hsp70-siRNA, respectively. Compared to control cells, the Hsp70-overexpressing ones were significantly resistant to hydrogen peroxide-induced DNA damage, while Hsp70-depleted cells showed an enhanced sensitivity. In addition, the "intracellular calcein-chelatable iron pool" was determined in the presence or absence of Hsp70 and found to be related to the sensitivity of nuclear DNA to H(2)O(2). It seems likely that the main action of Hsp70, at least in this system, is exerted at the lysosomal level, by protecting the membranes of these organelles against oxidative stress-induced destabilization. Apart from shedding additional light on the mechanistic details behind the action of Hsp70 during oxidative stress, our results indicate that modulation of cellular Hsp70 may represent a way to make cancer cells more sensitive to normal host defense mechanisms or chemotherapeutic drug treatment.
Free Radical Biology and Medicine 03/2007; 42(4):567-77. · 5.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The regulation of non-autonomous retrotransposition is not known. A recombinant bearing a hygromycin gene and a viral-like 30 (VL30) retrotransposon tagged with an enhanced green fluorescent protein (EGFP) gene-based retrotransposition cassette was constructed and used for detection of retrotransposition events. Transfection of this recombinant produced retrotransposition events, detected both by EGFP fluorescence and PCR analysis, in hygromycin-selected clones of two established simian virus 40 (SV40)-transformed mouse NIH3T3 cell lines but not in normal NIH3T3 cells. The retrotransposition potential of this recombinant, as a provirus, was studied in stably transfected NIH3T3 clones. Transfection of these clones with either a wild-type or a mutant LE1135T SV40 large T antigen gene, not expressing small t protein, induced retrotransposition events at high frequencies as measured by fluorescence-activated cell scanning (FACS). In addition, measuring retrotransposition frequencies over a period of nine days following infection with isolated SV40 particles, revealed that the frequency of retrotransposition was time-dependent and induced as early as 24 h, increasing exponentially to high levels (>10(-2) events per cell per generation) up to nine days post-infection. Furthermore, ectopic expression of a cloned MoMLV-reverse transcriptase gene also produced retrotransposition events and suggested that the large T antigen most likely acted through induction of expression of endogenous reverse transcriptase genes. Our results show a direct correlation between SV40-cell transformation and VL30 retrotransposition and provide for the first time strong evidence that SV40 large T antigen up-regulates the retrotransposition of VL30 elements.
Journal of Molecular Biology 08/2006; 361(3):450-61. · 4.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Retroviral reverse transcriptase (RT) plays a definite role in retroviral life cycle and is essential for the process of retrotransposition. We investigated the RNA expression of endogenous reverse transcriptases (enRTs) in the NIH3T3 mouse genome using, as a probe, a mixture of RT-PCR generated reverse transcriptase products potentially detecting a large number of RTs following treatment with different agents. We found that the expression of enRTs is induced approximately 500-fold following 5'-azacytidine-treatment. Amongst steroid hormones used such as estradiol, diethylstilbestrol, progesterone and dexamethasone only the latter was effective in inducing enRTs up to 4-fold at a concentration of 10(-7) M. Expression of a mouse dominant-negative form of p53 protein in cell clones resulted in induction of 20- to 50-fold, whereas C2-ceramide in a 4-fold induction at concentrations of 20-80 micro M. In a parallel analysis, the respective expression of the transposable viral-like 30 elements (VL30s) was also measured. Their expression was induced up to 50-fold by 5'-azacytidine, overexpression of the p53 gene and C2-ceramide at 80 micro M. It was also induced approximately 3- to 5-fold following estradiol, diethylstilbestrol or progesterone treatment and 30-fold by dexamethasone. Collectively, our results suggest that such stimuli inducing enRTs might play a role in the activation of transcription and retrotransposition of VL30.
International Journal of Oncology 11/2003; 23(4):1237-43. · 2.40 Impact Factor
