VL30 retrotransposition signals activation of a caspaseindependent 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 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.

Ectopic expression of clusterin/apolipoprotein J or Bcl-2 decreases the sensitivity of HaCaT cells to toxic effects of ropivacaine

Local anesthetics inhibit cell proliferation and induce apoptosis in various cell types. Ropivacaine, a unique, novel tertiary amine-type anesthetic, was shown to inhibit the proliferation of several cell types including keratinocytes. We found that Ropivacaine could inhibit the proliferation and induce apoptosis in an immortalized human keratinocyte line, HaCaT, in a dose- and time-dependent manner and with the deprivation of serum. The dose-dependent induction of apoptosis by ropivacaine was demonstrated by DNA fragmentation analysis and the proteolytic cleavage of a caspase-3 substrate — poly (ADP-ribose) polymerase (PARP). In addition, ropivacaine downregulated the expression of clusterin/ apoliporotein J, a protein with anti-apoptotic properties, in a dose-dependent manner, which well correlated with the induction of apoptosis of HaCaT cells. To investigate the role of clusterin/apoliporotein J in ropivacaine-induced apoptosis, HaCaT cells overexpressing clusterin/apoliporotein J were generated and compared to cells expressing the well estab- lished anti-apoptotic Bcl-2 protein. Ectopic overexpression of the secreted form of clusterin/apoliporotein J or Bcl-2 decreased the sensitivity of HaCaT cells to toxic effects of ropivacaine as demonstrated by DNA fragmentation, the proteolytic cleavage of PARP and by a reduction in procaspase-3 expression. Furthermore, the downregulation of endogenous clusterin/apolipoprotein J levels by ropivacaine suggested that this might be one mechanism by which ropivacaine induced cell death in HaCaT cells. In conclusion, the ability of ropivacaine to induce antiproliferative responses and to suppress the expression of the anti-apoptotic protein clusterin/apolipoprotein J, combined with previ- ously reported anti-inflammatory activity and analgesic property of the drug, suggests that ropivacaine may have poten- tial utility in the local treatment of tumors.