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

https://doi.org/10.1016/j.mad.2004.11.008Get rights and content

Abstract

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.

Introduction

In normal cells, cellular senescence or replicative senescence is reached after a finite number of cell division (Hayflick and Moorehead, 1961). As cells progress towards senescence, levels of p53, p21WAF1, and p16INK4a protein gradually increase, and finally cells are irreversibly growth arrested (Bringold and Serrano, 2000). The senescence process is likely triggered by telomere attrition which may be interpreted as a form of DNA damage (Campisi, 2001). A senescent state can be induced in cancer cells as well through activation of p53-p21WAF1/p16INK4a-Rb pathway via gene transduction or treatment of genotoxic chemicals (Hwang, 2002).

DNA damage stresses induce apoptotic cell death as well. Senescence and apoptosis, the two irreversible states of cellular life, were frequently shown to be induced by different doses of the same stress. Senescence is induced at subapoptotic doses, suggesting that more severe DNA damage induces apoptotic response, while less severe one causes cellular senescence (Luschen et al., 2000). However, it is largely unknown how the severity of DNA damage is sensed by cellular surveillance systems and activates different signaling pathways leading to the two cellular states.

It appears that p53 protein stands at the center of the complex signal transduction pathways for DNA damage responses. Activated p53 induces a variety of genes whose products, respectively, functions as an executer in DNA repair, G1 check point (p21WAF1), apoptosis (Bax1, PIG3, p53AIP1, POX, and Fas ligand), and autoregulation (MDM2) (Sionov and Haupt, 1999). At least 20 sites in the human p53 protein are modified in response to different stress signaling (Appella and Anderson, 2001). p53 is activated either directly by the ATM/ATR/DNA-PK kinases or indirectly through Chk2, a kinase activated by ATM, and is also regulated by acetylation/deacetylation at lys382 (Appella and Anderson, 2001, Brooks and Gu, 2003). In addition, p53 is negatively regulated through phosphorylation by Jun N-terminal kinase (JNK) (Fuchs et al., 1998). Phosphorylation at Ser15, Thr18, and Ser376, and dephosphorylation at Ser392 has been detected in fibroblasts undergoing replicative senescence (Webley et al., 2000). Meanwhile, phosphorylation at Ser46 was shown to be associated with induction of p53AIP1 and apoptosis (Oda et al., 2000). However, how different p53 phosphorylation and downstream gene selection are linked to each other to determine different cellular outcomes is mostly undetermined.

Furthermore, possible interactions between p53-downstream gene products and other cellular components in pathways toward different cellular outcomes are largely undetermined yet. For p53-induced apoptosis, activation of Fas signaling (Muller et al., 1997a, Muller et al., 1997b; Munsch et al., 2000) and caspase-9 pathway (Soengas et al., 1999) has been shown to be involved. And, recently, it was shown that, during apoptosis induced by DNA damage, caspase inhibition caused an induction of senescence in place of apoptosis, and this was accompanied by an upregulation of p21WAF1 which was otherwise suppressed (Rebbaa et al., 2003). In a simplest view, this might be interpreted that, upon DNA damage, the pathways for apoptosis and senescence may be initiated together, but only one is activated while the other is suppressed, and p21WAF1 is the key molecule that determines the direction.

DNA damage stresses cause an increase in cellular level of reactive oxygen species (ROS) (Buttke and Sandstrom, 1994, Jacobson, 1996). p53 activation is likely involved, since p53 induces several known genes involved in ROS generation such as PIG3 or POX (Polyak et al., 1997). Recently, it was reported that p53-induced senescence and ROS generation are subject to an inhibition by Bcl-xL, suggesting Bcl-xL is involved in the ROS generation pathway (Jung et al., 2004). However, not much is known for the nature of the molecules and pathways responsible for ROS production in cells. ROS disrupts the mitochondrial membrane potential, and therefore, have been proposed as an additional route by which p53 induce apoptosis (Johnson et al., 1996, Li et al., 1999). Addition of ROS or depletion of cellular antioxidants induces apoptosis (Buttke and Sandstrom, 1994). In contrast, many antioxidants and free-radical scavengers, such as N-acetyl-l-cysteine (NAC) inhibits or delays apoptosis (Mayer and Noble, 1994). And, sublethal doses of H2O2 (Hagen et al., 1997, Chen et al., 1998) or hyperoxia (von Zglinicki et al., 1995, Dumont et al., 2000) can force human fibroblasts to undergo a senescent state. Based on these and other observations, it has been generally believed that ROS induce apoptosis at a high level and senescence at an intermediate level, and cell proliferation at a low level. However, a correlation between degree of DNA damage stress, ROS level and cellular outcomes has not been established yet.

The status of p53 and its downstream gene products in the cells at apoptosis or senescence has been documented in numerous studies. But, during the induction process of apoptosis and senescence, the levels of these proteins as well as of cellular ROS are expected not static. Here, the cellular and molecular events during induction of either senescence or apoptosis were compared in a series of time course studies on MCF-7 cells treated with different doses of adriamycin. Cells in senescence condition, are typified with high level ROS accumulation, transient p53 activation, and sustained p21WAF expression, while those in apoptosis condition, were devoid of a significant ROS, prolonged p53 and E2F1 upregulation, and lack of p21WAF1. Furthermore, it was demonstrated that ATM/ATR activation is an upstream event required for both ROS accumulation and senescence expression.

Section snippets

Cell culture and chemicals

Human breast cancer cell line, MCF-7, was maintained in DMEM (Dulbecco's Modified Eagle Medium) (Invitrogen) containing 10% FBS. All chemicals were purchased from Sigma Chemical Co (St. Louis, MI, USA).

Western blotting analyses

Cell extracts were prepared by lysis in RIPA buffer (50 mM Tris–Cl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1mM PMSF, and 10 μg/ml leupeptin), and 15–30 μg proteins were separated by SDS-PAGE using 12% gel, and transferred to nitrocellulose membrane (Hybond ECL,

Adriamycin treatment conditions for induction of cellular senescence or apoptosis

First, conditions to induce senescence and apoptosis in MCF-1 cells by using adriamycin as a genotoxin were sought. Cells were treated with varying concentrations of adriamycin for 2, 4, or 8 h and further incubated for 5 days without adriamycin, and assayed for SA β-Gal activity, a marker for cellular senescence (Dimri et al., 1995). Since senescence phenotype expression is apparent only after 4–5 days post stimulation and should occur in the absence of initial stimulant by definition (Hwang,

Discussion

Described in this report are the time-course profiles of cellular and biochemical events accompanying senescence or apoptosis, which were separately induced by a pulse or continuous DNA damaging insult of adriamycin. In the dose-response studies, the presence of an intermediate state that is neither senescence or apoptosis was found, which indicates that senescence and apoptosis are cellular states induced by certain restricted degree of DNA damage, and should not be referred to as responses to

Acknowledgements

This study was supported by a grant from the Korea Science and Engineering Foundation (KOSEF) through the Center for Aging and Apoptosis Research at Seoul National University (R11-2002-001-04001-0).

References (49)

  • M.D. Jacobson

    Reactive oxygen species and programmed cell death

    Trends Biochem. Sci.

    (1996)
  • M.S. Jung et al.

    Bcl-xL and E1B-19K proteins inhibit p53-induced irreversible growth arrest and senescence by preventing reactive oxygen species-dependent p38 activation

    J. Biol. Chem.

    (2004)
  • S. Luschen et al.

    Sensitization to death receptor cytotoxicity by inhibition of fas-associated death domain protein (FADD)/caspase signalling. Requirement of cell cycle progression

    J. Biol. Chem.

    (2000)
  • S.K. Manna et al.

    Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappaB and activated protein-1

    J. Biol. Chem.

    (1998)
  • I. Muller et al.

    Effect of concentration on the cytotoxic mechanism of doxorubicin--apoptosis and oxidative DNA damage

    Biochem. Biophys. Res. Commun.

    (1997)
  • D. Munsch et al.

    Human and mouse Fas (APO-1/CD95) death receptor genes each contain a p53-responsive element that is activated by p53 mutants unable to induce apoptosis

    J. Biol. Chem.

    (2000)
  • K. Oda et al.

    p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53

    Cell

    (2000)
  • T. von Zglinicki et al.

    Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence?

    Exp. Cell Res.

    (1995)
  • M. Woloschak et al.

    Molecular and cellular responses to DNA damage in a murine pituitary adenoma cell line

    Mol, Cell Endocrinol.

    (1996)
  • S.O. Yoon et al.

    Dose effect of oxidative stress on signal transduction in aging

    Mech. Ageing Dev.

    (2002)
  • J. Yu et al.

    PUMA induces the rapid apoptosis of colorectal cancer cells

    Mol. Cell

    (2001)
  • E. Appella et al.

    Post-translational modifications and activation of p53 by genotoxic stresses

    Eur. J. Biochem.

    (2001)
  • Q.M. Chen et al.

    Molecular analysis of H2O2-induced senescent-like growth arrest in normal human fibroblasts: p53 and Rb control G1 arrest but not cell replication

    Biochem. J.

    (1998)
  • G.P. Dimri et al.

    A biomarker that identifies senescent human cells in culture and in aging skin in vivo

    Proc. Natl. Acad. Sci. USA

    (1995)
  • Cited by (0)

    View full text