Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells

BackgroundEnhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence.MethodsWe developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2.ResultsIn this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence.ConclusionGSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence

Iron chelation-induced senescence-like growth arrest in hepatocyte cell lines: association of transforming growth factor beta1 (TGF-beta1)-mediated p27Kip1 expression.

Iron is essential for cellular proliferation in all organisms. When deprived of iron, the growth of cells is invariably inhibited. However, the mechanism involved remains largely unclear. In the present study, we have observed that subcytotoxic concentrations of desferroxamine mesylate (DFO), an iron chelator, specifically inhibited the transition from G1 to S-phase of Chang cells, a hepatocyte cell line. This was accompanied by the appearance of senescent biomarkers, such as enlarged and flattened cell morphology, senescence-associated beta-galactosidase activity and reduced expression of poly(ADP-ribose) polymerase. Concomitantly, p27Kip1 (where Kip is kinase-inhibitory protein) was induced markedly, whereas other negative cell-cycle regulators, such as p21Cip1 (where Cip is cyclin-dependent kinase-interacting protein), p15INK4B and p16INK4A (where INK is inhibitors of cyclin-dependent kinase 4), were not, implying its association in the G1 arrest. Furthermore, the induction of p27Kip1 was accompanied by an increased level of transforming growth factor beta1 (TGF-beta1) mRNA. When neutralized with an anti-(TGF-beta1) antibody, p27Kip1 induction was completely abolished, indicating that TGF-beta1 is the major inducer of p27Kip1. Finally, DFO-induced senescence-like arrest was found to be independent of p53, since cell-cycle arrest was still observed with two p53-negative cell lines, Huh7 and Hep3B cells. In conclusion, DFO induced senescence-like G1 arrest in hepatocyte cell lines and this was associated with the induction of p27Kip1 through TGF-beta1, but was independent of p53

Three Novel cis-Acting Elements Required for Efficient Plus-Strand DNA Synthesis of the Hepatitis B Virus Genome

Synthesis of the relaxed-circular (RC) DNA genomes of hepadnaviruses by reverse transcriptase involves two template switches during plus-strand DNA synthesis. These template switches require repeat sequences (so-called donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. To determine cis-acting elements apart from the donor and acceptor sites that are required for plus-strand RC DNA synthesis by hepatitis B virus (HBV), a series of mutants bearing a small deletion were made and analyzed for their impact on the viral genome synthesis. We found three novel cis-acting elements in the HBV genome: one element, located in the middle of the minus strand, is indispensable, whereas the other two elements, located near either end of the minus strand, contribute modestly to the plus-strand RC DNA synthesis. The data indicated that the first element facilitates plus-strand RNA primer translocation or subsequent elongation during plus-strand RC DNA synthesis, while the last two elements, although distantly located on the minus strand, act at multiple steps to promote plus-strand RC DNA synthesis. The necessity of multiple cis-acting elements on the minus-strand template reflects the complex nature of hepadnavirus reverse transcription

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation*

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7

Mitoribosomal Deregulation Drives Senescence via TPP1-Mediated Telomere Deprotection

While mitochondrial bioenergetic deregulation has long been implicated in cellular senescence, its mechanistic involvement remains unclear. By leveraging diverse mitochondria-related gene expression profiles derived from two different cellular senescence models of human diploid fibroblasts, we found that the expression of mitoribosomal proteins (MRPs) was generally decreased during the early-to-middle transition prior to the exhibition of noticeable SA-β-gal activity. Suppressed expression patterns of the identified senescence-associated MRP signatures (SA-MRPs) were validated in aged human cells and rat and mouse skin tissues and in aging mouse fibroblasts at single-cell resolution. TIN2- and POT1-interaction protein (TPP1) was concurrently suppressed, which induced senescence, accompanied by telomere DNA damage. Lastly, we show that SA-MRP deregulation could be a potential upstream regulator of TPP1 suppression. Our results indicate that mitoribosomal deregulation could represent an early event initiating mitochondrial dysfunction and serve as a primary driver of cellular senescence and an upstream regulator of shelterin-mediated telomere deprotection