RESEARCH ARTICLE

Part II—mechanism of adaptation: A549 cells adapt to highconcentration of nitric oxide through bypass of cellcycle checkpoints

Madeeha Aqil & Zane Deliu & Kim M. Elseth &

Grace Shen & Jiaping Xue & James A. Radosevich

Received: 17 July 2013 /Accepted: 14 October 2013 /Published online: 17 November 2013# International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Previous work has shown enhanced survivalcapacity in high nitric oxide (HNO)-adapted tumor cells. InPart I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UVand X-ray radiation treatment. These cells exhibit increasedexpression of proteins involved in DNA damage recognitionand repair pathway, both the non-homologous end joiningpathway and homologous recombination. These includeKu80, DNA-PK, XLF ligase and MRN complex proteins.Further, the A549-HNO cells show high levels of ATM,ATR, Chk1 and Chk2, and phospho-p53. Activation of thesemolecules may lead to cell cycle arrest and apoptosis due toDNA damage. This is observed in parent A549 cells inresponse to NO donor treatment; however, the A549-HNOcells proliferate and inhibit apoptosis. Cell cycle analysisshowed slowed progression through S phase which will allowtime for DNA repair. Thus, to better understand the increasedgrowth rate in A549-HNO when compared to the parent cellline A549, we studied molecular mechanisms involved in cellcycle regulation in A549-HNO cells. During the initial timeperiod of NO donor treatment, we observe high levels ofcyclin/Cdk complexes involved in regulating various stagesof the cell cycle. This would lead to bypass of G1–S andG2–Mcheckpoints. The HNO cells also show much higherexpression of Cdc25A. Cdc25A activates Cdk moleculesinvolved in different phases of the cell cycle. In addition, there

is enhanced phosphorylation of the Rb protein in HNO cells.This leads to inactivation of Rb/E2F checkpoint regulatingG1–S transition. This may lead to faster progression in Sphase. Thus, all of these perturbations in HNO cells lead toaccelerated cell cycle progression and a higher growth rate.We also assessed expression of cell cycle inhibitors in HNOcells. Interestingly, the HNO cells show a significant declinein p21CIP1 at initial time points, but with prolonged exposure,the levels were much higher than those of the parent cells.This suggests an initial bypass of cell cycle checkpoints asp21CIP1 can inhibit the activity of all cyclin/Cdk complexes.p21CIP1 is also known to inhibit p53-induced apoptosis. Thiscould be important during later phases of the cell cycle toallow time for repair of damaged DNA and thus bettersurvival of HNO cells.

Keywords Nitricoxide .A549 .DNAdamage .Checkpoint .

DNA repair . Cell cycle . Apoptosis . Cyclin/Cdk . p21CIP1

Introduction

Cancer represents one of the best examples of a deregulatedcell cycle [1]. Proper progression through the cell cycle ismonitored by checkpoints that verify whether processes ateach phase of the cell cycle have been accurately completedbefore the cell enters the next phase. In eukaryotic cells, thethree major checkpoints are the G1–S, intra-S, and G2–Mcheckpoints [2]. These are mainly controlled by cyclins andcyclin-dependent kinases (Cdks), which sequentially act in G1to initiate the S phase and in G2 to initiate mitosis. Cdk activityrequires binding of specific cyclins which act as a regulatorysubunit. The expression of cyclins is tightly regulated duringdifferent phases of the cell cycle, thus regulating Cdk activityin a timely manner [3]. Cancer-associated mutations lead to

M. Aqil : Z. Deliu :K. M. Elseth :G. Shen : J. Xue :J. A. Radosevich (*)Department of Oral Medicine and Diagnostic Sciences, College ofDentistry, University of Illinois at Chicago, 801 S. Paulina St.,Chicago, IL 60612, USAe-mail: jrados@uic.edu

J. A. RadosevichJesse Brown VAMC, Chicago, IL 60612, USA

Tumor Biol. (2014) 35:2417–2425DOI 10.1007/s13277-013-1319-5

frequent deregulation of certain Cdk–cyclin complexes,resulting in continued proliferation or untimely entry into thecell cycle [1, 4, 5]. Cdk activity is regulated by two families ofcell cycle inhibitors: (1) the INK4 family includingp16INK4A, p15INK4B, and p18INK4C and (2) the Cip andKip (kinase inhibitor protein) family inhibitors includingp21CIP1, p27KIP1, and P57KIP2 [6]. Any assault on DNAintegrity due to endogenous or exogenous sources leads toactivation of DNA damage response proteins [7].

We have earlier shown in different adenocarcinoma andsquamous carcinoma cell lines that HNO adaptation leads toenhanced growth behavior and resistance to chemotherapyand radiation treatments [8–13]. In Part I of this two-part seriesof manuscripts, we have shown that HNO-adapted A549 cellsshow enhanced expression of key components of the non-homologous end joining (NHEJ) repair pathway andhomologous recombination compared to parent cells. Further,A549-HNO cells have induced expression of DNA damagecheckpoint kinases (Chk1 and Chk2) and phospho-p53(Ser15). Checkpoint kinases and phospho-p53 can regulatethe function and expression of cell cycle inhibitor moleculeswhich can in turn regulate Cdks. A549-HNO cells alsodemonstrated increased activation of anti-apoptotic pathways.Thus, in the present study we monitored the status of key cellcycle regulators in response to NO exposure in both parentaland HNO cells to understand the effect of activatedcheckpoint machinery in HNO cells on cell cycle progression.

Materials and methods

Cell culture and cell lines

Except where noted, all media and supplements werepurchased from Invitrogen (CA, USA). The high nitricox ide -adap ted A549 (A549-HNO) human lungadenocarcinoma cell line was previously prepared [14]. Inbrief, A549 cells were grown in RPMI-1640 mediasupplemented with 10 % fetal calf serum inactivated at56 °C for 30 min, 100 U/mL penicillin, 100 μg/mLstreptomycin, 2 mM L -glutamine, and 2.5 μg/mLamphotericin B solution. Cells were grown in a humidifiedincubator at 37 °C and 5 % CO2. Cells were graduallyintroduced to increasingly higher levels of the nitric oxide(NO) donor DETA-NONOate (at 50-μM increments) overthe course of approximately 65 days, reaching a finalconcentration of 600 μM DETA-NONOate. Due to the shorthalf-life of DETA-NONOate (approximately 38 h at 37 °Cand pH 7.4), media was refreshed every 2–3 days. As with ourprevious studies, the analogous A549 “parental” cell line,grown without additional NO donor (i.e., the original,commercially available cell line prior to the adaptationprocess), was also used in this study.

Western blot analysis

Western blot analysis was used to investigate the differences inthe cell cycle pathway between the A549 parental and A549-HNO cells. Cells were washed twice in cold 1× PBS and lysedin 1× RIPA buffer with protease inhibitor tablet (Roche,Germany). The lysate was sonicated and the concentration ofprotein was determined by the BioRAD assay (Bio-Rad,Hercules, CA, USA). Samples were prepared in a samplebuffer containing lithium dodecyl sulfate and were separatedin Tris–acetate polyacrylamide gels using a Tris–tricine–SDS–sodium bisulfite buffer, pH 8.2, as electrophoresis buffer andblotted to a polyvinylidene fluoride (PVDF) membrane [15].Primary antibodies, Cdc25A #3652, phospho-Cdc25C(Ser216) #4901, Cdc25C #4688, p21CIP1 #2947, p27KIP1#3698, p15INK4B #4822, phospho-Rb (Ser780) #9307,phospho-Rb (Ser795) #9301, phospho-Rb (Ser807/811)#9308, Rb #9309, CDK1 #9112, CDK2 #2546, cyclin B1#4138, cyclin E #4129, cyclin A #4656, CDK4 #2906,CDK6 # 3136, CDK7 #2916, and cyclin D3 #2936, are fromCell Signaling. Anti-β-actin clone AC-15 (Cat# A544) wasobtained from SIGMA (St. Louis, MO, USA). Secondary anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody#7074 and anti-mouse IgG HRP-linked antibody #7076 arealso from Cell Signaling (Boston, MA, USA).

Gene chip assay

The gene chip experiment was performed as described in PartI of this two-part series of papers [16].

Results

Our previous work shows enhanced DNA damage responsesignaling in A549-HNO cells compared to parent cells resultingin activation of checkpoint kinases and p53. The HNO cellsalso exhibited increased anti-apoptotic signaling andderegulation of the cell cycle. Here we characterized molecularmechanisms responsible for cell cycle modulation in these cellsby analyzing expression of various cyclins and cyclin-dependent kinases and the cell cycle activators and inhibitorsthat regulate Cdk activity and thus cell cycle progression.Figure 1 shows a schematic of cell cycle phases with differentregulator proteins.

Cdc25A is upregulated in A549-HNO in a NO-dependentmanner

An important positive regulator of the cell cycle is the dual-specificity phosphatase Cdc25. Cdc25 regulates the cell cyclethrough dephosphorylation and thereby activating cyclin-dependent kinases. In humans, there are three isoforms of

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Cdc25: Cdc25A, Cdc25B, and Cdc25C [17]. Cdc25Afunctions throughout the cell cycle. It activates both Cdk2–cyclin E (an S-phase kinase) and Cdk1–cyclin B (a keyregulator of G2–M transition) [18, 19]. In response to DNAdamage, Chk1 and Chk2 phosphorylate Cdc25A which israpidly degraded through the ubiquitin pathway [20, 21].Degradation of Cdc25A is important for activation of S andG2 cell cycle checkpoints in response to DNA damage [22].Cdc25B functions at the G2/M transition of the cell cycle [23]and Cdc25C acts during M phase to sustain the activity ofCdk1–cyclin B [24]. We have shown in our previous workthat enhanced activation of ataxia telangiectasia mutated(ATM) and ataxia telangiectasia and Rad3-related (ATR)kinases in A549-HNO cells leads to phosphorylation andactivation of downstream effector kinases, Chk1 and Chk2.It was observed that total Cdc25C levels were the same inA549 and A549-HNO cells. Also, it was constitutivelyphosphorylated at serine 216 in an NO-independent mannerin both parental and HNO cells until 8 h post NO treatment. At16 h the expression of Cdc25C and phospho-Cdc25C wasdiminished in parent cells, but was sustained in HNO cells(Fig. 2). Further comparing the levels of total and phospho-Cdc25 shows that almost all Cdc25C is present in thephosphorylated form. The expression level of Cdc25A wasalso assessed in these cells. Interestingly, it was highlyinduced in both parental and HNO cells in response to theNO donor. At 16 h the expression in HNO cells was very highcompared to that in parent cells (Fig. 2). Failure in Cdc25Adegradation leads to impairment in all three checkpoints. Thisresults in an accelerated entry of cells into S phase and mitosisand thus bypasses DNA damage and replication checkpoints.Sustained upregulation of Cdc25A in response to NO-inducedDNA damage might be one of the mechanisms for aggressiveproliferation of high NO cells.

G1 and G2 checkpoints are bypassed in A549-HNO cells

In the cell cycle, the Cdk4/Cdk6/cyclin D complex is associatedwithG1 phase. During lateG1 phase, theCdk2/cyclin E complexis activated which facilitates entry into S phase. The Cdk2/cyclinA complex is important for maintenance of S phase, whereasCdk1/cyclinA is important for G2 phase. For entry intoMphase,the Cdk1/cyclin B complex is activated, which is critical for theG2–M checkpoint and maintenance of M phase (Fig. 1) [3].HNO cells are able to proliferate under NO exposure, whichprompted the investigation of the comparison of the expressionpatterns of Cdks and cyclins between parental and HNO cells.

Cdk4 expression was strongly induced by the NO donor inparent cells and approached levels similar to those of HNOcells. In HNO cells, it remained high at all time points and didnot show much change with NO donor treatment (Fig. 3). In

Fig. 1 A schematicrepresentation of the cell cyclewith different regulatory proteins

Fig. 2 a Western blot and b densitometry analyses of Cdc25phosphatases in A549 (parent) vs. A549-HNO cells at early time pointsafter DETA-NONOate treatment

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contrast, the expression of Cdk6 decreased initially in bothparental and HNO cells following NO exposure; however, itincreased in HNO cells in a NO-dependent manner at 16 h.With prolonged NO treatment, the levels in HNO cellsdecreased compared to those in parent cells. Cyclin D3 wasupregulated in response to NO in parent cells. In HNO cells, itremained high at all time points independent of the NO donor.With prolonged treatment for 72 h, cyclin D3was high in HNOcells whereas Cdk4 levels were similar. The NO-responsiveexpression patterns of Cdk6 suggest that Cdk6/cyclin D3 mayspecifically operate in G1 phase under NO stress in A549 cells.

Next, we checked for expression of the cyclin E/Cdk2complex. Cdk2 was upregulated initially in response to NOtreatment in parent cells till 8 h but started decreasing at 16 h.In HNO cells, the Cdk2 expression was independent of NOexposure. With prolonged exposure, the levels were the same inboth cell types (Fig. 3). Cyclin E levels also increased in bothparent and HNO cells in response to NO exposure; however,

sustained expression was only maintained in the HNO cells at16 h as well as 72 h. The high level of cyclin E/Cdk2 complexsuggests that the G1–S checkpoint is impaired in the HNO cells.This can lead to an accelerated entry of HNO cells in S phase.

For maintenance of S phase, the cyclin A/Cdk2 complex isimportant; thus, we assessed cyclin A expression status [25].Cyclin A levels increased initially in response to NO treatmentin both parent andHNOcells; however, with prolonged exposureat 72 h, the levels were similar in both cell types (Fig. 3). Duringlate S phase, cyclin A/Cdk1 complexes are formed, and they areimportant for G2 phase, whereas cyclin B/Cdk1 complexes arecrucial for the G2–Mcheckpoint. Expression of Cdk1 and cyclinB increases in HNO cells in response to NO donor treatment,whereas the levels in the parent cells remain similar initially butdecrease slightly at 16 h (Fig. 3). However, with prolonged NOdonor treatment at 72 h, the Cdk1 and cyclin B1 expression isdiminished in HNO cells. Thus, at early time points during NOdonor treatment, i.e., before 24 h, the G1–S and G2–M

Fig. 3 a Western blot and b densitometry analyses of various cyclins and Cdk proteins in A549 (parent) vs. A549-HNO cells at early and late timepoints after DETA-NONOate treatment

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checkpoints are bypassed in HNO cells, suggesting enhancedproliferation of the HNO cells. On the other hand, withprolonged NO donor treatment, the A549-HNO cells showdiminished expression of cyclin B and Cdk1 which impliesactivation of the G2–M checkpoint, thus prolonging S phase.

We also investigated the level of expression for other Cdksthat are involved in transcription, like Cdk7. Cdk7 is at thecrossroad of transcription, DNA repair, and cell cycle controlby forming complexes with the general transcription factorTFIIH [26]. It was observed that Cdk7 was responsive to NOstimulation bymoderately increasing expression levels in bothparental and HNO cells (Fig. 3). With prolonged exposures at72 h, the levels of Cdk7 were slightly higher in HNO cells.

Enhanced Rb inactivation in A549-HNO cells

One important target of cyclin D/Cdk4/Cdk6 and cyclin E/Cdk2complexes is the retinoblastoma tumor suppressor protein (Rb2).It is also a key regulator of G1–S transition during the cell cycle.In its non-phosphorylated active state, pRb binds to E2F at thepromoters of E2F-regulated cell proliferation genes. In this way,Rb prevents assembly of transcriptional transactivationcomplexes and instead leads to assembly of other complexeswhich alter the local chromatin structure, thereby silencing geneexpression. In response to a positive growth signal, the activecyclin D/Cdk4 (or Cdk6) complexes phosphorylate Rb, causingrelease and hence activation of the E2F transcription factor.Induction of cyclin E by E2F initiates entry into S phase.Subsequent additional phosphorylation of Rb by the cyclin E–Cdk2 complex creates a positive feedback loop for E2F-drivengenes. We observed constitutive phosphorylation (Ser780,Ser795) and inactivation of Rb protein in both A549-HNO andparent cells (Fig. 4a). However, the phosphorylation of Rb atSer780/795 was enhanced in response to NO treatment in bothcell types. Sustained and further hyperphosphorylation of Rb atSer780/795 after 16 h of NO treatment was only observed in theHNO cells (Fig. 4). In contrast, phosphorylation of Rb at Ser807/811 occurred in a NO-dependent manner, which was exclusivelyinduced in response to NO exposure both in parental and HNOcells (Fig. 4). Interestingly, the total Rb levels were similar inparent and HNO cells in response to NO donor treatment up to16 h. However, with prolonged NO treatment, the levels of totalRb and phospho-Rb were much higher in HNO cells than inparent cells (Fig. 4). Thus, in high NO-adapted cells, the Rb-mediated G1–S restriction point is impaired due to enhancedphosphorylation and inactivation of Rb. This would result in anenhanced entry of cell in S phase in HNO cells.

ATM/ATR–p53–p21CIP1-operated G1 checkpoint remainsintact in HNO cells

To prevent entry into S phase with damaged DNA, cellstraversing G1 activate the checkpoint-transducing kinases

ATM/ATR and Chk1/Chk2 which, in turn, target two criticaleffectors operating in distinct branches of the G1 checkpoint:the Cdc25A phosphatase and the p53 transcription factor.Since the Cdc25A-operated G1 checkpoint is impaired inA549 cells, we questioned whether the p53-mediated G1checkpoint still operates in A549 cells. We have previouslyshown that phosphorylation of p53 at serine 15 and serine 46was enhanced in HNO cells but diminished in parental cellswith prolonged NO exposure.

The levels of the cell cycle inhibitor proteins p21CIP1,p27KIP1, and p15INK4B were also assessed. Interestingly,p21CIP1 was drastically upregulated in parental cells at the8 h time point compared to 0 h after NO treatment (Fig. 5),suggesting the activation of the G1 checkpoint in parentalcells. It was high in HNO cells initially and gradually reducedwith NO donor exposure. It was completely diminished underNO exposure at the 16 h time point in both parent and HNOcells. At prolonged exposure at 72 h, the HNO cells showedmuch higher levels of p21CIP1 compared to parent cells(Fig. 5). This is consistent with the cells attaining quiescenceand staying in G1 phase after longer exposure of NO. Theexpression level of p27KIP1 was reduced with extended NOtreatment in both parental and HNO cells although at amoderate level (Fig. 5a). The expression of the level ofp15INK4B was also moderately reduced both in parentaland HNO cells in response to NO treatment (Fig. 5).

Gene chip assay

The total gene expression profile of the A549 parent to theA549-HNO cells was compared by carrying out a high-throughput gene chip experiment. This resulted in a total of2,444 genes having a significant (more than 50 %) change inexpression. Among these, 1,561 were upregulated, and 873genes were downregulated. Some of the genes that wecharacterized in this study were also detected in the gene chipexperiment analysis. Table 1 provides a list of these geneswith their corresponding fold change values. The change inexpression in the gene chip experiment correlated with ouranalysis.

Discussion

High nitric oxide-adapted cells demonstrate increasedproliferation rate and enhanced ability to survive under harshconditions [27]. We have shown this ability for severaladenocarcinomas and squamous cell carcinomas cell lines[8–13]. Our previous work shows greater resistance ofA549-HNO cells to UV and X-ray exposure when comparedto the parent cells [10]. The HNO cells also demonstrateenhanced expression of gene products involved in DNAdamage detection, repair, and activation of anti-apoptotic

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pathways. As DNA damage response is known to modulatethe cell cycle, these observations prompted us to study themolecular mechanisms involved in cell cycle regulation ofHNO cells. Progression through the cell cycle involvescooperative action of specific cyclin–Cdk complexes indifferent phases. The Cdk activity is modulated by activatorsand inhibitor proteins [28]. The activator proteins include theCdc25 phosphatase family. There are three isoforms inhumans: Cdc25A, Cdc25B, and Cdc25C. All three areinvolved in regulating G1–S and G2–M checkpoints [24].An important target of Cdc25A is Cdk2 [29], which plays a

key role in late G1–S transition and S phase. Phosphorylationof Cdc25A by Chk1 or Chk2 targets it for ubiquitin-mediateddegradation [29, 30]. A very crucial point to consider is thatthe phosphorylation at different sites in Cdc25A regulates itsdifferent activities under various biological conditions. It isreported that Chk1- and Chk2-mediated phosphorylation ofCdc25A at serine 123 is a prerequisite for ubiquitylation anddegradation. On the other hand, cyclin B–Cdk1-mediatedphosphorylation at Ser17 and Ser115 during mitosis stabilizesCdc25A levels [31]. Interestingly, we observed NO-dependent upregulation and maintenance of high levels of

Fig. 4 a Western blot and bdensitometry analyses ofretinoblastoma protein in A549(parent) vs. A549-HNO cells atearly and late time points afterDETA-NONOate treatment. At72 h, phospho-Rb Ser780 was notdetermined (ND)

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Cdc25A in HNO cells. This might lead to an accelerated entryof HNO cells in S phase through bypass of the G1–Scheckpoint and also increased mitosis. High levels ofstabilized Cdc25A in mitosis are essential for continuedactivation of Cdk1 and thus on accelerated proliferation rate[24]. This could be one of the mechanisms implicated in theobserved high proliferation rate of HNO cells. Further,Cdc25A is also involved in regulating cell motility andinvasiveness. It has been shown in A549 cells thatdownregulation of Cdc25A through siRNA significantlyreduces cell invasiveness [32]. As observed in HNO cellsherein, and also reported in several other cancers, the HNOenvironment in tumors might mediate the upregulation ofCdc25A. This would result in increased invasiveness and

aggressive behavior observed in clinical tumors. In thisregard, a high frequency of Cdc25A overexpression isreported in several types of human cancers includingcarcinomas of the lung, breast, head and neck, and esophagus[33–35]. Moreover, correlation between Cdc25Aoverexpression and both clinically aggressive tumor behaviorand poor survival of patients has been reported [33, 34].Overexpression of Cdc25A in esophageal carcinoma isassociated with deeper tumor invasion, lymph nodemetastasis, tumor size, and poor survival [33]. The HNO cellsshowed sustained and constitutive phosphorylation ofCdc25C at Ser216, resulting in cytoplasmic sequestration.This prevents access to and activation of cyclin/Cdkcomplexes [36]. Interestingly, a recent report shows highlevels of nuclear phospho-Cdc25C (Ser216) in vulvarsquamous cell carcinoma [37]. In the nucleus, phospho-Cdc25C is unable to interact with 14-3-3 proteins whichnormally shuttle it back to the cytoplasm and thus can activatecyclin/Cdk complexes in the nucleus.

One of the crucial regulators of the cell cycle in G1 andG1–S phase transition is the tumor suppressor protein Rb. Inquiescent cells and early G1, Rb is hypophosphorylated andbound to E2F. This inhibits expression of E2F-regulated genesincluding, cyclin E and Cdc25A. Phosphorylation of Rb byboth Cdk4/Cdk6–cyclin D and Cdk2–cyclin E occurs in lateG1. This leads to the dissociation of Rb–E2F complexes andactivation of E2F genes [38]. We observed constitutive andhigh levels of hyperphosphorylated Rb protein at Ser780,Ser795, and Ser807 in A549-HNO cells. This may act as adriving force for continuation of the cell cycle in A549-HNOcells exposed to NO. We further investigated expression ofdifferent cyclin/Cdk complexes involved in maintenance ofvarious phases of the cell cycle. Enhanced expression ofphase-specific cyclin/Cdk complexes was observed in A549-HNO cells compared to parental cells, implying bypass of

Fig. 5 a Western blot and b densitometry analyses of cell cycleinhibitory proteins in A549 (parent) vs. A549-HNO cells at early andlate time points after DETA-NONOate treatment. At 72 h only p21CIP1expression status was determined

Table 1 Gene chip resultscomparing the A549 parent cellline to the A549-HNO cell line

Gene symbol Fold change in A549-HNO cellline compared to A549 parentcells

Gene description

Cyclin A2 +20 Cyclin A2 (CCNA2), mRNA, mitotic G2 checkpoint

Cyclin B1 +11.6 Cyclin B1 (CCNB1), mRNA, mitosis process

Cyclin B2 +94 Cyclin B2 (CCNB2), mRNA, mitosis process

Cyclin D3 +4.4 Cyclin D3 (CCND3), mRNA, cell cycle, G1 phase

Cdk1 +20.1 Cell division cycle 2, G1 to S and G2 to M (CDC2),transcript variant 1, mRNA

Cdk7 −2.3 Cyclin-dependent kinase 7 (MO15 homolog, Xenopuslaevis , Cdk-activating kinase) (CDK7), mRNA,positive regulation of transcription

Cdk9 +2.0 Cyclin-dependent kinase 9 (CDC2-related kinase)(CDK9), mRNA, cell proliferation, transcriptioninitiation and elongation from RNA polymerase IIpromoter, protein amino acid phosphorylation

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G1–S and G2–M checkpoints during early time points aftertreatment with NO donor. However, with prolonged NOdonor treatment, the HNO cells activated the intra-S phasecheckpoint by lowering Cdk1 levels and thus arrest of cells inS phase.

The expression of Cdk inhibitors which are negativeregulators of the cell cycle was also studied herein. Thereare two families of such inhibitors: the INK4 family and theCip/Kip family. The INK4 family includes INK4A, INK4B,INK4C, and INK4D. They specifically inhibit CDK4 andCDK6. The Cip/Kip family includes p21CIP1, p27KIP1,and P57KIP2. They inhibit all cyclins D, E, and A [39, 40].The expression level of p15INK4B was moderately reducedboth in parental and HNO cells in response to NO treatment.This may be one of the mechanisms involved in enhancedCdk4 and Cdk6 activity in G1 phase resulting inhyperphosphorylation and inactivation of Rb. Similarly,p27KIP1 was also slightly reduced in response to NO donortreatment in both parent and HNO cells, thus contributing toan enhanced proliferation rate. In contrast, the expression ofp21CIP1 significantly increased in the parent cells and thendeclined abruptly. In HNO cells, p21CIP1 levels decreasedgradually until 8 h and then decreased to negligible levels at16 h. It should be noted that at this time point the expression ofCdc25A is very high. This would result in bypass of allcheckpoints and thus an enhanced proliferation rate. Withprolonged NO donor treatment, p21CIP1 levels increased inHNO cells to very high levels. This could result in theinhibition of the cell cycle at G1, G2, or S phase dependingon which cyclin/Cdk complex it is binding and inactivating.However, it has been reported that the cyclin D3/Cdk6complex is not inactivated by p21CIP1 [41]. Thus, cellsutilizing this complex in G1 phase can still bypass high levelsof p21CIP1 and enter S phase [39]. High levels of p21CIP1can lead to both pro-apoptotic and anti-apoptotic signalingdepending upon the cellular context. It can bind to apoptoticsignaling kinase (ASK1) in the cytoplasm and inhibit theMAP kinase pathway [41]. It can also bind to procaspase 3and prevent its processing and activation [42]. Further,activated ERK2 can phosphorylate p21CIP1 leading to itscytoplasmic translocation and ubiquitin-mediated degradation[6]. Expression of p21CIP1 is transcriptionally regulated byp53. It is well known that high levels of p21CIP1 protect cellsfrom p53-dependent apoptosis during DNA damage andinduce cell cycle arrest [5]. Thus, inhibition of apoptosis andrestriction of HNO cells in S phase might be due to high levelsof p21CIP1. Sustained and enhanced ATR/Chk1/p53/p21CIP1 DNA damage checkpoint activation in A549-HNOcells may also correlate with enhanced DNA repair capacity tomaintain the genome integrity of A549-HNO cells. Wepropose that high expression of p21CIP1 in HNO cells withprolonged HNO treatment is one of the mechanisms toprevent p53-mediated apoptosis, which is normally seen in

the parent cells. This would lead to cell cycle arrest, in thiscase, in S phase for HNO cells, and provide time for DNArepair. Thus, during long-term NO adaptation, A549-HNOcells gradually acquired and adapted an enhanced cell cycleregulation network, which distinguishes HNO cells fromparental cells in response to NO exposure and also leads toimproved survival in harsh conditions.

It is well established that deregulation of the cell cycle is ahallmark of cancer development and progression.Consequently, several small molecules that target specific cellcycle regulatory proteins have been identified and are underclinical trials [43, 44]. Our study highlights and furthercontributes to the importance of DNA damage checkpointsand cell cycle regulation in providing resistance to tumor cellsthat are HNO expressers. HNO tumors show poor prognosisin the clinic. Thus, specific molecules identified in our study,for example, p21CIP1, can be used as a potential biomarker toprofile patients with resistant tumors to be able to provide thebest possible drug combinations. Further, specific inhibitorscan be designed that target the ATR/Chk1/p53/p21CIP1 axisin resistant tumor cells to impair their DNA repair ability andmake them more susceptible to radiation and chemotherapy.

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