Protein cysteine sulfinic acid reductase (sulfiredoxin) as a regulator of cell proliferation and drug response

Abstract

Sulfiredoxin (Srx) is one of a family of low molecular weight sulfur containing proteins linked with maintenance of cellular redox balance. One function of Srx is the reduction of cysteine sulfinic acid to sulfenic acid in proteins subject to oxidative stress. Other redox active protein families have multiple functions in regulating redox and controlling proliferation/death pathways; increased Srx has been linked with oncogenic transformation. To explore the biological functions of Srx in tumors, we established cell lines that overexpress Srx. Enhanced levels of Srx promoted cell proliferation and enhanced cell death following cisplatin. Srx overexpression triggered an alteration in expression and phosphorylation of cell cycle regulators p21, p27 and p53; stabilized the phosphatase PTEN and, importantly, interacted directly with, and enhanced the activity of, phosphatase PTP1B. In turn, this promoted Src kinase activity by dephosphorylating its inhibitory tyrosine residue (Y530). Srx expression was stimulated by cell exposure to certain growth factors. These data support a role for Srx in controlling the phosphorylation status of key regulatory kinases through effects upon phosphatase activity with an ultimate effect on pathways that influence cell proliferation.

Introduction

Thiol disulfide regulation is critical in normal physiology and aberrant redox balance has been linked with a number of human pathologies including, aging, neurodegenerative disorders, cardiovascular disease and cancer (Finkel, 2003; Rhee et al., 2005). In human cancers, it has been proposed that elevated levels of reactive oxygen species (ROS) are causal in tumorigenesis and progression (Finkel, 2003; Gius and Spitz, 2006) and redox-targeted therapeutics have been used to increase tumor sensitivity to conventional clinical treatments (Gius and Spitz, 2006; Tew, 2007). Cysteine is one of the least common amino acids in mammalian proteins and in certain proteins (such as NFκB, cJun, actin, some phosphatases) redox regulation of cysteine residues regulates their structure, function and subcellular localization (Martinez-Ruiz and Lamas, 2007; Tew, 2007; Townsend, 2007).

The sulfur residue in cysteine can be present in multiple chemical forms (Jacob et al., 2004) including, thiol (–SH), deprotonated thiolate (–S⁻), disulfide bond (–S—S–) and complexed in metal coordination (for example, in zinc finger). In addition, cysteine can be nitrosylated, S-glutathionylated and oxidized to sulfenic acid (–SOH), sulfinic acid (SO₂H), and sulfonic acids (–SO₃H). These multiple forms have created an evolutionary opportunity that facilitates stress-signaling pathways that converge through the regulation of sulfur oxidation states. Of significance is the relative reversibility. Forward and reverse reaction kinetics frequently mimic those for protein phosphorylation and dephosphorylation. Recently, reversible reduction of protein cysteine sulfinic acid to sulfenic acid and removal of S-glutathionylation (deglutathionylation) from low pKa cysteine residues was shown to be mediated by sulfiredoxin (Srx; Biteau et al., 2003; Findlay et al., 2005, 2006). Both nucleotide and amino acid sequences of Srx show high levels of conservation across species, and even phyla (Biteau et al., 2003; Basu and Koonin, 2005). Presently, peroxiredoxin is the only direct Srx target reported. Based on current sequence databases, Srx appears to be present in eukaryotes and cyanobacteria, whereas homologs of the peroxiredoxin family members have been found in Archeae and Eubacteria. Moreover, functional roles for Srx seem to be different in yeast and mammals (Basu and Koonin, 2005; Bondareva et al., 2007). Oxidative stress can markedly upregulate Srx transcript and protein levels. Srx was also found to be induced by glucose and cAMP analogues in insulin secreting cells (Glauser et al., 2007) and by nitric oxide in primary murine macrophages (Diet et al., 2007). Deletion of yeast Srx reduces the organism’s tolerance to H₂O₂ (Biteau et al., 2003; Bozonet et al., 2005; Vivancos et al., 2005; Demasi et al., 2006). Plant Srx (Arabidopsis thaliana Srx) is also involved in photo-oxidative stress response (Liu et al., 2006; Rey et al., 2007) and mammalian Srx can restore the peroxidase activity of peroxiredoxins (Chang et al., 2004; Jacob et al., 2004; Jonsson et al., 2005; Lee et al., 2005, 2006; Woo et al., 2005; Jeong et al., 2006; Rhee et al., 2007). Invaluable information has been gained from structural data of Srx and its interacting partners (Jonsson et al., 2005; Lee et al., 2005, 2006; Jonsson et al., 2008). The earlier studies of multistage carcinogenesis in mouse epidermal cells demonstrated that Srx transcript levels were induced in the transformed and tumor promoter sensitive cells, but not in cells refractory to transformation (Sun et al., 1995).

Srx appears to function in a coordinate manner with other key redox regulators. Activities of many cellular proteins contribute to redox homeostasis, but several systems constitute the regulatory core including the thioredoxin system (thioredoxin, thioredoxin reductase and NADPH; Holmgren et al., 2005; Lillig and Holmgren, 2007), glutathione system (GSH/GSSG/glutathione-S-transferases (GST); Townsend, 2007), protein disulfide isomerases (Hatahet and Ruddock, 2007) and peroxiredoxins (Jonsson et al., 2007; Rhee et al., 2007). Reduction of protein disulfide by thioredoxin and glutaredoxin is essential for many cellular processes. Thiol regeneration of the oxidized cysteines formed during the reduction process at the active sites of thioredoxin and glutaredoxin is catalysed by an oxidoreductase flavoenzyme, either thioredoxin reductase or glutathione reductase, respectively. The required electrons are transferred from NADPH. In addition, glutaredoxin, in conjunction with cellular GSH/GSSG ratios and GSTs, regulate S-glutathionylation and deglutathionylation processes (Gallogly and Mieyal, 2007; Tew, 2007). It is apparent that Srx expression patterns are species specific and functionalities may differ. For example, whereas yeast induces peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induce Srx as well as other redox relevant mRNAs such as glutamate cysteine ligase, GSTs and metallothioneins (Bondareva et al., 2007). The functions of peroxiredoxins in H₂O₂ scavenging and signaling require thioredoxin and Srx. Dysregulation of these systems are likely to have significant consequences to redox regulation and may have importance in the etiology of a variety of diseases (Findlay et al., 2005; Townsend et al., 2008).

These observations together with our earlier results showing that Srx reversed S-glutathionylation of phosphatases impacting their catalytic activity (Findlay et al., 2005, 2006; Townsend et al., 2006) provided the basis for the present studies to ascertain what role(s) Srx may have in regulating proliferation of tumor cells. We previously reported that PTP1B is cysteine S-glutathionylated in vitro by radicals induced by PABA/NO in the presence of GSH and Srx can promote the deglutathionylation and restore the PTP1B phosphatase activity. In current studies, ectopic overexpression of Srx in lung cancer cells promotes tumor cell growth and increased drug sensitivity and correlates with changes in key proteins controlling cell cycle and drug response patterns. Moreover, Srx can bind to, regulate the stability and restore the activity of specific phosphatases and this is contributory to the observed phenotype.

Results

Overexpression of Srx promotes cell proliferation

A Flag-tagged human Srx-expressing construct was generated and in transient transfection assays was expressed in Hek293 cells at levels detected by both anti-Flag antibody and anti-Srx antibody (Figure 1a). Control vector and expression construct were transfected and stable clones selected. A549-Srx cells had a faster growth rate compared with A549-vector control cells (Figure 1b). A time-course growth graph indicated that the difference primarily occurred in the exponential phase (Figure 1c). Flow cytometry demonstrated that A549-Srx cells have a smaller subpopulation of cells in G₁ and more in S and G₂ phases (Figure 1d). Thus, overexpression of Srx promoted clonal expansion and cell growth, partially driven by changes of cell-cycle profiles.

Figure 1.

Stable cell-line establishment and cell growth. (a) The expression constructs were transfected into Hek293 cells and sulfiredoxin (Srx) expression was detected using anti-Flag and anti-Srx monoclonal antibodies (left two panels). The right panels show expression of Srx in stable cell lines. The top two bands on the second immunoblot (IB) from left are cross-reactive with unknown cellular proteins. (b) Cells (5000) were plated on a 60mm plate and allowed to grow for 7 days. Cell colonies were stained with Sulforhodamine B (SRB). (c) 5 × 10⁴ cells were subcultured in a six-well plate (9.4 cm²). Cells were fixed at each time point, stained with SRB and mean ± s.d. of three experiments were plotted. Asterisk (*) denotes statistical significance (Student’s t-test, which is also plotted as growth curve in the insert panel, white-line (A549-Vector) and black-line (A549-Srx). (d) A typical cell cycle profile (mean of three experiments ± s.d.) with flow cytometry profiles is shown below.

Overexpression of Srx alters key regulators of cell cycle and drug sensitivity

These changes prompted us to examine the expression levels of some of the proteins involved in regulating the cell cycle. Immunoblots (IB) indicated that there were different expression patterns of p27, p21 and p53 in A549-vector versus A549-Srx cells (Figure 2a). Interestingly, clinical studies have demonstrated that low levels and activity of p27, p21 and p53 correlate with poor response rates in lung cancer patients (Hirabayashi, 2002; Singhal et al., 2005; Zhu et al., 2006). We found that p27 levels were significantly lower in A549-Srx cells in basal and cisplatin treated groups. Conversely, p21 and p53 protein levels were elevated in the A549-Srx relative to A549-vector cells. These changes were not demonstrated at the transcript level (using real-time RT-PCR; Supplementary Figure 1). As p27 and p21 act as cyclin-dependent kinase inhibitors and prevent abnormal cell-cycle progression, it is conceivable that low p27 expression could confer a proliferation advantage on the A549-Srx cells. The relevance of the elevated p21 is less clear, but is discussed below in more detail. The relevance of cellular redox balance in determining response to certain chemotherapeutic drugs has been well documented (Fruehauf and Meyskens, 2007). Lung cancer is therapeutically managed with cisplatin (NCI Cancer Bulletin 4(19), 2007). A549-Srx cells had a dose-dependent increased sensitivity to cisplatin and an average of 32% sub-G₁ subpopulation (dead cells) versus 15% in A549-Vector (Figure 2).

Figure 2.

Detection of cell-cycle regulators and sensitivity to DNA damage. (a) The protein level of various cell-cycle regulators was measured by immunoblot (IB) identified at the right side of the panel. (b) Cells at 50–70% confluence were treated with different doses of cisplatin and viable cells stained at 48 h with SRB and presented as mean ± s.d. calculated from three independent experiments. (c) Cells were treated with 3 µg/ml cisplatin for 24 h and dead cells quantified as the sub-G₁ subpopulations (mean ± s.d.) from three independent samples. Flow cytometry profiles of a representative assay are displayed below.

Srx is involved in cellular redox regulation and growth factor signaling

Deletion of yeast Srx reduces its tolerance to hydrogen peroxide and Srx has been shown to restore the functional cysteine residues of peroxiredoxins (Prxs; Biteau et al., 2003; Bozonet et al., 2005; Vivancos et al., 2005; Demasi et al., 2006). We therefore examined whether overexpression of Srx would reduce levels of ROS in mammalian cells. Contrary to expectation, DCF (5- and 6-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester) staining showed that A549-Srx cells had higher basal levels of ROS than control 0cells (Figure 3a). This was further tested and confirmed in the breast cancer line, MCF7 overexpressing Srx by retroviral gene transfer (Figure 3b). Cisplatin and H₂O₂ induced time-dependent ROS production in A549-Srx cells (Figure 3c). Mechanistically, we speculate that higher levels of Srx may alter redox patterns, for example, through enhancing the recycling the cysteine sulfinic acid forms of Prxs, with the consequence that more hyperoxidized Prxs can be reactivated. However, this could interfere with cellular redox homeostasis leading to the paradoxical pattern of ROS seen at later time points. There was no indication of changes in the cellular ratios of GSH/GSSG in A549-Vector and A549-Srx cells (data not shown).

Figure 3.

Measurement of cellular reactive oxygen species (ROS). (a) Stable A549 cells and virally transduced and (b) MCF7 cells were pulse loaded with CM-H₂DCF-DA and mean fluorescence intensity (MFI) measured using flow cytometry. Data are presented as mean ± s.d. of three batches of cells. Sulfiredoxin (Srx) protein expression in MCF7 cells is shown below. (c) Stably transfected A549 cell lines were treated with cisplatin or hydrogen peroxide (H₂O₂) for the indicated times and induced MFI calculated as normalized values relative to each basal MFI. Plots represent mean ± s.d. of three experiments.

A variety of growth factors are known to cause changes in redox balance associated with stimulation of proliferation. Srx transcript levels are positively regulated by growth factors such as epidermal growth factor and insulin (Figure 4a). The quantitative extent of stimulation, as evaluated by activation of extracellular signal-regulated kinase (ERK), is essentially equivalent to growth stimulatory concentrations of H₂O₂ (Figure 4b). Perhaps this reflects a feedback associated with growth factor stimulated oxidation, or is an independent branch of signaling downstream of growth factor exposure.

Figure 4.

Regulation of sulfiredoxin (Srx) by growth factors or oxidation. (a) Relative transcript levels were assessed from total RNA, which was extracted from control and treated cells and used for real-time PCR. The results are presented as ζ = Power (2, −ΔΔCt). All treatments caused a statistically significant induction of Srx transcripts relative to control (P < 0.05). Doses were epidermal growth factor (10 nm), insulin (200 ng/ml) and all treatments were for 2 h. (b) The corresponding endogenous Srx was detected using immunoblot (IB) and the stimulating effect by growth factors was examined by IB of mitogen-activated protein kinase (MAPK) activation.

The influence of Srx on phosphorylation patterns of key growth regulatory proteins

p27 is primarily regulated at the protein level by post-translational mechanisms including phosphorylation-mediated ubiquitin proteasome degradation (Chu et al., 2007; Grimmler et al., 2007; Kaldis, 2007). We detected reduced p27 at the transcript levels in A549-Srx cells (Supplementary Figure 1). We designed experiments to test the model proposed in Figure 5a. To date, at least 10 protein kinases have been shown to differentially phosphorylate seven critical residues in p27 including serine-10 by Akt and mitogen-activated protein kinase, tyrosines-74/88/89 by Src family kinases, and threonines-157/187/198 by Cdk1/2 (Kaldis, 2007). Phosphorylation of these residues promotes p27 degradation. We were particularly interested in the tyrosine phosphorylation pattern in p27. Anti-phosphotyrosine IB of p27 confirmed that p27 is tyrosine-phosphorylated. There was a modest but reproducible increased level of tyrosine phosphorylation in Srx overexpressing cells (1.1 vs 1.0 absolute units; Figure 5b). Further experiments were performed to test these results. IP-coupled Src kinase assays confirmed that p27 is a substrate of Src kinase (Chu et al., 2007; Grimmler et al., 2007) and indicated that Src kinase from A549-Srx cells had a higher kinase activity toward p27 (1.5 vs 1.1 absolute units; Figure 5c). These data imply that either (a) the Src family kinases responsible for the p27 tyrosine phosphorylation are more active in A549-Srx cells or (b) dephosphorylation is diminished.

Figure 5.

Regulation of Src kinase activity by sulfiredoxin (Srx). (a) Proposed model for Srx involvement in regulation of Src kinase and p27. Srx promotes the reversal of cysteine modified PTP1B to its reduced and enzymatically active form; this enhances the basal Src kinase activity through dephosphorylating the C-terminal inhibitory tyrosine-530 by PTP1B, increasing its kinase activity toward p27 at the three tyrosine residues, leading to degradation mediated by an enhanced p27 tyrosine phosphorylation mechanism (bold arrows on figures indicate increase or decrease). (b) p27 was immunoprecipitated from cells using rabbit anti-p27 antibody (sc-817, Santa Cruz) and immunoblotted with mouse anti-phosphotyrosine (61–5800, Zymed/Invitrogen) and HRP-conjugated anti-mouse (715035-150, Jackson ImmunoResearch) antibodies. (c) Src kinase assay. Src kinase was immunoprecipitated from the cells and its kinase activity was assayed as phosphorylation of recombinant p27 in vitro. Bands on the top panel show phosphorylated p27 (the slower mobility band appears to be multiple phosphorylated p27) and bands on the lower panel show protein Coomassie staining of the kinase-assay gel. The nonspecific band on the lower panel was from the immunoprecipitation (IP)-antibody. (d) Phosphorylation of Src kinase at tyrosine-530 (pY530) and tyrosine-419 (p-Y419) was detected by immunoblot. The relative IB band intensities (measured by ImageJ) are numerically shown on the top of each panel where an asterisk (*) denotes a statistically significant value compared to vector controls.

Src kinase activity is critically regulated by phosphorylation at two tyrosine residues (Y530 and Y419), with opposing effects (Yeatman, 2004; Roskoski, 2005). More than 90% of Src in certain cells is phosphorylated at Y530 and phosphorylation of this residue impedes substrate access and is thereby inhibitory, whereas phosphorylation at Y419 is required for kinase activity. A549-Srx cells have reduced phosphorylation at Y530 (0.59 vs 1.1 absolute units) and slightly increased phosphorylation of Y419 (1 vs 1.1 absolute units; Figure 5d) relative to A549-vector cells. These results provide a mechanistic explanation for the increased Src kinase activity in A549-Srx cells. Thus, for Src kinase activation, different phosphatases have been identified as candidates for the dephosphorylation of residue Y530. Among these, PTP1B appears to be prominent. In breast cancer cell lines, more than 50% of the Y530 dephosphorylation of Src was catalysed by PTP1B (Yeatman, 2004; Roskoski, 2005). We previously reported that Srx promoted the deglutathionylation of PTP1B and restored its enzymatic activity post oxidative and nitrosative stress (Findlay et al., 2006).

Srx effects on phosphatases

Protein expression levels of other phosphatases were estimated by IB. Although there were comparable levels of PTP1B, expression of phosphatase and tensin homolog (PTEN) was increased in A549-Srx cells (1.4 fold; Figure 6a). PTEN regulates phosphorylation/activation of Akt. Increased PTEN levels in A549-Srx cells corresponded to a lower activation of phosphorylation of Akt at serine-473 (about threefold). This is consistent with the principle that reversal of protein oxidation by Srx can prevent protein degradation resulting from terminal oxidation. In contrast to the downregulation of Akt signaling, there was a 1.6-fold increase in Erk phosphorylation in Srx overexpressing cells (Figure 6a). Involvement of PTP1B in activation of the Erk pathway has been linked with integrin signaling and Ras GTPase-activating protein (p120RasGAP, Dube and Tremblay, 2005). Although PTP1B protein levels were not altered in A549-Srx cells, we interrogated qualitative changes in PTP1B that might influence kinase pathways. Endogenous PTP1B immunoprecipitated from Srx overexpressing cells had significantly elevated enzymatic activity compared with vector control cells (Figure 6b). Using AMS (4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid) labeling (Biteau et al., 2003), we determined that at least two of the five conserved cysteine residues in PTP1B were modified at basal culture conditions (Figure 6c). Srx overexpressing cells have a reduced intensity of the top band in the AMS-labeling assay, suggesting fewer multiple-cysteine modifications.

Figure 6.

Sulfiredoxin (Srx) targets PTP1B. (a) Cell lysates were prepared and immunoblotted to evaluate the effect of Srx expression on PTP1B and phosphatase and tensin homolog (PTEN). Corresponding activation through phosphorylation of Erk and Akt was also examined. The numbers on the top of each panel are the relative band intensities of phosphorylation after normalization for protein. An asterisk (*) denotes a statistically significant value compared to vector controls. (b) Overexpression of Srx enhances PTP1B activity (P < 0.05). Endogenous PTP1B was immunoprecipitated from cell lysates and PTP1B activity was determined as the amount of released phosphate from a phosphopeptide substrate, detected using Malachite Green Phosphate Detection Kit (arbitrary units). (c) Cell lysates were immunoprecipitated with anti-Srx antibody and co-precipitated PTP1B was detected by immunoblot (right panel). Protein cysteine modifications were detected by AMS labeling (left panel). Cells were fixed with TCA and free cellular thiols blocked with N-ethylmaleimide (NEM) followed by reduction with dithiothreitol (DTT). AMS labels those cysteine residues that have been reduced by DTT. The three bands represent PTP1B with different cysteine modifications. The lowest band represents PTP1B with a single cysteine modified whereas the higher bands reflect multiple cysteine modifications. PTP1B without AMS labeling (right panel) runs as a single band on SDS–(PAGE).

In earlier studies we showed that PTP1B was deglutathionylated in vitro by Srx. In the present report we have validated via coimmunoprecipitation that protein–protein interactions between PTP1B and Srx occur in cells (Figure 6c). Collectively, these results suggest that overexpression of Srx positively regulates the stability and activity of certain phosphatases and that these might contribute to the observed differences in kinase phosphorylation patterns.

Discussion

A growing body of evidence indicates that a variety of low molecular weight thiol-containing proteins have functions that influence cell signaling, proliferation and apoptosis pathways. Our present data show that overexpression of Srx can produce alterations in cell proliferation/growth rate and drug sensitivity. At least one component of this is the thiol-mediated regulation of kinase/phosphatase cascades, as demonstrated by our observation that Srx interacts with PTP1B and promotes its phosphatase activity to increase Src kinase activity. Srx overexpression does not influence PTP1B protein levels. Moreover, our observations that Srx mRNA and protein are regulated by growth factor signaling events and that Srx overexpression increases basal cellular ROS levels, both initially and after prolonged periods of exposure to stress, also indicates a possible signaling function for the protein. These results are reminiscent of the multiple functions of other redox-regulating proteins. For example, Grx, Prx, Trx and GST family members have a variety of cell functions and for Grx, the protein can act as a reversible initiator of S-glutathionylation (Shelton et al., 2005). In addition, GSTπ has ligand-binding properties that regulate Jun N-terminal kinase activity (Adler et al., 1999; Wang et al., 2001) and thioredoxin can interact with, and regulate ASK1 (Saitoh et al., 1998). In each case, the protein–protein interactions can be disrupted by stress inducing drugs to activate the respective kinases and cause subsequent effects on proliferative and apoptotic pathways (Tew, 2007). Most of the major signaling pathways controlled by phosphorylation are influenced to a certain degree by cellular redox conditions. In many instances this is achieved through reversible S-glutathionylation of critical phosphatases. This provides a potentially important congruence of sulfur and phosphorus biochemistry in these regulatory pathways and provides a context for Srx within these pathways.

Higher PTP1B phosphatase activity in Srx overexpressing cells and oxidation of multiple cysteine residues in PTP1B under basal culture conditions (Figure 6) suggests that restoration of a functional protein cysteine residue is one important aspect of Srx functions. PTP1B is a member of cysteine-based phosphatases, which share a common catalytic motif Cys-X5-Arg (Barford et al., 1994; Hunter, 1995; Alonso et al., 2004; Tonks, 2006), in which the catalytic cysteine, as a consequence of the positive electrostatic potentials exerted from the adjacent microenvironment, is present as a thiolate anion (–S⁻). This can be oxidized readily by cellular activities associated with redox cycling (Nakamura et al., 1997; Denu and Dixon, 1998). More than 30 years ago, Czech et al. (1974), documented the involvement of sulfhydryl oxidation in insulin signaling, which more recent studies have shown to be mediated through one of the cysteine residues of PTP1B. It is interesting to note that insulin positively regulates Srx transcription and protein expression (Figure 4), which in turn can promote a negative regulatory activity on insulin signaling. This may provide a new path for drug development to target diseases such as diabetes with symptomatic dysregulation of insulin signaling. It is worth mentioning that only certain oxidized cysteine species can be reversed in proteins such as Prxs and PTP1B. An increasing degree of oxidation converts susceptible cysteine residues to thiyl radicals and sulfenic, sulfinic and sulfonic acids. Sulfonic acid modifications are terminal and generally lead to ubiquitination and degradation of the protein. Cysteine sulfinates cannot be reduced by dithiothreitol (DTT), or in a cell by facile reduction with glutathione or other small molecule thiols. Reversal can be achieved with Srx (Shelton et al., 2005) and possibly by sestrin (Budanov et al., 2004). The predominant form of cysteine oxidation in PTP1B is a cyclic sulfenylamide species, in which the sulfur atom of the catalytic cysteine is covalently linked to the main chain nitrogen of an adjacent serine residue (Salmeen et al., 2003). The sulfenylamide species in PTP1B can be reduced by reductants such as DTT and glutathione. PTP1B is also modified by S-glutathionylation, which is a reversible post-translational modification (Barrett et al., 1999; Findlay et al., 2006). It is likely that in vivo a sulfenic acid intermediate (Cys-OH) exists before the sulfenylamide species and that this is accompanied by S-glutathionylation. The sulfenic form of cysteine is unstable and highly reactive and also serves to inactivate the normal catalytic cysteine function because the thiol sulfur atom loses its nucleophilicity toward the substrate phosphoester bonds when Cys-SH is oxidized to Cys-SOH. Protein Cys-SOH modifications are reversible by cellular small molecule reductants and redox proteins such as Prxs. PTEN is also subject to oxidation through an intramolecular disulfide bond between the catalytic and a vicinal cysteine residue (Leslie et al., 2003). Enzymatic activity can be restored by reduction of the disulfide to the respective thiol.

Our results show that enhanced expression of Srx reduced levels of p27 and increased p21 and p53. These findings are significant because epidemiological studies have suggested that these proteins are most important to lung cancer prognosis and drug response (Hirabayashi, 2002; Singhal et al., 2005; Zhu et al., 2006). p27 is a cell-cycle inhibitor and low p27 correlates with a poor survival rate in lung cancer patients. We observed reduced basal p27 transcript levels in A549-Srx cells (Supplementary Figure 1) and this may partially explain the low p27 protein levels in A549-Srx cells. p27 protein levels are primarily regulated by post-translational mechanisms including phosphorylation-mediated ubiquitination that leads to proteasome degradation (Kaldis, 2007). Srx seems to influence the phosphorylation status of p27 by increasing Src kinase activity through promoting PTP1B activities as discussed above. In spite of sequence similarities and functional complementation between p21 and p27, p21 is mainly regulated at the transcriptional level (el-Deiry et al., 1993; Supplementary Figure 1). As the basal protein level of p53 is also elevated in the Srx transfected cells, one possible linkage to increased p21 may be through p53. The elevated p53 in A549-Srx cells may also be a factor in increasing sensitivity to cisplatin. One question is why p53 levels are maintained at a higher level? As we observe more aneuploidy in A549-Srx cells (data not shown), it may be that higher Srx levels shorten the duration of sulfinic acid recycling and interrupt an established cellular redox state. In turn, this could lead to genomic instability and the consequent activation of p53. Reversible inhibition of p53 activity by S-glutathionylation at cysteines has been reported (Velu et al., 2007) and increased Srx may also promote active p53 through a deglutathionylation mechanism. Srx shares remarkable sequence and structural similarities with the bacterial ParB protein which is involved in bacterial chromosome partitioning (Basu and Koonin, 2005) and this may imply a possible, yet to be determined, role for Srx in maintaining genomic stability, perhaps through interactions with p53.

Direct interaction with cellular proteins may be an additional mechanism that contributes to Srx regulation of cell proliferation and drug sensitivity. Peroxiredoxins (Prx-I, -II, -III and -IV) physically interact with Srx (Woo et al., 2005). It seems likely that Srx in addition to Prxs, actin and PTP1B could have other interacting partners. Protein structure based modeling and cocrystallization of Srx-Prx suggest that the hydrophobic surface interactions and structural rearrangements between Srx and its interacting partners are required for bringing the Srx catalytic cysteine close to the cysteine sulfinic acid residue (Lee et al., 2006; Jonsson et al., 2008). In this respect, it is possible that Srx targeting of PTP1B may be related directly to the presence of a sulfinic acid residue or other types of modified cysteine species on the phosphatase and that some conserved motifs may mediate the initial interaction. Srx may target multiple proteins with modified cysteines and this targeting may be regulated, for example, by cellular GSH/GSSG balance, as Srx-catalysed reactions consume ATP and ATP hydrolysis kinetics are markedly affected by GSH/GSSG (Jeong et al., 2006).

In summary, Srx expression impacts phosphatase stability and activity through reduction of oxidized protein cysteine residues. At least one of the mechanisms involves protein–protein interactions between Srx and PTP1B. In turn this influences the phosphorylation state of a number of growth-regulating kinases. The net effect is that chronic increase in Srx levels translates into enhanced cell proliferation rates and enhanced sensitivity to the anticancer drug cisplatin. By engaging multiple cellular processes in an integrated manner and discriminating between normal and malignant cells, manipulation of cellular redox conditions could provide a viable strategy for novel cancer drug discovery. Srx appears to bridge multiple key redox-regulating systems and further understanding of its cellular function(s) could implicate it as a plausible drug target.

Materials and methods

Reagents

Cell culture media of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (10-092-CM), DMEM (15-017-CM) and G418 Sulfate (30-234-CR) were purchased from Mediatech Inc., Manassas, VA, USA. Antibodies of p21 (187, sc-817), p27 (C-19, sc-528), p53 (DO-1, sc-126), p-Erk (E-4, sc-7383), Erk2 (sc-1549) and protein A/G plus Agarose beads (sc-2003) from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Flag Immunoprecipitation Kit (FlagIPT-1), NEM (E-1271, N-ethylmaleimide) and general chemicals from Sigma, St Louis, MO, USA. CM-H₂DCFDA (C6827), calcium phosphate transfection kit (K2780) and AMS (A484, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid) were from Invitrogen, Carlsbad, CA, USA. FuGene-6 transfection reagent was from Roche, Indianapolis, IN, USA. Src antibody kit (no. 9935) was from Cell Signaling Technology, Danvers, MA, USA. Rabbit-IgG (011-000-003), HRP-conjugated donkey anti-mouse (715-035-150) and anti-rabbit (711-035-152) were from Jackson ImmunoResearch, West Grove, PA, USA. Anti-glutathione (101-A-100) antibody was obtained from Virogen, Watertown, MA, USA. Mouse monoclonal anti-Srx antibodies were produced in the MUSC core facility under the protocol approved by the Institute for Laboratory Animal Research committee. Full-length human Srx recombinant protein was used as immunogen in mice and hybridomas was produced using standard protocol. The generated monoclonal antibody recognizes endogenous and ectopically-expressed human Srx proteins in IB and IP. Cross reaction of the antibody with other species has not been systematically tested.

Cell culture and clone selection

A549 cells (a non-small cell lung cancer derived cell line, from American Type Culture Collection, Manassas, VA, USA), MCF7 and Hek293 cells were cultured in DMEM/F12 and DMEM medium, respectively, supplemented with 10% fetal bovine serum and 100 IU/ml penicillin per 100 µg/ml streptomycin. A549 cells were transfected with pcDNA3-Flag-Srx where the Flag-tag is fused to the N-terminus of Srx cDNA in frame. G418 selection (100 µg/ml) started 48 h post transfection and was maintained for 15 days before switching to regular culture medium. Flag-Srx expression was examined by IB using anti-Flag and anti-Srx antibodies. Expression of Srx by the retroviral system (pQCXIP, Clontech, Mountain View, CA, USA) followed the manufacturer’s protocol.

Immunoblot and immunoprecipitation

Cells were washed with ice-cold phosphate buffered saline (PBS), lysed in Triton lysis buffer and processed for IB and immunoprecipitation (IP; Lei et al., 2002). Lysates were kept at −80 °C until use. In a typical assay, 20–50 µg total proteins were used for IB and 200–500 µg for IP. Cell lysates or IP-products were resolved using standard SDS–polyacrylamide gel electrophoresis (PAGE), semi-dry transferred onto PVDF membrane (Bio-Rad, Hercules, CA, USA), blocked in standard Tris-Buffered Saline-0.15% Tween-20 (TBST) supplemented with 5% non-fat-dry milk or 5% bovine serum albumin (Fraction V, BP1600-100, Sigma). Primary antibody was incubated in TBST at 4 °C for 16 h and followed by peroxidase-conjugated secondary antibody incubation for 1–3 h at room temperature. The membrane was developed using the ECL system (170–5040, Bio-Rad). IB band intensity was quantitated with ImageJ software and Student’s t-tests were performed for evaluating density difference of bands.

Kinase assay

p27 cDNA was cloned into pET28b (Novagen, Gibbstown, NJ, USA) protein expression vector at Nde I—EcoR I sites, transformed into BL21 (DE3) pLysS (no. 44–0307, Invitrogen). p27 protein was purified using Ni-NTA agarose beads according to the manufacture’s protocol (no. 30210, Qiagen) and confirmed by IB. Cells were lysed in Triton lysis buffer and Src was immunoprecipitated using anti-Src antibody (36D10, no. 2109, Cell Signaling Technology). Kinase assay was performed using p27 as substrate (0.5–1 µg) in a reaction containing 20mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 5mm MnCl₂, 0.1mm Na-orthovanadate, 0.1mm ATP and 50 µm [γ-³²P] ATP (370 kBq/nmol). The assay product was resolved on SDS–PAGE and phosphorylation detected by autoradiography.

IP-coupled phosphatase activity assay

Cells were lysed in CHAPS lysis buffer (0.3% CHAPS, 20mm Tris, 1mm EDTA, and 137mm NaCl) and PTP1B was immunoprecipitated using polyclonal rabbit anti-PTP1B antibody (R&D Systems, Minneapolis, MN, USA, AF1366) and PTP1B phosphatase activity was measured as the amount of released phosphate from phosphopeptide substrate (R&D Systems, ES006) using Malachite Green Phosphate Detection Kit (R&D Systems, DY996).

Flow cytometry and cell viability assay

For cell cycle and sub-G₁ flow cytometry assays, cells were trypsinized, fixed with 70% ethanol, stained with 50 µg/ml propidium iodide and 2 µg/ml RNase A in PBS, analysed using a Becton Dickinson instrument. Data presented are non-gated total population cells. Sulforhodamine B staining following the Sigma Tox-6 protocol determined cell viability. The cell-cycle profile at basal culturing condition was performed at 24–40 h post plating with a cell density around 70–80%.

ROS measurement

Cells were treated with 0.6mm hydrogen peroxide or 2 µg/ml cisplatin. At the indicated time points, CM-H₂DCFDA (C6827, Molecular Probe, Eugene, OR, USA) was loaded to a final concentration of 5 µm and incubated for 30 min. Mean fluorescence intensity was acquired using Becton Dickinson, Franklin Lakes, NJ, USA flow cytometry analysis for nongated total populations of cells.

Real-time PCR

Total RNA was extracted using RNA STAT0-60 (no. CS-110, Tel-Test, Friendswood, TX, USA) or Nucleospin RNAII Kit (no. 635992, Macherey-Nagel, Mountain View, CA, USA). cDNA was prepared using Invitrogen RT–PCR Kit based on SuperScript III reverse transcriptase (no. 18080-093) and oligo (dT) 12–18 (18418-012). Real-time PCR was performed using iQ SYBR Green Supermix (no. 170–8891, Bio-Rad) and iCycler-MyiQ (Bio-Rad) system using the condition of 45 cycles and two-step PCR at 95 °C for 10 s and 60 °C for 25 s. Primers were designed using Primer3 software and validated manually and experimentally. The housekeeping gene succinate dehydrogenase primers are TGGGAACAAGAGGG CATCTG (forward) and TCACCACTGCATCAAATCATG.