Eun Seong Hwang

Senescence‐associated β‐galactosidase is lysosomal β‐galactosidase

Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence‐associated β‐galactosidase (SA‐β‐gal), which is defined as β‐galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA‐β‐gal and its cellular roles in senescence are not known. We demonstrate here that SA‐β‐gal activity is expressed from GLB1, the gene encoding lysosomal β‐D‐galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive GM1‐gangliosidosis, which have defective lysosomal β‐galactosidase, did not express SA‐β‐gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small‐hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA‐β‐gal. GLB1 mRNA depletion also prevented expression of SA‐β‐gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA‐β‐gal induction during senescence was due at least in part to increased expression of the lysosomal β‐galactosidase protein. These results also indicate that SA‐β‐gal is not required for senescence.

Inactivation of Interferon Regulatory Factor-1 Tumor Suppressor Protein by HPV E7 Oncoprotein IMPLICATION FOR THE E7-MEDIATED IMMUNE EVASION MECHANISM IN CERVICAL CARCINOGENESIS

In studying biological roles of interferon regulatory factor (IRF)-1 tumor suppressor in cervical carcinogenesis, we found that HPV E7 is functionally associated with IRF-1. Binding assays indicate a physical interaction between IRF-1 and HPV E7in vivo and in vitro. The carboxyl-terminal transactivation domain of IRF-1 was required for the interaction. Transient co-expression of E7 significantly inhibits the IRF-1-mediated activation of IFN-β promoter in NIH-3T3 cells. Co-transfection of E7 mutants reveals that the pRb-binding portion of E7 is necessary for the E7-mediated inactivation of IRF-1. It was next determined whether histone deacetylase (HDAC) is involved in the inactivation mechanism as recently suggested, where the carboxyl-terminal zinc finger domain of E7 associates with NURD complex containing HDAC. When trichostatin A, an inhibitor of HDAC, was treated, the repressing activity of E7 was released in a dose-dependent manner. Furthermore, the mutation of zinc finger abrogates such activity without effect on the interaction with IRF-1. These results suggest that HPV E7 interferes with the transactivation function of IRF-1 by recruiting HDAC to the promoter. The immune-promoting role of IRF-1 evokes the idea that our novel finding might be important for the elucidation of the E7-mediated immune evading mechanism that is frequently found in cervical cancer.

A comparative analysis of the cell biology of senescence and aging

Various intracellular organelles, such as lysosomes, mitochondria, nuclei, and cytoskeletons, change during replicative senescence, but the utility of these changes as general markers of senescence and their significance with respect to functional alterations have not been comprehensively reviewed. Furthermore, the relevance of these alterations to cellular and functional changes in aging animals is poorly understood. In this paper, we review the studies that report these senescence-associated changes in various aging cells and their underlying mechanisms. Changes associated with lysosomes and mitochondria are found not only in cells undergoing replicative or induced senescence but also in postmitotic cells isolated from aged organisms. In contrast, other changes occur mainly in cells undergoing in vitro senescence. Comparison of age-related changes and their underlying mechanisms in in vitro senescent cells and aged postmitotic cells would reveal the relevance of replicative senescence to the physiological processes occurring in postmitotic cells as individuals age.

Nicotinamide-induced Mitophagy EVENT MEDIATED BY HIGH NAD+/NADH RATIO AND SIRT1 PROTEIN ACTIVATION

Background: Nicotinamide treatment decreases mitochondrial content and helps cells maintain high mitochondrial quality. Results: Metabolically enhanced NAD+/NADH ratio and chemically induced SIRT1 activation similarly decreased mitochondrial content, increased autophagy, and induced mitochondrial fragmentation. Conclusion: Mitochondrial content is modulated by high NAD+/NADH ratio and mechanisms that involve SIRT1 activation. Significance: Elevation of NAD+/NADH ratio may promote cellular health by facilitating mitochondrial autophagy. Active autophagy coupled with rapid mitochondrial fusion and fission constitutes an important mitochondrial quality control mechanism and is critical to cellular health. In our previous studies, we found that exposure of cells to nicotinamide causes a decrease in mitochondrial content and an increase in mitochondrial membrane potential (MMP) by activating autophagy and inducing mitochondrial fragmentation. Here, we present evidence to show that the effect of nicotinamide is mediated through an increase of the [NAD+]/[NADH] ratio and the activation of SIRT1, an NAD+-dependent deacetylase that plays a role in autophagy flux. The [NAD+]/[NADH] ratio was inversely correlated with the mitochondrial content, and an increase in the ratio by the mobilization of the malate-aspartate shuttle resulted in autophagy activation and mitochondrial transformation from lengthy filaments to short dots. Furthermore, treatment of cells with SIRT1 activators, fisetin or SRT1720, induced similar changes in the mitochondrial content. Importantly, the activators induced mitochondrial fragmentation only when SIRT1 expression was intact. Meanwhile, MMP did not increase when the cells were treated with the activators, suggesting that the change in MMP is not induced by the mitochondrial turnover per se and that elevation of the [NAD+]/[NADH] ratio may activate additional mechanisms that cause MMP augmentation. Together, our results indicate that a metabolic state resulting in an elevated [NAD+]/[NADH] ratio can modulate mitochondrial quantity and quality via pathways that may include SIRT1-mediated mitochondrial autophagy.

Dinstinct ROS and biochemical profiles in cells undergoing DNA damage-induced senescence and apoptosis

Cellular senescence and apoptosis are both caused by DNA damage stresses, and their severity appears to decide between the two cellular outcomes. In recent studies, it is suggested that these two states may be closely linked and be switched by certain molecular determinants such as p21WAF1 and caspase (Abdelhadi, 2003). However, it is unknown how the pathways to senescence and apoptosis are determined. In addition, although DNA damage stresses frequently accompany cellular accumulation of reactive oxygen species (ROS), how ROS are involved in the decision between the two pathways is unknown. In the present study, MCF-7 cells were induced to senescence or apoptosis by the treatment of varying doses of adriamycin. And, through a series of time course studies, ROS generation profiles and changes in the status of the proteins involved in growth regulation and apoptosis were determined. Significant levels of ROS were produced in senescing cells but not in apoptotic cells. Therefore, senescence is associated with ROS accumulation, but apoptosis is caused independently of ROS. In addition, cells in these two states exhibited quite distinct time course profiles of the proteins, p53, p21WAF1, and E2F1.

Antiproliferative effects of retinoic acid/interferon in cervical carcinoma cell lines: Cooperative growth suppression of IRF-1 and p53

Retinoids and interferons have been implicated in the growth regulation of cervical cancer cells. However, the molecular mechanisms are not fully defined. To analyze detailed mechanisms, HPV‐positive (HeLa, CaSki), HPV‐negative (C33A, HT‐3) and non‐cervical Cos‐1 cell lines were treated with 1 μM all‐trans‐retinoic acid (RA) and/or 10 ng/ml interferon‐γ (IFN‐γ). The growth of RA‐treated HeLa cells was less effectively suppressed than that of IFN‐γ‐treated ones. A combination of RA and IFN‐γ leads to an additive antiproliferative effect on the cell growth. CaSki cell growth was also inhibited by IFN‐γ but was little stimulated by RA treatment, and the IFN effect was attenuated when IFN‐γ was combined with RA. HPV‐negative C33A and HT‐3 cells, which are defective in p53 and Rb, and Cos‐1 cells were weakly or not responsive to all combined treatments. The molecular mechanism underlying the differential effects of RA/IFN on HeLa and C33A cells was investigated. Combined RA/IFN‐γ treatment caused a marked increase in the level of IFN regulatory factor‐1 (IRF‐1) in HeLa, whereas no induction of IRF‐1 was observed in C33A, consistent with the findings that IFN signaling is functional in HeLa but is defective in C33A cells. The increase of p53 in HeLa cells might account for the down‐regulation of HPV‐18 E6 gene expression by RA/IFN‐γ. Furthermore, RA/IFN‐γ treatment resulted in the concurrent induction of p21WAF1 CDK inhibitor and dephosphorylation of Rb protein. Transient co‐expression of IRF‐1 and p53 led to the cooperative activation of the p21WAF1 promoter. Our results indicate that 2 transcription factors, increased in response to RA/IFN‐γ, cooperate functionally to regulate the cell cycle through the activation of a common p21WAF1 gene in HeLa cells. Int. J. Cancer 85:416–423, 2000. ©2000 Wiley‐Liss, Inc.

Nicotinamide extends replicative lifespan of human cells

We found that an ongoing application of nicotinamide to normal human fibroblasts not only attenuated expression of the aging phenotype but also increased their replicative lifespan, causing a greater than 1.6‐fold increase in the number of population doublings. Although nicotinamide by itself does not act as an antioxidant, the cells cultured in the presence of nicotinamide exhibited reduced levels of reactive oxygen species (ROS) and oxidative damage products associated with cellular senescence, and a decelerated telomere shortening rate without a detectable increase in telomerase activity. Furthermore, in the treated cells growing beyond the original Hayflick limit, the levels of p53, p21WAF1, and phospho‐Rb proteins were similar to those in actively proliferating cells. The nicotinamide treatment caused a decrease in ATP levels, which was stably maintained until the delayed senescence point. Nicotinamide‐treated cells also maintained high mitochondrial membrane potential but a lower respiration rate and superoxide anion level. Taken together, in contrast to its demonstrated pro‐aging effect in yeast, nicotinamide extends the lifespan of human fibroblasts, possibly through reduction in mitochondrial activity and ROS production.

Down-regulation of Sp1 Activity through Modulation of O-Glycosylation by Treatment with a Low Glucose Mimetic, 2-Deoxyglucose

2-Deoxyglucose (2-DG), a nonmetabolizable glucose analogue, blocks glycolysis at the phosphohexose isomerase step and has been frequently used as a glucose starvation mimetic in studies of a wide variety of physiological dysfuctions. However, the effect of 2-DG on protein glycosylation and related signal pathways has not been investigated in depth. In HeLa, an HPV18-positive cervical carcinoma line, 2-DG treatment down-regulates human papillomavirus early gene transcription. This down-regulation was also achieved by low glucose supply or hypoxia, suggesting that this is a response commonly modulated by cellular glucose or energy level. We investigated how 2-DG and low glucose affect transcriptional activity. Human papillomavirus gene transcription was only marginally affected by the inhibition of ATP synthesis or the supplementation of pyruvate to 2-DG-treated cells, suggesting that poor ATP generation is involved only to a limited extent. 2-DG treatment also inhibited activation of p21 WAF1 promoter, which is controlled by p53 and/or Sp1. In a reporter assay using p21 WAF1 promoter constructs, 2-DG exerted a strong inhibitory effect on Sp1 activity. DNA binding activity of Sp1 in 2-DG-treated HeLa cells was intact, whereas it was severely impaired in cells incubated in a low glucose medium or in hypoxic condition. Unexpectedly, Sp1 was heavily modified with GlcNAc in 2-DG-treated cells, which is at least partially attributed to the inhibitory effect of 2-DG on N-acetyl-β-d-glucosaminidase activity. Our results suggest that 2-DG, like low glucose or hypoxic condition, down-regulates Sp1 activity, but through hyper-GlcNAcylation instead of hypo-GlcNAcylation.

Nicotinamide enhances mitochondria quality through autophagy activation in human cells

Nicotinamide (NAM) treatment causes a decrease in mitochondrial respiration and reactive oxygen species production in primary human fibroblasts and extends their replicative lifespan. In the current study, it is reported that NAM treatment induces a decrease in mitochondrial mass and an increase in membrane potential (ΔΨm) by accelerating autophagic degradation of mitochondria. In the NAM‐treated cells, the level of LC3‐II as well as the number of LC3 puncta and lysosomes co‐localizing with mitochondria substantially increased. Furthermore, in the NAM‐treated cells, the levels of Fis1, Drp1, and Mfn1, proteins that regulate mitochondrial fission and fusion, increased and mitochondria experienced dramatic changes in structure from filaments to dots or rings. This structural change is required for the decrease of mitochondrial mass indicating that NAM accelerates mitochondrial autophagy, at least in part, by inducing mitochondrial fragmentation. The decrease in mitochondria mass was attenuated by treatment with cyclosporine A, which prevents the loss of mitochondrial membrane potential by blocking the mitochondrial permeability transition, suggesting autophagic degradation selective for mitochondria with low ΔΨm. All these changes were accompanied by and dependent on an increase in the levels of GAPDH, and are blocked by inhibition of the cellular conversion of NAM to NAD+. Taken together with our previous findings, these results suggest that up‐regulation of GAPDH activity may prolong healthy lifespan of human cells through autophagy‐mediated mitochondria quality maintenance.

Replicative senescence and senescence-like state induced in cancer-derived cells

Studies on the replicative senescence and premature senescence induced by various stresses in normal somatic cells have provided important clues on the role of telomere shortening and mechanisms involved in aging processes and carcinogenesis. Recent work revealed that cancer cells also are induced to undergo replicative senescence state via telomere shortening as well as to enter a senescence-like state by the activation of cell cycle inhibitory pathways. Although less relevant in terms of aging physiology, studies on these phenomena in cancer cells have yielded important information on telomerase regulation and the roles of tumor suppressors in senescence and immortalization, and are expected to generate valuable anti-cancer strategies. Several features of the phenotypes specific for the senescent and senescence-like states induced in cancer cells are discussed.