Tinzaparin inhibits VL30 retrotransposition induced by oxidative stress and/or VEGF in HC11 mouse progenitor mammary cells: Association between inhibition of cancer stem cell proliferation and mammosphere disaggregation.

Tinzaparin is an anticoagulant and antiangiogenic drug with inhibitory properties against tumor growth. VEGF stimulates angiogenesis, while an association between reactive oxygen species (ROS) and angiogenesis is involved in tumor progression. The present study aimed to investigate the effect of tinzaparin on VL30 retrotransposition­positive mouse HC11 mammary stem­like epithelial cells, previously reported to be associated with induced mammosphere/cancer stem cell (CSC) generation and tumorigenesis. Under 24 h serum starvation, 15.2% nominal retrotransposition frequency was increased to 29%. Additionally, while treatment with 3­12 ng/ml VEGF further induced retrotransposition frequency in a dose­dependent manner (up to 40.3%), pre­incubation with tinzaparin (2 IU/ml) for 0.5­4 h reduced this frequency to 18.3% in a time­dependent manner, confirmed by analogous results in NIH3T3 fibroblasts. Treatment with 10­40 pg/ml glucose oxidase (GO) for 24 h induced HC11 cell retrotransposition in a dose­dependent manner (up to 82.5%), while a 3 h pre­incubation with tinzaparin (1 or 2 IU/ml) elicited a 13.5 or 25.5% reduction in retrotransposition, respectively. Regarding tumorigenic VL30 retrotransposition­positive HC11 cells, treatment with 2 IU/ml tinzaparin for 5 days reduced proliferation rate in a time­dependent manner (up to ~55%), and after 3 weeks, disaggregated soft agar­formed foci, as well as low­adherent mammospheres, producing single mesenchymal­like cells with a ~50% reduced retrotransposition. With respect to the VL30 retrotransposition mechanism: While 12 ng/ml VEGF increased the level of VL30 and endogenous reverse transcriptase (enRT) transcripts ~1.41­ and ~1.16­fold, respectively, subsequent tinzaparin treatment reduced both endogenous/ROS­ and VEGF­induced levels 1.15­ and 0.40­fold (VL30) and 0.60­ and 0.52­fold (enRT), respectively. To the best of our knowledge, these data demonstrate for the first time, the novel inhibition activity of tinzaparin against ROS­ and VEGF­induced VL30 retrotransposition, and the proliferation and/or aggregation of mouse HC11 mammosphere/tumor­initiating CSCs, thus contributing to the inhibition of VL30 retrotransposition­induced primary tumor growth.

VL30 retrotransposition is associated with induced EMT, CSC generation and tumorigenesis in HC11 mouse mammary stem­like epithelial cells.

Retrotransposons copy their sequences via an RNA intermediate, followed by reverse transcription into cDNA and random insertion, into a new genomic locus. New retrotransposon copies may lead to cell transformation and/or tumorigenesis through insertional mutagenesis. Methylation is a major defense mechanism against retrotransposon RNA expression and retrotransposition in differentiated cells, whereas stem cells are relatively hypo­methylated. Epithelial­to­mesenchymal transition (EMT), which transforms normal epithelial cells into mesenchymal­like cells, also contributes to tumor progression and tumor metastasis. Cancer stem cells (CSCs), a fraction of undifferentiated tumor­initiating cancer cells, are reciprocally related to EMT. In the present study, the outcome of long terminal repeat (LTR)­Viral­Like 30 (VL30) retrotransposition was examined in mouse mammary stem­like/progenitor HC11 epithelial cells. The transfection of HC11 cells with a VL30 retrotransposon, engineered with an EGFP­based retrotransposition cassette, elicited a higher retrotransposition frequency in comparison to differentiated J3B1A and C127 mouse mammary cells. Fluorescence microscopy and PCR analysis confirmed the specificity of retrotransposition events. The differentiated retrotransposition­positive cells retained their epithelial morphology, while the respective HC11 cells acquired mesenchymal features associated with the loss of E­cadherin, the induction of N­cadherin, and fibronectin and vimentin protein expression, as well as an increased transforming growth factor (TGF)­ß1, Slug, Snail­1 and Twist mRNA expression. In addition, they were characterized by cell proliferation in low serum, and the acquisition of CSC­like properties indicated by mammosphere formation under anchorage­independent conditions. Mammospheres exhibited an increased Nanog and Oct4 mRNA expression and a CD44+/CD24­/low antigenic phenotype, as well as self­renewal and differentiation capacity, forming mammary acini­like structures. DNA sequencing analysis of retrotransposition­positive HC11 cells revealed retrotransposed VL30 copies integrated at the vicinity of EMT­, cancer type­ and breast cancer­related genes. The inoculation of these cells into Balb/c mice produced cytokeratin­positive tumors containing pancytokeratin­positive cells, indicative of cell invasion features. On the whole, the findings of the present study demonstrate, for the first time, to the best of our knowledge, that stem­like epithelial HC11 cells are amenable to VL30 retrotransposition associated with the induction of EMT and CSC generation, leading to tumorigenesis.

Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production.

This study evaluated in situ expression of the nisA gene by an indigenous, nisin A-producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A-mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

Copy-number variation analysis in familial nonsyndromic congenital anomalies of the kidney and urinary tract: Evidence for the causative role of a transposable element-associated genomic rearrangement.

Most congenital anomalies of the kidney and urinary tract (CAKUT) are sporadic, but familial occurrence has been described, suggesting a genetic contribution. Copy­number variations (CNVs) were detected in patients with CAKUT to identify possible novel genomic regions associated with CAKUT. CNVs were investigated in 7 children with CAKUT from three unrelated families using array comparative genomic hybridization: female monozygotic twins with bilateral duplex collecting system/vesicoureteral reflux (VUR)/unilateral renal hypodyspasia (URHD); two male siblings with VUR/URHD; 3 male second cousins, one with bilateral VUR/URHD, one with bilateral VUR and one with ureterovesical junction obstruction (UVJO). Five patients had a normal constitution of CNVs, one had a duplication of 0.2 Mb on the 5q­arm (5q23.3), probably unrelated to CAKUT, and one with UVJO had a 1.4 Mb deletion on the 17q­arm (17q12) which includes a known CAKUT gene, HNF1B. The phenotype of HNF1B deletion was extended including renal magnesium wasting. A higher coverage in transposable elements (TEs) was found in the deleted region compared with the expected density in any random genomic region. Notably, the 5' breakpoint was mapped within a solo long terminal repeat (LTR) sequence. Moreover, highly similar members of solo LTR and mammalian interspersed repetitive (MIR) elements, as well as nucleotide sequence microhomology were detected at the breakpoint regions. In conclusion, the deletion detected in one patient suggests this genomic imbalance as causative for UVJO. A not very well known phenotype of HNF1B deletion resulting in both low urinary tract malformations and renal wasting of magnesium was described. The high load in TEs of the deleted region, the presence of highly similar elements, and the microhomology found at breakpoint regions may have contributed to the generation of the deletion. CNV analysis could reveal novel causative genomic regions in patients with CAKUT, and further studies in larger cohorts are needed.

Genomic analysis of mouse VL30 retrotransposons.

BACKGROUND: Retrotransposons are mobile elements that have a high impact on shaping the mammalian genomes. Since the availability of whole genomes, genomic analyses have provided novel insights into retrotransposon biology. However, many retrotransposon families and their possible genomic impact have not yet been analysed. RESULTS: Here, we analysed the structural features, the genomic distribution and the evolutionary history of mouse VL30 LTR-retrotransposons. In total, we identified 372 VL30 sequences categorized as 86 full-length and 49 truncated copies as well as 237 solo LTRs, with non-random chromosomal distribution. Full-length VL30s were highly conserved elements with intact retroviral replication signals, but with no protein-coding capacity. Analysis of LTRs revealed a high number of common transcription factor binding sites, possibly explaining the known inducible and tissue-specific expression of individual elements. The overwhelming majority of full-length and truncated elements (82/86 and 40/49, respectively) contained one or two specific motifs required for binding of the VL30 RNA to the poly-pyrimidine tract-binding protein-associated splicing factor (PSF). Phylogenetic analysis revealed three VL30 groups with the oldest emerging ~17.5 Myrs ago, while the other two were characterized mostly by new genomic integrations. Most VL30 sequences were found integrated either near, adjacent or inside transcription start sites, or into introns or at the 3' end of genes. In addition, a significant number of VL30s were found near Krueppel-associated box (KRAB) genes functioning as potent transcriptional repressors. CONCLUSION: Collectively, our study provides data on VL30s related to their: (a) number and structural features involved in their transcription that play a role in steroidogenesis and oncogenesis; (b) evolutionary history and potential for retrotransposition; and (c) unique genomic distribution and impact on gene expression.

An interstitial 4q31.21q31.22 microdeletion associated with developmental delay: case report and literature review.

The 4q deletion syndrome phenotype consists of growth failure and developmental delay, minor craniofacial dysmorphism, digital anomalies, and cardiac and skeletal defects. We have identified an inversion (inv(1)(q25.2q31.1)) and an interstitial deletion in a boy with developmental delay using array-comparative genomic hybridization. This de novo deletion is located at 4q31.21q31.22 (145,963,820- 147,044,764), its size is 0.9-1.1 Mb, and it contains 7 genes (ABCE1, OTUD4, SMAD1, MMAA, C4orf51, ZNF827, and ANAPC10) as well as 5 retrotransposon-derived pseudogenes. Bioinformatic analysis revealed that while small copy number variations seem to have no impact on the phenotype, larger deletions or duplications in the deleted region are associated with developmental delay. Additionally, we found a higher coverage in transposable element sequences in the 4q31.21q31.22 region compared to that of the expected repeat density when regarding any random genome region. Transposable elements might have contributed to the reshaping of the genome architecture and, most importantly, we identified 3 L1PA family members in the breakpoint regions, suggesting their possible contribution in the mechanism underlying the appearance of this deletion. In conclusion, this is one of the smallest deletions reported associated with developmental delay, and we discuss the possible role of genomic features having an impact on the phenotype.

Abnormal DLK1/MEG3 imprinting correlates with decreased HERV-K methylation after assisted reproduction and preimplantation genetic diagnosis.

Retrotransposons participate in cellular responses elicited by stress, and DNA methylation plays an important role in retrotransposon silencing and genomic imprinting during mammalian development. Assisted reproduction technologies (ARTs) may be associated with increased stress and risk of epigenetic changes in the conceptus. There are similarities in the nature and regulation of LTR retrotransposons and imprinted genes. Here, we investigated whether the methylation status of Human Endogenous Retroviruses (HERV)-K LTR retrotransposons and the imprinting signatures of the DLK1/MEG3. p57(KIP2) and IGF2/H19 gene loci are linked during early human embryogenesis by examining trophoblast samples from ART pregnancies and preimplantation genetic diagnosis (PGD) cases and matched naturally conceived controls. Methylation analysis revealed that HERV-Ks were totally methylated in the majority of controls while, in contrast, an altered pattern was detected in ART-PGD samples that were characterized by a hemi-methylated status. Importantly, DLK1/MEG3 demonstrated disturbed methylation in ART-PGD samples compared to controls and this was associated with altered HERV-K methylation. No differences were detected in p57(KIP2) and IGF2/H19 methylation patterns between ART-PGD and naturally conceived controls. Using bioinformatics, we found that while the genome surrounding the p57(KIP2) and IGF2/H19 genes differentially methylated regions had low coverage in transposable element (TE) sequences, the respective one of DLK1/MEG3 was characterized by an almost 2-fold higher coverage. Moreover, our analyses revealed the presence of KAP1-binding sites residing within retrotransposon sequences only in the DLK1/MEG3 locus. Our results demonstrate that altered HERV-K methylation in the ART-PGD conceptuses is correlated with abnormal imprinting of the DLK1/MEG3 locus and suggest that TEs may be affecting the establishment of genomic imprinting under stress conditions.

Arsenic induces VL30 retrotransposition: the involvement of oxidative stress and heat-shock protein 70.

Arsenic is an environmental contaminant with known cytotoxic and carcinogenic properties, but the cellular mechanisms of its action are not fully known. As retrotransposition consists a potent mutagenic factor affecting genome stability, we investigated the effect of arsenic on retrotransposition of an enhanced green fluorescent protein (EGFP)-tagged nonautonomous long terminal repeat (LTR)-retrotransposon viral-like 30 (VL30) in a mouse NIH3T3 cell culture-retrotransposition assay. Flow cytometry analysis of assay cells treated with 2.5-20µM sodium arsenite revealed induction of retrotransposition events in a dose- and time-dependent manner, which was further confirmed as genomic integrations by PCR analysis and appearance of EGFP-positive cells by UV microscopy. Specifically, 20µM sodium arsenite strongly induced the VL30 retrotransposition frequency, which was ~90,000-fold higher than the natural one and also VL30 RNA expression was ~6.6-fold. Inhibition of the activity of endogenous reverse transcriptases by efavirenz at 15µM or nevirapine at 375µM suppressed the arsenite-induced VL30 retrotransposition by 71.16 or 79.88%, respectively. In addition, the antioxidant N-acetyl-cysteine reduced the level of arsenite-induced retrotransposition, which correlated with the rescue of arsenite-induced G2/M cell cycle arrest and cell toxicity. Treatment of assay cells ectopically overexpressing the human heat-shock protein 70 (Hsp70) with 15µM sodium arsenite resulted in an additional ~4.5-fold induction of retrotransposition compared with normal assay cells, whereas treatment with 20µM produced a massive cell death. Our results show for the first time that arsenic both as an oxidative and heat-shock mimicking agent is a potent inducer of VL30 retrotransposition in mouse cells. The impact of arsenic-induced retrotransposition, as a cellular response, on contribution to or explanation of the arsenic-associated toxicity and carcinogenicity is discussed.

H2O2 signals via iron induction of VL30 retrotransposition correlated with cytotoxicity.

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.

VL30 retrotransposition signals activation of a caspase-independent and p53-dependent death pathway associated with mitochondrial and lysosomal damage.

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.

Retrotransposon RNA expression and evidence for retrotransposition events in human oocytes.

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.

Vanadium induces VL30 retrotransposition at an unusually high level: a possible carcinogenesis mechanism.

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.

SV40 large T antigen up-regulates the retrotransposition frequency of viral-like 30 elements.

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.