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...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 PUMAa 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-acetylcysteiue 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-specilic 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.展开更多
In addition to shaping genome diversification over evolutionary time, L1 retrotransposition alters gene expression as well. The most notable gene altering process involves insertional mutagenesis. The aim of the study...In addition to shaping genome diversification over evolutionary time, L1 retrotransposition alters gene expression as well. The most notable gene altering process involves insertional mutagenesis. The aim of the study was the examination of both nuclear L1 expression levels and cellular localization in cancer cell lines, PBMCs from healthy volunteers and PBMCs from cancer patients. L1 was detected by FISH in chromosome preparations. L1 probe was custom-made using end-point PCR against L1-ORF2 and conjugated with FITC. It was found that cancer cell lines and clinical samples from cancer patients contained significantly elevated levels of L1 per nucleus compared to healthy volunteers. Cytoplasmic L1 was also increased in the above mentioned samples denoting that cancer could be associated with increased L1 activation and mobility. Our results may provide a novel cancer diagnostic marker and highlight the possibility of cytoplasmic L1 inhibition as a therapeutic intervention for cancer.展开更多
文摘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 PUMAa 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-acetylcysteiue 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-specilic 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.
文摘In addition to shaping genome diversification over evolutionary time, L1 retrotransposition alters gene expression as well. The most notable gene altering process involves insertional mutagenesis. The aim of the study was the examination of both nuclear L1 expression levels and cellular localization in cancer cell lines, PBMCs from healthy volunteers and PBMCs from cancer patients. L1 was detected by FISH in chromosome preparations. L1 probe was custom-made using end-point PCR against L1-ORF2 and conjugated with FITC. It was found that cancer cell lines and clinical samples from cancer patients contained significantly elevated levels of L1 per nucleus compared to healthy volunteers. Cytoplasmic L1 was also increased in the above mentioned samples denoting that cancer could be associated with increased L1 activation and mobility. Our results may provide a novel cancer diagnostic marker and highlight the possibility of cytoplasmic L1 inhibition as a therapeutic intervention for cancer.
