Typical Cell Signaling Response to Ionizing Radiation:DNA Damage and Extranuclear Damage

Hui Yu

Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas78229, USA

Introduction

Radiation-induced bystander-type biological responses were first described in overlooked literature in the late 1940s.At that time, most radiobiologists still believed that only the directly irradiated cells suffered the effects of radiation exposure through direct ionization or the action of water radiolysis products.Recently, the finding of Nagasawa and Little[1]in 1992 sparked people’s interest in radiation-induced bystander effect.Their results showed that when the monolayer cells were exposed to low-dose α-particles, some cells (30% of the cells) showed biological damage in sister chromatid exchanges (SCEs), and less than 1% of the cells were estimated to undergo a nuclear traversal based on the microdosimetric principle.Though not recognized initially, in the past 17 years, the significance of radiationinduced bystander effects has been widely accepted.Occurrences of the bystander effect after various qualities,doses, and dose-rates of radiation have been recently demonstrated in studies of bothin vitroand a fewin vivomodels.Bystander communication has been shown both in the systems where irradiated cells are in contact with each other through gap junction pathways and in the systems where the cells are at considerable distances apart from each other via secreted factors[2].It i compelling to speculate that, when cells are in clos contact, signaling processes mediated through solubl factors in the medium may play a predominant rol Several soluble factors have been considered as potenti candidates in the bystander response.However, ver little is known about the nature of the signalin mediators, their targets in non-irradiated cells, the mechanism of maintaining sustained communication, o the duration of the communication after irradiation.

Overall, bystander effects are manifested as th expression of a wide range of endpoints, such a mutagenesis, chromosomal aberrations, micronucleatio neoplastic transformation, proliferation, and different ation.Radiation-induced bystander effects refer to th responses of cells that were not subjected to ionizin radiation (IR) exposure.In other words, the damage caused by radiation in irradiated cells are augmented b subsequent damage to non-irradiated bystander cell These bystander cells may have been neighbors o irradiated cells or may have been physically separate but subject to soluble secreted signals from irradiate cells.

Surviving tumor cells at the treatment site afte radiation therapy may elicit signaling mechanisms tha may be responsible for clonal selection, tumor ce proliferation/tumor growth, and metastasis.Hence, it i imperative to understand the relationship between tumo re-growth and those altered responses following radiation exposure.If this was to occur, either the cells hit by radiation should be viable and non-responsive to radiation, which is very unlikely, or a sub group of tumor cells should develop resistance and maintain functional integrity to elicit communication in both irradiated and non-targeted bystander neighboring cells.This might allow the cancer cells that are surviving the radiation exposure to develop a clone (clonal selection), re-grow(tumor cell proliferation and growth), and cause tumor relapse at the treatment site.Simultaneous angiogenic support through intercellular communication between the surviving tumor cells and the surrounding endothelium would further augment the tumor growth and increase the risk of distant metastasis.

DNA Damage and Activation of Nuclear Sensory Protein

There are many types of DNA damage induced by radiation, such as single-strand breaks (SSBs) and doublestrand breaks (DSBs), sugar and base modifications, and DNA-protein cross-links[3].The damaged DNA can be not only recognized by the sensory proteins, leading to recruitment of DNA repair enzymes, but also generate signals to delay cell cycle progression until the DNA damage is repaired.Some important proteins implicated in the surveillance to DNA damage and the activation of the damage checkpoint and cell cycle arrest are phosphatidylinositol 3-kinase Family (PI3K-like kinase),like ataxia telangiectasia mutated (ATM), and ATM,Rad3-related protein (ATR).ATM, a serine/threonine-specific protein kinase, is named for the disease, ataxia telangiectasia, caused by mutations of ATM[4].Activated ATM can phosphorylate and regulate various downstream target proteins, including tumor suppressor proteins P53 and breast cancer type 1 susceptibility protein (BRCA1), checkpoint kinase 2(CHK2), checkpoint proteins RAD17 and RAD9, and DNA repair protein Nibrin (NBS1), leading to cell cycle arrest, DNA repair or apoptosis[5].ATR is also a serine/threonine-specific protein kinase, and mutations in ATR are responsible for a rare human disease, Seckel syndrome.ATR has a similar function to ATM that once it is activated, it can phosphorylate downstream proteins like serine/threonine-protein kinase (CHK1), initiating a signal transduction cascade that culminates in cell cycle arrest[6,7].However, there is difference between ATM and ATR on the kinetics of activation and the types of damage to which they respond best.ATM is preferred in response to DSBs, while ATR is activated in response to persistent single-stranded DNA, which usually occur at stalled replication forks as an intermediate in DNA detection and repair pathways such as nucleotide excision repair(NER) and homologous recombination[8].Recent studies also support that ATM is the main determinant of the early cell cycle checkpoint response to IR-induced damage, whereas ATR responds later to processed damage induced by IR[9,10].P53 is a tumor suppressor protein, and is usually called as "the guardian of the genome" to describe its important role in conserving stability by preventing genome mutation.In normal cells,P53 is highly unstable due to the fact that Mdm2 (Hdm2 in humans) binds to P53 to promote its ubiquitylation and destruction in proteasomes, so P53 usually presents at very low concentration.DNA damage activates the protein kinases that cause the phosphorylation of P53,then reduce its binding to Mdm2 and decrease the P53 degradation.As a result, P53 accumulates to high concentration level and stimulate the gene transcription.The P53 functions through two main mechanisms: It can activate DNA repair protein and/or promote transcription of genes that induce cell cycle arrest(especiallyp21, it is transcriptionally activated by P53,and can suppress G1/S-Cdk and S-Cdk complexes, and keep the cell cycle arrest in G1).Alternatively, if the DNA damage cannot be reparable any more, it can initiate apoptosis, the programmed cell death (Figure 1)[11].

It is not limited that, ATR, ATM, CHk1, CHk2 as we mentioned above, are implicated in the genome integrity checkpoint or other responses to several forms of DNA damage (induced by either ultraviolet (UV), IR or chemical agents, such as hydrogen peroxide)[12,13], another group of protein kinase, mitogen-activated protein kinase(MAPK), including c-Jun N-terminal kinases (JNK1/3), extracellular-signal-regulated kinases (ERK1/2),ERK5 and P38 mitogen-activated protein kinases (p38 MAPK), can also respond to several types of stress, such as membrane damage, oxidative stress, osmotic shock,and heat shock, through transcriptionally activatingp53.

Extranuclear Damage and Activation

IR can directly interact with water, then generate small amounts of reactive oxygen species (ROS), which are amplified by mitochondria, generating large amount of ROS and reactive nitrogen species (RNS).ROS and RNS can inhibit protein tyrosine phosphatase (PTPase)activities.PTPase can remove phosphate groups from phosphorylated tyrosine residues on proteins.PTPase and tyrosine kinase work together to regulate the phosphorylation state balance of many important tyrosine phosphorylation signaling molecules.Hence, the inhibition of PTPase induced by radiation through the ROS and RNS increase the potential of tyrosine phosphorylation of the downstream proteins.Recent data also showed that the epidermal growth factor receptor(EGFR; ErbB-1; HER1 in humans) can be rapidly activated by IR in many tumor cellsin vitro[14,15].EGFR, a family of four structurally related receptor tyrosine kinases, is high affinity cell surface receptors for various growth factors, cytokines and hormones.Insufficient EGFR signaling in humans is associated with the development of neurodegenerative diseases[16], while somatic mutant EGFR are associated with aggressive tumor growth in a number of cancers[17].

The signals can be broadcasted from the cell surface EGFR to the other parts of the cell through the transducer proteins, among which a very important one is Ras protein.Ras proteins belong to the Ras superfamily of monomeric GTPase.Like other guanosine-5'-triphosphate (GTP)-binding proteins, Ras functions as a cycling transition between two conformations, activated and inactivated forms, respectively RAS-GTP and RAS-GDP.The inactivated Ras is tightly bound to guanosine diphosphate (GDP), and guanine nucleotide exchange factors (GEFs) can activate Ras by stimulating it to give up GDP, and uptake GTP from the cytosol at the same time.GTPase-activating proteins (GAPs) can inactivate Ras by increasing the rate of hydrolysis of its bound GTP.Overactive mutant Ras can be found in about 30% human tumors, because it is resistant to GAPs to avoid the hydrolysis of bound GTP, and keep the active state which can promote the development of tumor cells.Active Ras can in turn activate many downstream signaling pathways.One of novel types is MAPK serine/threonine phosphorylation pathway.Multiple pathways involving many proteins structurally and functionally function differently (Figure 1).The active Ras activates MAP-kinase-kianse-kianse (MAPKKK), which is a serine/threonine protein kinase.It is recruited from the cytosol as inactivated state to the cytosolic face of the plasma membrane as activated state, which then activates the MAP-kinase-kianse (MAPKK).MAPKK is a dual function kianse that can phosphorylate both tyrosine and threonine residues on its substrate MAPK, and this unusual feature can make sure MAPK is specifically activated by MAPKK.Activated MAPK in turn phosphorylates lots of downstream proteins including other gene kinases and gene regulatory proteins resulting in protein activitation and gene expression change(Figure 2).

Figure 1.Signaling pathways involving many proteins structurally and functionally induced by IR.

Bystander Effect

Immense advances have been made in cancer treatment by radiotherapy.Currently, one of the major problems that still remain is the recurrent tumor relapse after treatment of primary tumor.A newly emerging phenomenon called “bystander effect” is responsible for tumor relapse after treatment.Tissue/cells at treatment site (including tumor-associated endothelial cells, normal tissue and remnant cancer cells) that initially survive irradiation may elicit factors.These factor(s) may transduce and alter the physiology of the neighboring bystander cells[18-22]by paracrine feedback signaling that may pre-dispose the cells to carcinogenic processes[23]in normal tissue, and tumor cell growth in surviving cancer cells leading to tumor recurrence at later time.Reports from our laboratory demonstrated that radiation at doses used in fractionated radiotherapy could selectively induce the DNA-binding activity of nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)[24-26]and the occurrence of a positive feedback loop between tumor necrosis factor-alpha (TNF-α) and NF-κB.Other published reports also revealed the possible occurrence of autocrine loop mechanisms upon radiation exposure[27-29].NF-κB-dependent cellular functions include cytoprotection, oxidative stress response,anti-apoptosis, and transformation[30-33], so occurrence of processes such as signal amplification through cytokine mediator and NF-κB upon radiation exposure may ultimately cause cells at the treatment site to undergo tumor cell proliferation.

Figure 2.Typical MAPK signaling pathway.

TNF-α Stimulates NF-κB Activation

Depending upon cell type/strain, TNF-α exerts its function through two different pathways with opposite effects[34]: promotion or attenuation of apoptosis in target cells.Attenuation of the apoptotic pathway may largely be attributed to the capacity of TNF-α to initiate a preferred NF-κB-dependent signaling.Recently this mechanism has been demonstrated bothin vitrousing human primary cells[35]andin vivoin hepatic ischemiareperfusion[36].The initial steps in TNF-α pathway to activate NF-κB are fairly well understood[37].Interaction of TNF-α with one of the two distinct surface receptors TNFR1 (also called P55;)[38]leads to the recruitment of tumor necrosis factor receptor type 1-associated death domain protein (TRADD) [but not Fas-associated protein with death domain (FADD)] to the intracellular portion of the receptor[39].Receptor-bound TRADD now recruits both protein kinase designated receptor-interacting protein (RIP) and the ring/zinc finger protein TNF receptor associated factor 2 (TRAF2)[40,41].These two adaptor proteins are thought to interact with NF-κB-inducing kinase (NIK)[42]which can directly phophorylate and activate IκB kinases[42-44].IκB kinases then phosphorylate IκB Family members, which dissociate and undergo proteolytic degradation and release active NF-κB complex.Concurrently,TNF-α-generated free radicals[45]could also be involved in activating NF-κB directly through other mechanisms.

NF-κB is an inducible transcriptional regulator,ubiquitously expressed by human and other mammalian cells[46-49].Our group[24-26]and others[50]have demonstrated that radiation at doses ranging from 0.1 to 2 Gy could activate NF-κB.Of several transcription factors evaluated in different mammalian cells, NF-κB is the one that is profoundly activated in irradiated cell population[51,52].This pathway once is induced, it will proceed via phosphorylation of the inhibitor subunits IκB-α and IκB-β[29,53,54].The phosphorylation of inhibitor subunits is followed by ubiquitination and degradation,resulting in the release of active NF-κB.Upon activation,NF-κB translocates into the nucleus, binds sequence-specifically to the promoter/enhancer region of various target genes and transactivates their expression[55-60].Recent progress in molecular cloning analysis has disclosed the presence of one or more NF-κB binding site in the promoter/ enhancer region of TNF-α[31,55,57].NF-κB-mediated TNF-α expression by several inducers has been clearly demonstrated by several laboratories.Also, a recent study has shown a positive autocrine loop in human mast cells stimulated with anti-IgE that augments the activation of NF-κB and initiates the NF-κB-TNF-α feedback cycle[27].In addition,a study demonstrating autocrine effects of fibroblast growth factor (bFGF) in repair of radiation damage of endothelial cells and a study showing an increased mRNA and protein levels of TNF-α that remain in elevated levels as long as 5 days post-exposure clearly indicated the possibility of a feedback mechanism in which TNF-α induces activation of NF-κB, which in turn regulates TNF-α expression.

NF-κB Regulates Survival Advantage

The active form of NF-κB could induce transcription of antiapoptotic gene products[61-65]and initiate transcriptional activation of telomerase.The antiapoptotic pathway, in conjunction with telomerase activation through TNF-α-mediated NF-κB activation in response to radiation could thus impart survival advantage in the bystander cells.

NF-κB induces activation of apoptosis inhibitor protein family.Transfection studies as well as embryonic lethality of NF-κB p65 knockout mice, as a consequence of extensive apoptosis in the liver, strongly support the anti-apoptotic role of NF-κB[66].Further cells lacking the p65 subunit of NF-κB or cells over-expressing an IκB-α mutant showed enhanced susceptibility to apoptosis[67].Activation of NF-κB and the duration of the activation are likely to dictate the anti-apoptotic pathway through several downstream effectors/mediators including cellular inhibitors of apoptosis (cIAPs)[61-63,67]and bcl family proteins[68,69].cIAPs, in general, can inhibit apoptosis induced by both death receptors and a mitochondria-dependent pathway.cIAP such as surviving expressed in a cell cycle-dependent manner at the G2/M phase of the cell cycle[70,71].Surviving thus inhibits the normal apoptotic pathway at this check point[72,73]and allows the cells to progress through mitosis and continue cell division.NF-κB also controls the B-cell lymphoma (bcl) family of proteins that inhibit apoptosis through B-cell lymphoma-extra large (bcl-xL) proteins.Substantial evidence indicated that NF-κB could regulate these proteins at the transcriptional level by binding to their promoter/enhancer region[74,75]or indirectly through CRE/SP-1 cooperative regulation[74].Studies have indicated that, NF-κB- mediated BCL-2 expression may be initiated through TNF-α signaling[76].Since these factors must act in concert, studying the alterations in a single protein may not yield complete information.Mice lacking single apoptotic factor fail to exhibit massive apoptosis and often are believed to function through compensatory mechanism[77].Accumulating evidence points out that upregulation of telomerase allows cells to escape from senescence and proliferate indefinitely.Ectopic expression of telomerase catalytic subunit telomerase reverse transcriptase (hTERT) in telomerase-negative cells is sufficient to restore telomerase activity and extend their life span[78].On the other hand, introduction of dominant-negative hTERT into immortalized cells limits their growth.In addition,the relationship between telomerase activation and apoptosis is apparent and an inverse relationship of telomerase activity and programmed cell death has been well documented[79-82].Recently, it was shown that activation of NF-κB by phorbol 12-myristate 13-acetate(PMA) enhanced the activity of the native TERT promoter in mouse hepatoma cells[83].Thus, it is evident that NF-κB regulates transcription of the telomerase catalytic subunit.Other than direct transactivation of hTERT, it is possible that NF-κB may also regulate telomerase activity through several other mechanisms; for example, NF-κB activation of Myc expression may lead to subsequent increase in TERT expression[84].Alternatively, by interfering with the repressors of TERT activity (Wilm’s tumor 1 suppressor gene), Mad, and histone deacetylase, NF-κB may maintain persistent telomerase activity[84].

Collectively, these studies strongly support the positive feedback loop.However, there has been no study undertaken to correlate the activation of NF-κB, induction of TNF-α that leads to bystander effect resulting in genomic instability, and survival advantage after exposure to clinically relevant doses of radiation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

1.Nagasawa H, Little JB.Induction of sister chromatid exchanges by extremely low doses of alpha-particles.Cancer Res 2002; 52:6394-6.

2.Azzam EI, de Toledo SM, Gooding T, et al.Intercellular communication is involved in the bystander regulation of gene expression in human cells exposed to very low fluences of alpha particles.Radiat Res 1998;150:497-504.

3.Bourguignon MH, Gisone PA, Perez MR, et al.Genetic and epigenetic features in radiation sensitivity part I: Cell signalling in radiation response.Eur J Nucl Med Mol Imaging 2005; 32:229-46.

4.Shiloh Y.ATM.From phenotype to functional genomics--and back.Ernst Schering Res found Workshop 2002; 36:51-70.

5.Kastan MB, Lim DS.The many substrates and functions of ATM.Nat Rev Mol Cell Biol 2000; 1:179-86.

6.Brown EJ, Baltimore D.Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance.Genes Dev 2003; 17:615-28.

7.Cha RS, Kleckner N.ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones.Science 2002; 297:602-6.

8.Zou L, Elledge SJ.Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.Science 2003; 300:1542-8.

9.Nyberg KA, Michelson RJ, Putnam CW.Toward maintaining the genome:DNA damage and replication checkpoints.Annu Rev Genet 2002;36:617-56.

10.Zhou BB, Elledge SJ.The DNA damage response: Putting checkpoints in perspective.Nature 2000; 408:433-9.

11.Hehnly H, Doxsey S.Polarity sets the stage for cytokinesis.Mol Biol Cell 2012; 23:7-11.

12.Bell S, Klein C, Muller L, et al.P53 contains large unstructured regions in its native state.J Mol Biol 2002; 322:917-27.

13.Bates S, Phillips AC, Clark PA, et al.p14ARF links the tumor suppressors RB and p53.Nature 1998; 395:124-5.

14.Tombes RM, Auer KL, Mikkelsen R, et al.The mitogen-activated protein(MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic.Biochem J 1998; 330:1451-60.

15.Kavanagh BD, Lin PS, Chen P, et al.Radiation-induced enhanced proliferation of human squamous cancer cells in vitro: A release from inhibition by epidermal growth factor.Clin Cancer Res 1995; 1:1557-62.

16.Bublil EM, Yarden Y.The EGF receptor family: Spearheading a merger of signaling and therapeutics.Curr Opin Cell Biol 2007; 19:124-34.

17.Paez JG, Janne PA, Lee JC, et al.EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy.Science 2004; 304:1497-500.

18.Clutton SM, Townsend KM, Walker C, et al.Radiation-induced genomic instability and persisting oxidative stress in primary bone marrow cultures.Carcinogenesis 1996; 17:1633-9.

19.Azzam EI, De Toledo SM, Spitz DR, et al.Oxidative metabolism modulates signal transduction and micronucleus formation in bystander cells from alpha-particle-irradiated normal human fibroblast cultures.Cancer Res 2002; 62:5436-42.

20.Nagar S, Smith LE, Morgan WF.Characterization of a novel epigenetic effect of ionizing radiation: The death-inducing effect.Cancer Res 2003;63:324-8.

21.Munro AJ.Bystander effects and their implications for clinical radiotherapy.J Radiol Prot 2009; 29:A133-42.

22.Nagasawa H, Cremesti A, Kolesnick R, et al.Involvement of membrane signaling in the bystander effect in irradiated cells.Cancer Res 2002;62:2531-4.

23.Boothman DA, Meyers M, Odegaard E, et al.Altered G1 checkpoint control determines adaptive survival responses to ionizing radiation.Mutat Res 1996; 358:143-53.

24.Mohan N, Meltz ML.Induction of nuclear factor kappa B after low-dose ionizing radiation involves a reactive oxygen intermediate signaling pathway.Radiat Res 1994;140:97-104.

25.Prasad AV, Mohan N, Chandrasekar B,.Activation of nuclear factor kappa B in human lymphoblastoid cells by low-dose ionizing radiation.Radiat Res 1994; 138:367-72.

26.Mohan N, Sadeghi K, Reiter RJ, et al.The neurohormone melatonin inhibits cytokine, mitogen and ionizing radiation induced NF-kappa B.Biochem Mol Biol Int 1995; 37:1063-70.

27.Coward WR, Okayama Y, Sagara H, et al.NF-kappa B and TNF-alpha: A positive autocrine loop in human lung mast cells? J Immunol 2002;169:5287-93.

28.Haimovitz-Friedman A, Vlodavsky I, Chaudhuri A.Autocrine effects of fibroblast growth factor in repair of radiation damage in endothelial cells.Cancer Res 1991; 51:2552-8.

29.Grilli M, Chiu JJ, Lenardo MJ.NF-kappa B and rel: Participants in a multiform transcriptional regulatory system.Int Rev Cytol 1993; 143:1-62.

30.Wu J, Dent P, Jelinek T, et al.Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate.Science 1993;262:1065-9.

31.Wang CY, Mayo MW, Baldwin AS Jr.TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-kappaB.Science 1996;274:784-7.

32.Lenardo MJ, Baltimore D.NF-kappa B: A pleiotropic mediator of inducible and tissue-specific gene control.Cell 1989; 58:227-9.

33.Szarek E, Cheah PS, Schwartz J, et al.Molecular genetics of the developing neuroendocrine hypothalamus.Mol Cell Endocrinol 2010; 323:115-23.

34.Chen Q, Casali B, Pattacini L, et al.Tumor necrosis factor-alpha protects synovial cells from nitric oxide induced apoptosis through phosphoinositide 3-kinase akt signal transduction.J Rheumatol 2006; 33:1061-8.

35.Relic B, Bentires-Alj M, Ribbens C, et al.TNF-alpha protects human primary articular chondrocytes from nitric oxide-induced apoptosis via nuclear factor-kappaB.Lab Invest 2002; 82:1661-72.

36.Teoh N, Leclercq I, Pena AD, et al.Low-dose TNF-alpha protects against hepatic ischemia-reperfusion injury in mice: Implications for preconditioning.Hepatology 2003; 37:118-28.

37.Liu ZG, Hsu H, Goeddel DV, et al.Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death.Cell 1996; 87:565-76.

38.Tartaglia LA, Goeddel DV.Two TNF receptors.Immunol Today 1992;13:151-3.

39.Hsu H, Xiong J, Goeddel DV.The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation.Cell 1995; 81:495-504.

40.Hsu H, Shu HB, Pan MG, et al.TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways.Cell 1996;84:299-308.

41.Hsu H, Huang J, Shu HB, et al.TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex.Immunity 1996;4:387-96.

42.Lee FS, Hagler J, Chen ZJ, et al.Activation of the IkappaB alpha kinase complex by MEKK1, a kinase of the JNK pathway.Cell 1997; 88:213-22.

43.Karin M, Delhase M.JNK or IKK, AP-1 or NF-kappaB, which are the targets for MEK kinase 1 action? Proc Natl Acad Sci USA 1998; 95:9067-9.

44.Malinin NL, Boldin MP, Kovalenko AV, et al.MAP3K-related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1.Nature 1997; 385:540-4.

45.Singh N, Khanna N, Sharma H, et al.Insights into the molecular mechanism of apoptosis induced by TNF-alpha in mouse epidermal JB6-derived RT-101 cells.Biochem Biophys Res Commun 2002; 295:24-30.

46.Yang JQ, Zhao W, Duan H, et al.v-ha-RaS oncogene upregulates the 92-kDa type IV collagenase (MMP-9) gene by increasing cellular superoxide production and activating NF-kappaB.Free Radic Biol Med 2001; 31:520-9.

47.Baeuerle PA, Baltimore D.I kappa B: A specific inhibitor of the NF-kappa B transcription factor.Science 1988; 242:540-6.

48.Schreck R, Rieber P, Baeuerle PA.Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1.EMBO J 1991; 10:2247-58.

49.Baeuerle PA, Baltimore D.Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor.Cell 1988; 53:211-7.

50.McBride WH, Pajonk F, Chiang CS, et al.NF-kappa B, cytokines,proteasomes, and low-dose radiation exposure.Mil Med 2002; 167:66-7.

51.Brach MA, Hass R, Sherman ML, et al.Ionizing radiation induces expression and binding activity of the nuclear factor kappa B.J Clin Invest 1991;88:691-5.

52.Sahijdak WM, Yang CR, Zuckerman JS, et al.Alterations in transcription factor binding in radioresistant human melanoma cells after ionizing radiation.Radiat Res 1994; 138:S47-51.

53.Ghosh S, Baltimore D.Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B.Nature 1990; 344:678-82.

54.Libermann TA, Baltimore D.Activation of interleukin-6 gene expression through the NF-kappa B transcription factor.Mol Cell Biol 1990;10:2327-34.

55.Osborn L, Kunkel S, Nabel GJ.Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B.Proc Natl Acad Sci USA 1989; 86:2336-40.

56.Cogswell JP, Godlevski MM, Wisely GB, et al.NF-kappa B regulates IL-1 beta transcription through a consensus NF-kappa B binding site and a nonconsensus CRE-like site.J Immunol 1994; 153:712-23.

57.Collart MA, Baeuerle P, Vassalli P.Regulation of tumor necrosis factor alpha transcription in macrophages: Involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B.Mol Cell Biol 1990;10:1498-506.

58.Shakhov AN, Collart MA, Vassalli P, et al.Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages.J Exp Med 1990;171: 35-47.

59.Ho YS, Howard AJ, Crapo JD.Molecular structure of a functional rat gene for manganese-containing superoxide dismutase.Am J Respir Cell Mol Biol 1991; 4:278-86.

60.Wu H, Lozano G.NF-kappa B activation of p53.A potential mechanism for suppressing cell growth in response to stress.J Biol Chem 1994; 269:20067-74.

61.Chu ZL, McKinsey TA, Liu L, et al.Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control.Proc Natl Acad Sci USA 1997; 94:10057-62.

62.Stehlik C, de Martin R, Kumabashiri I, et al.Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis.J Exp Med 1998;188:211-6.

63.Messadi DV, Doung HS, Zhang Q, et al.Activation of NFkappaB signal pathways in keloid fibroblasts.Arch Dermatol Res 2004; 296: 125-33.

64.Heckman CA, Mehew JW, Boxer LM.NF-kappaB activates bcl-2 expression in t(14;18) lymphoma cells.Oncogene 2002; 21:3898-908.

65.Opipari AW Jr, Hu HM, Yabkowitz R, et al.The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity.J Biol Chem 1992;267:12424-7.

66.Sonenshein GE.Rel/NF-kappa B transcription factors and the control of apoptosis.Semin Cancer Biol 1997; 8:113-9.

67.Wang CY, Mayo MW, Korneluk RG, et al.NF-kappaB antiapoptosis:Induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation.Science 1998; 281:1680-3.

68.Sumitomo M, Tachibana M, Nakashima J, et al.An essential role for nuclear factor kappa B in preventing TNF-alpha-induced cell death in prostate cancer cells.J Urol 1999; 161:674-9.

69.Beg AA, Baltimore D.An essential role for NF-kappaB in preventing TNF-alpha-induced cell death.Science 1996; 274:782-4.

70.Kato K, Takeuchi H, Miyahara N, et al.Distinct orders of GalNAc incorporation into a peptide with consecutive threonines.Biochem Biophys Res Commun 2001; 287:110-5.

71.Li F, Ambrosini G, Chu EY, et al.Control of apoptosis and mitotic spindle checkpoint by survivin.Nature 1998; 396:580-4.

72.Ambrosini G, Adida C, Altieri DC.A novel anti-apoptosis gene, survivin,expressed in cancer and lymphoma.Nat Med 1997; 3:917-21.

73.Grossman D, Kim PJ, Schechner JS, et al.Inhibition of melanoma tumor growth in vivo by survivin targeting.Proc Natl Acad Sci USA 2001;98:635-40.

74.Ciuffreda L, Del Bufalo D, Desideri M, et al.Growth-inhibitory and antiangiogenic activity of the MEK inhibitor PD0325901 in malignant melanoma with or without BRAF mutations.Neoplasia 2009; 11:720-31.

75.Dong QG, Sclabas GM, Fujioka S, et al.The function of multiple IkappaB :NF-kappaB complexes in the resistance of cancer cells to taxol-induced apoptosis.Oncogene 2002; 21:6510-9.

76.Tamatani M, Che YH, Matsuzaki H, et al.Tumor necrosis factor induces bcl-2 and bcl-x expression through NFkappaB activation in primary hippocampal neurons.J Biol Chem 1999; 274:8531-8.

77.Karin M, Lin A.NF-kappaB at the crossroads of life and death.Nat Immunol 2002; 3:221-7.

78.Wang Z, Yi J, Li H, et al.Extension of life-span of normal human fibroblasts by reconstitution of telomerase activity.Shi Yan Sheng Wu Xue Bao 2000;33:129-40.

79.Burger AM, Double JA, Newell DR.Inhibition of telomerase activity by cisplatin in human testicular cancer cells.Eur J Cancer 1997; 33:638-44.

80.Kondo S, Tanaka Y, Kondo Y, et al.Antisense telomerase treatment:Induction of two distinct pathways, apoptosis and differentiation.FASEB J 1998; 12:801-11.

81.Mandal M, Kumar R.Bcl-2 modulates telomerase activity.J Biol Chem 1997; 272:14183-7.

82.Fu W, Begley JG, Killen MW, et al.Anti-apoptotic role of telomerase in pheochromocytoma cells.J Biol Chem 1999; 274:7264-71.

83.Yin L, Hubbard AK, Giardina C.NF-kappa B regulates transcription of the mouse telomerase catalytic subunit.J Biol Chem 2000; 275:36671-5.

84.Wang J, Xie LY, Allan S, et al.Myc activates telomerase.Genes Dev 1998;12:1769-74.