human epidermal cell protein responses to arsenite treatment in culture

Chemico-Biological Interactions 155 (2005) 43–54

Human epidermal cell protein responses toarsenite treatment in culture

Chan Leea, Young Moo Leeb, Robert H. Ricec,∗

a Department of Food Science and Technology, Chung-Ang University, Ansung, Republic of Koreab Molecular Structure Facility, University of California, Davis, CA 95616-8656, USA

c Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, CA 95616-8588, USA

Received 30 October 2004; received in revised form 14 April 2005; accepted 14 April 2005Available online 17 May 2005

Abstract

Study of the responses of target cells in culture is anticipated to help understand the mechanisms by which inorganic arseniccauses pathological effects in vivo. Treatment of human epidermal cells with arsenic has been shown to produce a myriad ofchanges in gene transcription. Present work focused on finding the extent of arsenite-induced changes in the protein patternand whether global effects on protein sulfhydryls were evident. First, examining the profile of protein expression by two-dimensional gel electrophoresis indicated that≈40% of the 300 distinct protein spots that were monitored changed by at leasttwo-fold in amount all through a 9-day exposure period. Second, examining soluble extracts of the treated cells by ActivatedThiol Sepharose column chromatography gave little indication of change in the overall protein thiol content. Finally, amongt mes of theg y the cellso scriptionals sponses top©

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he 10 proteins identified that showed prominent changes in amount as a result of treatment for 1 or 4 days, enzylycolytic pathway were seen to be substantially elevated as a result of treatment, suggesting decreased utilization bf oxidative phosphorylation. Since these changes were more conspicuous at the protein level than in previous trantudies, the results emphasize the importance of proteomic analysis to complement transcriptional analysis of cell reerturbation by arsenic.2005 Elsevier Ireland Ltd. All rights reserved.

eywords:Activated Thiol Sepharose; Keratinocytes; Mass spectrometry

. Introduction

A widely encountered environmental contaminant,rsenic is a human carcinogen for the skin, bladder,

∗ Corresponding author. Tel.: +1 530 752 5176;ax: +1 530 752 3394.E-mail address:[email protected] (R.H. Rice).

lung and likely other organs, and it produces other dterious health effects[1]. Many humans are clearsuffering from exposure to hazardous concentrain their water supply[2] and display skin manifesttions [3,4]. Considerable uncertainty exists aboutmechanism by which arsenic acts and the shape odose–response relation, which complicates its regtion and the setting of exposure standards[5]. To help

009-2797/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2005.04.004

44 C. Lee et al. / Chemico-Biological Interactions 155 (2005) 43–54

evaluate the risk posed by moderate exposures com-monly consumed in drinking water, further mechanisticunderstanding is essential. Animal models are startingto provide insights into arsenic action[6], and cell cul-tures offer prospects for detailed analysis of target cellresponses.

As a target cell type, the keratinocyte is a goodsubject for mechanistic investigations. This cell typeis readily cultivated under conditions that optimizegrowth while permitting display of essential featuresof the differentiation program, which arsenic treatmentsuppresses[7]. The suppression is symptomatic of aglobal effect on the cells[8,9], with substantial changesin transcription of up to 10% of the expressed genes.Altered transcription in target cells is clearly importantfor the adverse effects of exposure, but the changes forwhich arsenic is responsible presumably are mediatedby its interaction with proteins and the resulting per-turbation of protein levels and functions. Alterationsof transcription may be reflected in altered protein lev-els in general, but the relation often is not simple,and large discrepancies between mRNA and proteinamounts are sometimes observed[10,11]. Thus a pro-teomic approach to describing arsenic effects, comple-menting transcriptional analysis, promises to provideuseful additional information. The present study indi-cates this approach will provide valuable insight intounderstanding arsenic effects on cellular function.

Among the major mechanisms proposed for arsenicaction [12,13], generation of oxidative stress in cellsi form y-g rela-t ithc gea er-a ica hi-o ents dro-g tiontc raf-fit e-ig mayu or

intermolecular disulfides, likely with glutathione. Theextent to which this happens in cells treated with arsenicat nonlethal concentrations could indicate the extent towhich the cellular protein disulfide reduction machin-ery is affected, a potential target in view of the observedinhibition of thioredoxin by arsenicals[21].

Employed for studying proteins with available thi-ols, Activated Thiol Sepharose consists of a mixeddisulfide of 2-thiopyridine and glutathione, where theamine of�-glutamic acid of the latter is attached tothe agarose resin. When applied to the resin, proteinthiols can displace 2-thiopyridine moieties, formingmixed disulfides with glutathione, thereby becomingcovalently attached to the resin, and can be released byreducing agents[22]. This approach has proven usefulin the purification of a variety of structural proteins andenzymes[23], characterization of signaling proteinssuch as the glucocorticoid receptor[24], probing struc-tures of protein complexes of thrombospondin[25] orof nuclear matrix proteins[26], and identification ofthe specific protein thiols that become attached to theresin[27]. Present work explores use of the resin for as-sessing arsenite effects on protein thiols in conjunctionwith two-dimensional gel electrophoretic separations.

2. Materials and methods

2.1. Cell culture

edk tedfi e ofD ple-mct eratas ter-v artedj mesa daysa sus-p n-t taili 64,

s prominent, and may be particularly importantethylated arsenicals[14]. Generation of reactive oxen has been demonstrated directly in cells at

ively high arsenic concentrations that are lethal whronic exposure[15], and evidence for such damat low concentration is increasing, including in ktinocytes[16]. A likely mechanism by which arsenffects cell behavior is through binding to vicinal tls in critical regulatory proteins. However, a rectudy indicates that arsenite inhibits pyruvate dehyenase (and likely other targets) at lower concentra

hrough generation of reactive oxygen[17]. Arseniteould plausibly target proteins involved in electron tcking or transport such as NAD(P)H oxidases[18] orhose in the mitochondria[19], the latter organelles bng active in reducing AsV to AsIII[20]. Sulfhydrylroups in proteins to which arsenic does not bindndergo oxidation to yield intramolecular disulfides

A continuous line of spontaneously immortalizeratinocytes (SIK) derived and minimally deviarom human epidermis[28] were grown with lethallyrradiated 3T3 feeder layer support in a 3:1 mixturulbecco-Vogt Eagle’s and Ham’s F-12 media supented with 5% fetal bovine serum, 0.4�g/ml hydro-

ortisone, 5�g/ml insulin, 5�g/ml transferrin, 20 pMriiodothyronine, 0.18 mM adenine, 10 ng/ml choloxin, and 10 ng/ml epidermal growth factor[29]. Cellst passages 25–35 were treated without or with 2�Modium arsenite with a medium change at 3-day inals. For most experiments, arsenite treatment stust before confluence and continued for various tis indicated. The cells were usually harvested 2–3fter a medium change by scraping from dishes andended in ice cold 0.1 M Tris–HCl buffer (pH 8.0) co

aining 10 mM EDTA and a protease inhibitor cockncluding antipain–HCl, bestatin, chymostatin, E-

C. Lee et al. / Chemico-Biological Interactions 155 (2005) 43–54 45

leupeptin, pepstatin, phosphoramidon, PefablocSC andaprotinin (Roche, Mannheim, Germany). They weresonicated for 30 s and centrifuged briefly to removecellular debris at 10,000×g for 20 min. Supernatantswere stored at−80◦C. Protein concentrations were de-termined with Coomassie G-250 (Bio-Rad, Richmond,CA) using bovine serum albumin as the standard[30].

2.2. Fractionation of proteins by thiol affinitychromatography

Proteins in clarified cell extracts were transferred to0.1 M Tris–HCl, pH 7.5–1 mM EDTA and low molec-ular weight thiol compounds were removed by gel fil-tration on Sephadex G-25. Aliquots (0.5 ml) were de-gassed and fractionated by affinity chromatography us-ing a 1 cm× 5 cm column of Activated Thiol Sepharose4B (Amersham Biosciences, Piscataway, NJ). Afterpermitting the sample to interact with the resin for30 min at 4◦C, the column was rinsed until no fur-ther protein eluted, and covalently bound moleculeswere then eluted with 10 mM dithiothreitol in 0.1 MTris–HCl containing 1 mM EDTA.

2.3. Two-dimensional gel electrophoresis

IPG strips (13 cm) were hydrated in 250�l of buffer(5M urea, 2M thiourea, 4% CHAPS, 0.05% TritonX-100, trace of bromphenol blue) containing protein(150�g), 1% fresh dithiothreitol and 0.5% (v/v) am-p so-e , Pis-c2 V,t to8 for1 8,6 mld henf c-e wast cry-l 00,A /gel.A al.,1 PD-Q n ar-s g for

variation between gels and to compare matched spotintensities for protein quantification. The time coursewas performed once in its entirety, and time points atdays 1 and 4 were repeated several times each, givingnearly identical patterns, in the process of generatingsufficient material for the identifications. As a check ofreproducibility, the fraction of proteins that increasedor decreased after 1 day of treatment was 21± 4% or26± 4%, respectively, in three independent trials; spot12 (HSP27) in the treated cultures over the 5-day timecourse varied in intensity by± 12% (values are givenas mean + standard deviation).

2.4. Identification of proteins by peptide massmapping

Protein spots of interest in the gel were excised fol-lowed by digestion with trypsin (Promega, MadisonWI, USA) using a standard in-gel-digestion procedure.Extracted tryptic peptides were desalted using a C18ZipTip (Millipore, MA) then eluted with 50% acetoni-trile/0.1% trifluoroacetic acid (TFA). A 1�l aliquot ofthe eluant was mixed with an equal volume of ma-trix solution (saturated�-cyano-4-hydroxycinnamicacid in 0.1% TFA-50% acetonitrile in water) and an-alyzed with a Bruker Biflex III (Brucker Daltonics,Bremen, Germany) matrix-assisted laser desorptionionization time-of-flight mass spectrometer (MALDI-TOF) equipped with a nitrogen 337 nm laser. The massspectra were acquired in the reflectron mode. Internalm uto-d pro-c ppmo ryp-t mand ascots al-gm ases.A achs e-t red tot resis.

2m

assm <63

holines (pH 3–10). Isoelectric focusing (IPGphor Ilectric Focusing System, Amersham Biosciencesataway, NJ) was carried out at 20◦C starting with0 V for 12 h, increasing gradually over 1 h to 150

hen 500 V over 1 h, 1000 V for 1 h and finally rising000 V for 20 h. The IPG strips were then soaked5 min in equilibration buffer (50 mM Tris–Cl, pH 8.M urea, 30% glycerol (v/v), 2% SDS (w/v), 2.5 mg/ithiothreitol and trace of bromphenol blue) and t

or 15 min in this solution containing 45 mg/ml iodoatamide. Separation in the second dimension

hen performed using polyacrylamide gels (10% aamide, 0.27% bisacrylamide) in SDS (Hoefer SE 6mersham Biosciences, Piscataway, NJ) at 40 mAfter the proteins were stained with silver (Merril et984), the gel image was analyzed with Bio Raduest software to match the protein pattern betweeenite treated and untreated cultures by correctin

ass calibration was performed with two trypsin aigestion fragments (842.5 and 2211.1 Da). Thisedure typically results in mass accuracies of 50r better. Measured monoisotopic masses of t

ic peptides were used as inputs to search huatabases or the NCBInr database using the Mearch engine with a probability based scoringorithm (http://www.matrixscience.com/). Up to oneissed tryptic cleavage was considered in most cmass accuracy of 50 ppm or lower was used for e

earch. TheMr and isoelectric point of each protein dermined through the database search were compahe corresponding values observed by electropho

.5. De novo sequencing of peptides by tandemass spectrometry (MS/MS)

Those proteins not identifiable by peptide mapping alone because of low “protein scores” (

http://www.matrixscience.com/


and p> 0.05, thus statistically not significant) wereidentified by de novo sequencing of peptides followedby Blast search. Aliquots of tryptic peptides werecleaned and concentrated using POROS R2 resin (Per-ceptive Biosystems, Framingham, MA) in a microcol-umn following the method described in the Protanamanual (Protana, Odense, Denmark). Tryptic pep-tides were then analyzed by a hybrid nanospray/ESI-Quadrupole-TOF-MS and MS/MS in a QSTAR massspectrometer (Applied Biosystems Inc., Foster City,CA). Peptides in 5% formic acid–methanol (50:50)were sprayed from the gold-coated capillary. Argongas was used as the collision gas. De novo sequencingof peptides was carried out using the QSTAR software(Analyst QS) and confirmed by a manual interpretationof MS/MS spectra.

3. Results

3.1. Overall changes in protein pattern

SIK cultures treated with 2�M arsenite just as theyreached confluence were examined for protein changesover the course of 9 days by two-dimensional gel elec-trophoresis. Although this concentration suppressestheir differentiation, it does not lead to cell loss[7]. Asshown inFig. 1, approximately 300 distinct spots ofrelatively abundant proteins were monitored, of which

F withs aysi two-d sitiesw fromu a leastt

≈40% were seen to undergo at least a two-fold changein silver staining intensity at each time point. The num-ber of changes was at least as great at 1 day of treatmentas at any other time point, and the fraction of proteinsthat decreased (21± 6%) did not differ significantlyfrom the number that increased (19± 5%). Overall, theprotein profiles through the time course were compa-rable under given conditions, although some proteinsappeared and others disappeared in accord with the dif-ferentiation program.

3.2. Fractionation of proteins by thiol affinitychromatography

When degassed and applied to the column, a sub-stantial portion of the soluble cell protein bound co-valently to the column. In repeated experiments, therelative amounts that bound or passed through exhib-ited some variation, possibly due to residual oxygen inthe samples. As shown inFig. 2, however, this frac-tionation divided the protein approximately in half in-dependently of the arsenite treatment of the cultures.This pre-fractionation step reduced the proteome com-plexity by about 50%.

F tedT t ei-t cova-l reswa ree ex-p

ig. 1. Alteration of protein expression in SIK cultures treatedodium arsenite (2�M). Cultures were treated for the number of d

ndicated, at which time the soluble proteins were submitted toimensional gel electrophoresis and silver stained. Spot intenere quantitated in comparison to those examined in parallelntreated cultures. Those spots increasing or decreasing by

wo-fold are indicated as changing.

ig. 2. Fractionation of proteins from SIK cultures using Activahiol Sepharose 4B. Illustrated are the fractions of protein tha

her passed directly through the column (unbound) or becameently bound and were eluted with dithiothreitol (bound). Cultuere treated for 1 day with (gray bars) or without (black bars) 2�Mrsenite. The means and standard deviations are shown for theriments.


Fig. 3. Silver stained protein profiles in two-dimensional gels after thiol affinity chromatography. SIK cultures treated with arsenic for 4 days(starting at confluence) were analyzed in parallel to untreated controls. Major proteins (most differentially expressed) are indicated by arrows andenumerated. The proteins were excised, in-gel digested with methylated trypsin, and identified by MALDI-TOF mass spectrometry. Isoelectricpoint (pH 3–10) and molecular weight scales (kDa) are indicated on the top and the right side of the gels, respectively. (A, C) Treated witharsenic; (B, D) untreated; (A, B) proteins not bound to the resin; (C, D) eluted from the resin with dithiothreitol.

The pattern of proteins eluted from the thiol affin-ity resin was examined by two-dimensional gel elec-trophoresis, as shown inFig. 3. Under the conditionsemployed, where overloading the gels was avoided,typically several dozen protein spots were separatedwell enough and in amounts sufficient for ready identi-fication by mass spectrometry. Samples from culturestreated with arsenite for 1 and 4 days were processedin this way. In each case, the effect of arsenic treatment

was most obvious in the profile obtained among theproteins that were not retained by the column. Com-parison of panels A and B, from samples treated for4 days, revealed at least nine protein spots (numbered6–14, indicated by arrows) that were visible only inarsenic-treated samples. A smaller number of differ-ences were noted among the proteins that bound to theresin (panels C and D). Panel C had two spots not visi-ble in D, and one in D was not visible in C. In addition,


two sentinel (marker) spots (numbers 15 and 16) areindicated in these panels that were used for orientation(actin and annexin A2).

3.3. Protein identification

Among the distinct protein spots separated inthe two-dimensional gels, 19 of the most prominentones showing marked changes in intensity wereprocessed and identified after fractionation by thiolaffinity chromatography. As illustrated inTable 1, fiveproteins were identified in the samples treated for 1day. Of these, three were greatly diminished (HSP70,calreticulin, protein disulfide isomerase), one wassubstantially increased by treatment (HSP27), and onewas a marker protein (glutamate dehydrogenase). Ofthe nine identified after 4 days of treatment, seven wereconsiderably induced (annexin 1, phosphoglyceratemutase, HSP27, triose phosphate isomerase, fructosebisphosphate aldolase, peroxiredoxin, glyceraldehyde-3-phosphate dehydrogenase), and two were markerproteins (actin, annexin A2). Four of the inducedproteins were detected as two or three distinctspots.

4. Discussion

While subsequent compensatory adjustments orother downstream changes may have occurred, thet wo-d ag-n ay.T n oft icu-l entsi nalt tedi n ofr re-s t thep nor-m nce)f ges,b tudeo thatc pro-t ects

additionally the time required for arsenate reduction toarsenite. Study of the relative rate of flux of arsenateand arsenite into keratinocytes and the rate of arsenatereduction to arsenite rationalizes the three-fold greaterpotency of the latter[31].

Because cells typically synthesize many thousandsof proteins, fractionation prior to two-dimensional gelelectrophoresis is useful in improving the resolutionobtained. Activated Thiol Sepharose proved useful byreducing the complexity of the soluble proteins approx-imately by half. In addition, it permitted an overall as-sessment of the effect of arsenite treatment on proteinsulfhydryls, at least among those available for bind-ing to the column. In principle, this type of analysiscould be extended to include insoluble proteins, includ-ing those (if any) that were lost from the soluble frac-tion during arsenic treatment due to increased disulfidebonding. However, in view of the lack of substantialeffect of treatment on the proportion of protein bind-ing to the column, the insoluble protein fraction seemsunlikely to be more sensitive overall. Certain proteinsmay be particularly sensitive to the treatment or couldbe partially affected but not evident in this analysis.Nevertheless, present findings indicate that whateveroxidative stress arsenic generates is not sufficient toproduce a detectable global loss of free thiols amongcellular proteins.

Present findings are consistent with reports that ar-senic treatment leads to increased glutathione levels inkeratinocytes[32], which may compensate for ongo-i pe-c teso ida-t tusi xire-d nasea bysT per-o in al c-ug cifict teint sinep rea eame

ime course of altered protein spots visualized by timensional gel electrophoresis indicates that the mitude of the perturbation was maximal within 1 dhis observation indicates that future examinatio

he cultures at even earlier time points will be partarly valuable for understanding the sequence of evn the cellular response to treatment. A transcriptioime course obtained by DNA microarray suggesnitial changes occurred in response to generatioeactive oxygen[9], which could drive subsequentponses and might be demonstrable more directly arotein level. In that study, arsenate treatment ofal human epidermal cells (approaching conflue

or 2 days produced only a small number of chanut this number increased by an order of magniver the next several days. While it is reasonablehanges in transcription resulted from effects onein function, the delay in response to arsenate refl

ng oxidation. This conclusion may well be tissue sific, but it points to the importance in keratinocyf protein targets that are especially sensitive to ox

ion. Alteration of the identified proteins in thiol stas uncertain but seems likely, since at least perooxin and glyceraldehyde-3-phosphate dehydrogere known to be highly sensitive to oxidation evenhort treatment of cells with hydrogen peroxide[33].his phenomenon could account for the markedxiredoxin decrease in the treated cells, resulting

owering of their capacity to benefit from its molelar chaperone function under stress[34]. Lack of alobal change in protein thiol status points to spe

argets that are especially sensitive. Identifying proargets, possibly including susceptible protein tyrohosphatases[35], could elucidate pathways that affected by arsenite and help rationalize downstrffects.


Table 1Characteristics of proteins identified in two-dimensional gels

Spota Protein M.W.(kDa)

Bb Density(untreated)

Density(treated)

Sequencecoverage(matchedpeptides)c

Identified sequence

1 Glutamate dehydrogenase 62 − 71 44 14% (5/8) 1893.99 IIKPCNHVLSLSFPIR1425.57 DDGSWEVIEGYR1915.91 GFIGPGIDVPAPDMSTGER1748.89 TFVVQGFGNVGLHSMR1737.88 HGGTIPIVPTAEFQDR

2 HSP27 21 − 0 14 MS/MS 481.30 SWDPFR595.60 NHFAPDELTVK

3 HSP70 71 − 116 5 17% (8/8) 1566.81 ITPSYVAFTPEGER1888.06 VTHAVVTVPAYFNDAQR1816.09 IINEPTAAAIAYGLDKR2165.03 IEIESFYEGEDFSETLTR1512.75 AKFEELNMDLFR1313.60 FEELNMDLFR1934.06 DNHLLGTFDLTGIPPAPR1974.92 IEWLESHQDADIEDFK

4 Calreticulin 63 − 282 9 MS/MS 1410.57 EQFLDGDGWTSR1219.64 GQTLVVQFTVK2391.63 IDNSQVESGSLEDDWDFLPPK1800.86 IKDPDASKPEDWDER1559.64 DPDASKPEDWDER2760.36 IDDPTDSKPEDWDKPEHIPDPDAK1019.51 LKEEEEDK

5 Protein disulfide isomerase 55 − 324 5 MS/MS 601.80 EADDLVNWLK

6 Annexin 1 35 − 0 54 18% (4/5) 1262.51 TPAQFDADELR1702.85 GLGTDEDTLIEILASR1739.65 SEDFGVNEDLADSDAR1550.76 GTDVNVFNTILTTR

7 Annexin 1 35 − 0 107 32% (8/12) 2140.89 QAWFIENEEQEYVQTVK2354.20 GGPGSAVSPYPTFNPSSDVAALHK1605.93 ALTGHLEEVVLALLK1262.57 TPAQFDADELR1702.89 GLGTDEDTLIEILASR1739.69 SEDFGVNEDLADSDAR1678.91 KGTDVNVFNTILTTR1550.80 GTDVNVFNTILTTR

8 Glyceraldehyde-3-phosphatedehydrogenase

36 − 10 73 21% (4/9) 1613.88 LVINGNPITIFQER2276.04 WGDAGAEYVVESTGVFTTMEK2595.39 VIHDNFGIVEGLMTTVHAITATQK1763.81 LISWYDNEFGYSNR


36 − 37 71 37% (8/14) 1613.88 LVINGNPITIFQER2277.09 WGDAGAEYVVESTGVFTTMEK1833.96 IISNASCTTNCLAPLAK2595.48 VIHDNFGIVEGLMTTVHAITATQK1411.75 GALQNIIPASTGAAK795.40 LTGMAFR1530.76 VPTANVSVVDLTCR1763.79 LISWYDNEFGYSNR


Table 1 (Continued)



Density(treated)


Identified sequence


36 − 0 61 19% (5/9) 1613.99 LVINGNPITIFQER1411.75 GALQNIIPASTGAAK795.44 LTGMAFR1530.83 VPTANVSVVDLTCR1763.88 LISWYDNEFGYSNR

11 Phosphoglycerate mutase 29 − 0 44 46% (9/12) 312.74 HGESAWNLENR1980.20 FSGWYDADLSPAGHEEAK2136.28 FSGWYDADLSPAGHEEAKR1780.02 DAGYEFDICFTSVQK1936.17 DAGYEFDICFTSVQKR2417.45 SYDVPPPPMEPDHPFYSNISK2425.54 YADLTEDQLPSCESLKDTIAR1150.73 VLIAAHGNSLR2115.31 NLKPIKPMQFLGDEETVR

12 HSP27 21 − 0 31 25% (3/9) 960.42 DWYPHSR1163.57 LFDQAFGLPR1783.91 VSLDVNHFAPDELTVK

13 HSP27 21 − 0 70 MS/MS 595.67 VSLDVNHFAPDELTVK538.32 LSSGVSELR582.00 FDQAFGLP

14 Triose phosphateisomerase

27 − 0 52 47% (9/12) 954.47 FFVGGNWK

1541.83 KQSLGELIGTLNAAK2191.98 VPADTEVVCAPPTAYIDFAR1585.72 DCGATWVVLGHSER1614.84 RHVFGESDELIGQK1458.67 HVFGESDELIGQK1807.97 VAHALAEGLGVIACIGEK1602.82 VVLAYEPVWAIGTGK1466.70 TATPQQAQEVHEK

15 Actin 41 + 293 225 46% (12/29) 976.45 AGFAGDDAPR1198.65 AVFPSIVGRPR1171.50 HQGVMVGMGQK1515.68 IWHHTFYNELR1954.06 VAPEEHPVLLTEAPLNPK3183.64 TTGIVMDSGDGVTHTVPIY-

EGYALPHAILR1132.48 GYSFTTTAER1790.87 SYELPDGQVITIGNER2343.08 KDLYANTVLSGGTTMYPGIADR2215.05 KLYANTVLSGGTTMYPGIADR795.48 IIAPPER2730.34 KYSVWIGGSILASLSTFQQMWISK

16 Annexin A2 39 + 106 115 55% (17/22) 1844.91 LSLEGDHSTPPSAYGSV K1086.44 AYTNFDAER1542.82 GVDEVTIVNILTN R1111.50 QDIAFAYQR1650.92 SALSGHLETVILGLLK


Table 1 (Continued)



Density(treated)


Identified sequence

1222.51 TPAQYDASELK1777.88 GLGTDEDSLIEIICSR1244.59 TNQELQEINR1811.90 TDLEKDIISDTSGDFR1225.53 DIISDTSGDFR2065.00 RAEDGSVIDYELIDQDAR1908.89 AEDGSVIDYELIDQDAR880.42 DLYDAGVK1035.47 WISIMTER1460.64 SYSPYDMLESIR2838.50 GDLENAFLNLVQCIQNKPLYFADR1421.61 SLYYYIQQDTK

17 Fructose bisphosphatealdolase

40 + 0 18 22% (5/12) 1647.20 LQSIGTENTEENRR1342.89 ADDGRPFPQVIK2107.79 IGEHTPSALAIMENANVLAR1676.23 FSHEEIAMATVTALR2228.66 YTPSGQAGAAASESLFVSNHAY

18 Fructose bisphosphatealdolase

40 + 7 26 31% (6/8) 2087.92 VNPCIGGVILFHETLYQK1342.57 ADDGRPFPQVIK2272.97 GVVPLAGTNGETTTQGLDGLSER2106.95 IGEHTPSALAIMENANVLAR3178.06 YASICQQNGIVPIVEPEILPDGDHDLKR1675.72 FSHEEIAMATVTALR

19 Peroxiredoxin 22 + 81 30 40% (8/13) 908.69 IGHPAPNFK1751.31 KQGGLGPMNIPLVSDPK1107.78 TIQADYGVLK894.62 ADEGISFR920.67 GLFIIDDK1211.88 QITVNDLPVGR819.59 SVDETLR1196.81 LVQAFQFTDK

a Spots 1–5 were taken from samples exposed to arsenic for 1 day (seeSupplementary File), while 6–19 were from samples exposed for 4days (Fig. 3). Marker spots 1, 15 and 16 changed little in amount but were used to confirm proper orientation of the gels. Spot 2 correspondedin location to spot 12.

b The sign indicates whether the proteins were (+) or were not (−) bound to the Activated Thiol Sepharose column.c MS/MS indicates de novo sequencing of the peptides shown.

On the order of 10,000 proteins are expressed in agiven cell type, and the dynamic range of their con-centrations typically spans five orders of magnitude.The present analysis was limited to proteins of rela-tively high abundance, thus necessarily omitting criti-cal regulatory proteins that are low in abundance andthat will require more focused efforts to study. Resultsof the present limited survey of prominent changes (10proteins) were compared to transcriptional changes ob-served by DNA microarray[8,9]. Although the changesevident after 1 day of treatment appeared to reflect

stress, as the microarray studies suggested, none wereeasily predictable. For example, arsenic has long beenknown to induce certain heat shock proteins in a vari-ety of cell types[36]. Present experiments (Fig. 3andTable 1) revealed a substantial induction of HSP27 af-ter 1 or 4 days of exposure, a heat shock protein knownto be phosphorylated in keratinocytes and found, ashere, in multiply charged forms[37]. Previously, ker-atinocytes cultured in the present system showed smallincreases (<2-fold) in the mRNA[9] instead of thelarge fold increase in the protein here. Substantial in-


duction of HSP70 was observed in mouse liver[38]or cultured human keratinocytes[39] with high arsen-ite exposure (300�mol/kg or 50�M, respectively). Atthe much lower levels employed in the present work(2�M), HSP70 mRNA showed changes in DNA mi-croarray studies that were insignificant[8] or small[9],but the protein was suppressed >95% after 1 day oftreatment in this work. Calreticulin and protein disul-fide isomerase also were suppressed >95% after 1 dayin this work, but their mRNA levels changed little inthe DNA microarray work.

Effects of such changes on cell function are un-certain but potentially profound. Calreticulin, locatedprimarily in the endoplasmic reticulum and reportedto have chaperone activity there[40], binds calciumion and thereby appears to function in calcium regu-lation, which can affect many cellular processes. Itsimportance has been demonstrated in cardiac develop-ment, where its loss appears to prevent the action ofthe calcium dependent phosphatase, calcineurin[41].The critical role of calcium ion in keratinocyte differen-tiation [42] raises the possibility that the dramatic de-crease induced by arsenic in present experiments couldcontribute to the observed suppression of differentia-tion in the cells. In the thioredoxin superfamily, proteindisulfide isomerase catalyzes protein folding and actsas a chaperone in the endoplasmic reticulum[43]. If itsloss resulted in accumulation of improperly folded pro-teins in the endoplasmic reticulum, induction of mRNAfor this protein and others such as BiP would have beeneb oar-r p-e micr anyt sec 1 dayw enta

s oft ifiede rede heed , buti s isn on.T d is

the upregulation of enzymes in anaerobic glycolysis.Dramatic increases in glyceraldehyde-3-phosphatedehydrogenase, phosphoglycerate mutase, triose phos-phate isomerase and fructose bisphosphate isomeraseall point to a decreased dependence of the cells onoxidative phosphorylation. In a possibly analogousphenomenon in yeast exposed to 1 mM cadmium ion,numerous proteins that were induced participated incarbohydrate metabolism, interpreted as a sulfur spar-ing response involving a switch to proteins with lowersulfur content[48]. The hypothesized driving force inyeast, upregulation of glutathione levels, does occur inkeratinocytes treated with arsenic, but the response inthe present case more plausibly reflects the well knownability of arsenite to inhibit the pyruvate dehydrogenasecomplex, thereby interfering with generation of NADHthrough oxidative phosphorylation[17]. In higher ani-mals, glycolytic enzymes are induced transcriptionally[49] and allosterically[50] by hypoxic conditions,which paradoxically can involve oxidative stress[51].Little transcriptional response has been noted in thearsenite-treated keratinocytes[8,9], however, suggest-ing a contribution from altered translational efficiencyor protein stability, perhaps due to the observedsubstantial inhibition of degradative activity of pro-teosomes in arsenite-treated tissue slices and culturedcells[52]. A more comprehensive study of the proteinchanges, also shedding light on the general correspon-dence between transcriptional and protein effects, mayreveal signaling pathways that distinguish actions ofa tiveo

A

cellc forg wass andE

A

ticlec /j

xpected as part of the unfolded protein response[44],ut this phenomenon was not observed by micray [9]. However, a lack of reduction of the improrly folded proteins might not evolve the endoplaseticulum-derived oxidative stress that can accompheir accumulation[45]. The long term effects of thehanges are not clear, since the changes seen atere transitory (except for HSP27) and not promint later times.

A protein that was greatly increased after 4 dayreatment is annexin 1, a component of the cornnvelope synthesized in differentiating cultupidermal cells[46]. This protein is upregulated in tpidermis in inflammatory conditions[47], possiblyue to stress such as increased reactive oxygen

t is expressed in a variety of cell types and thuot a specific marker of keratinocyte differentiatihe most striking feature of the changes observe

rsenite from those of other generators of reacxygen.

cknowledgments

We thank Qin Qin for invaluable assistance inulture and Michelle Salemi and Dr. Young Jin Leeenerous help in mass spectrometry. This workupported by USPHS grants AR27130, ES05707S04699.

ppendix A. Supplementary data

Supplementary data associated with this aran be found, in the online version, atdoi:10.1016.cbi.2005.04.004.

http://dx.doi.org/10.1016/j.cbi.2005.04.004

http://dx.doi.org/10.1016/j.cbi.2005.04.004


References

[1] National Research Council, Arsenic in Drinking Water, Na-tional Academy Press, Washington, 2001.

[2] D.K. Nordstrom, Worldwide occurrences of arsenic in groundwater, Science 296 (2002) 2143–2144.

[3] D.N.G. Mazumdur, R. Haque, N. Ghosh, B.K. De, A. Santra,D. Chakraborty, A.H. Smith, Arsenic levels in drinking waterand prevalence of skin lesions in West Bengal, India, Int. J.Epidemiol. 27 (1998) 871–877.

[4] T. Kadono, T. Inaoka, N. Murayama, K. Ushijima, M. Nagano,S. Nakamura, C. Watanabe, K. Tamaki, R. Ohtsuka, Skin man-ifestations of arsenicosis in two villages in Bangladesh, Int. J.Dermatol. 41 (2002) 841–846.

[5] A. Schoen, B. Beck, R. Sharma, E. Dube, Arsenic toxicity atlow doses: epidemiological and mode of action considerations,Toxicol. Appl. Pharmacol. 198 (2004) 253–267.

[6] M.P. Waalkes, J. Liu, J.M. Ward, B.A. Diwan, Animal modelsfor arsenic carcinogenesis: inorganic arsenic is a transplacen-tal carcinogen in mice, Toxicol. Appl. Pharmacol. 198 (2004)377–384.

[7] B.A. Jessen, Q. Qin, M.A. Phillips, D.L. Phillips, R.H. Rice,Keratinocyte differentiation marker suppression by arsenic: me-diation by AP1 response elements and antagonism by tetrade-canoylphorbol acetate, Toxicol. Appl. Pharmacol. 174 (2001)302–311.

[8] H.K. Hamadeh, K.J. Trouba, R.P. Amin, C.A. Afshari, D. Ger-molec, Coordination of altered DNA repair and damage path-ways in arsenite-exposed keratinocytes, Toxicol. Sci. 69 (2002)306–316.

[9] M.A. Rea, J.P. Gregg, Q. Qin, M.A. Phillips, R.H. Rice, Globalalteration of gene expression in human keratinocytes by inor-ganic arsenic, Carcinogenesis 24 (2003) 747–756.

[10] G.S. Chen, T.G. Gharib, D.G. Thomas, C.-C. Huang, D.E.Misek, R.D. Kuick, T.J. Giordano, M. Iannettoni, M.B. Or-

eIF-96–

[ cter-004)

[ odesenic

[ inte-

[ ode03)

[ fe,attri-

. 278

[ iu,e inRes.

[17] T. Samikkannu, C.-H. Chen, L.-H. Yih, A.S.S. Wang, L.S.-Y.T.-C. Chen, K.-Y. Jan, Reactive oxygen species are involved inarsenic trioxide inhibition of pyruvate dehydrogenase activity,Chem. Res. Toxicol. 16 (2003) 409–414.

[18] K.R. Smith, L.R. Klei, A. Barchowsky, Arsenite stimulatesplasma membrane NADPH oxidase in vascular endothelialcells, Am. J. Lung Cell. Mol. Physiol. 280 (2001) L442–L449.

[19] E. Corsini, L. Asti, B. Viviani, M. Marinovich, C.L. Galli,Sodium arsenate induces overproduction of interleukin-1a inmurine keratinocytes: role of mitochondria, J. Invest. Derma-tol. 113 (1999) 760–765.

[20] B. Nemeti, Z. Gregus, Mitochondria work as reactors in reduc-ing arsenate to arsenite, Toxicol. Appl. Pharmacol. 182 (2002)208–218.

[21] S. Lin, W.R. Cullen, D.J. Thomas, Methylarsenicals and ar-sinothiols are potent inhibitors of mouse liver thioredoxin re-ductase, Chem. Res. Toxicol. 12 (1999) 924–930.

[22] K. Brocklehurst, J. Carlsson, M.P.J. Kierstan, E.M. Crook, Co-valent chromatography by thiol-disulfide interchange, Meth.Enzymol. 34 (1974) 531–544.

[23] T.P. Bradshaw, R.B. Dunlap, Characterization of the cova-lent chromatography of thymidylate synthase on thiopropyl-Sepharose 6B, Biochim. Biophys. Acta 1163 (1993) 165–175.

[24] T. Idziorek, B. Sablonniere, P. Formstecher, V. Dumur, M.Dautrevaux, Covalent chromatography by thiol-disulfide inter-change of the highly purified non-transformed rat liver gluco-corticoid receptor, J. Steroid Biochem. 23 (1985) 593–597.

[25] M.V. Speziale, T.C. Detwiler, Reduction of a disulfide bondof thrombospondin in the supernatant solution of activatedplatelets, Arch. Biochem. Biophys. 286 (1991) 546–550.

[26] N. Stuurman, A. Floore, A. Colen, L. de Jong, R. van Driel,Stabilization of the nuclear matrix by disulfide bridges: identi-fication of matrix polypeptides that form disulfides, Exp. CellRes. 200 (1992) 285–294.

[27] T.A. Egorov, A. Svenson, L. Ryden, J.A. Carlsson, A rapid andomport,

[ ay-ur-Mol.

[ hy-es in

[ an-in-8–

[ .G.andtes,

[ ofd hu-Tox-

ringer, S.M. Hanash, D.G. Baer, Proteomic analysis of5A in lung adenocarcinoma, Proteomics 3 (2003) 4504.

11] K. Rautajoki, T.A. Nyman, R. Lahesmaa, Proteome charaization of human T helper 1 and 2 cells, Proteomics 4 (284–92.

12] K.T. Kitchin, Recent advances in arsenic carcinogenesis: mof action, animal model systems and methylated arsmetabolites, Toxicol. Sci. 172 (2001) 249–261.

13] T.G. Rossman, Mechanism of arsenic carcinogenesis: angrated approach, Mutation Res. 533 (2003) 37–65.

14] K.T. Kitchin, S. Ahmad, Oxidative stress as a possible mof action for arsenic carcinogenesis, Toxicol. Lett. 137 (203–13.

15] L. Liu, J.R. Trimarchi, P. Navarro, M.A. Blasco, D.L. KeeOxidative stress contributes to arsenic-induced telomeretion, chromosome instability, and apoptosis, J. Biol. Chem(2003) 31998–32004.

16] H. Shi, L.G. Hudson, W. Ding, S. Wang, K.L. Cooper, S. LY. Chen, X. Shi, K.J. Liu, Arsenite causes DNA damagkeratinocytes via generation of hydroxyl radicals, Chem.Toxicol. 17 (2004) 871–878.

specific method for isolation of thiol-containing peptides frlarge proteins by thiol-disulfide exchange on a solid supProc. Acad. Sci. U.S.A. 72 (1975) 3029–3033.

28] R.H. Rice, K.E. Steinmann, L.A. deGraffenried, Q. Qin, N. Tlor, R. Schlegel, Elevation of cell cycle control proteins ding spontaneous immortalization of human keratinocytes,Biol. Cell 4 (1993) 185–194.

29] B.L. Allen-Hoffmann, J.G. Rheinwald, Polycyclic aromaticdrocarbon mutagenesis of human epidermal keratinocytculture, Proc. Acad. Sci. U.S.A. 81 (1984) 7802–7806.

30] M.M. Bradford, A rapid and sensitive method for the qutitation of microgram quantities of protein utilizing the prciple of protein-dye binding, Anal. Biochem. 72 (1976) 24254.

31] T.J. Patterson, M. Ngo, P.A. Aronov, T.V. Reznikova, PGreen, R.H. Rice, Biological activity of inorganic arsenicantimony reflects oxidation state in cultured keratinocyChem. Res. Toxicol. 16 (2003) 1624–1631.

32] M. Shuliga, S. Chouchane, E.T. Snow, Upregulationglutathione-related genes and enzyme activities in cultureman cells by sublethal concentrations of inorganic arsenic,icol. Sci. 70 (2002) 183–192.


[33] J.W. Baty, M.B. Hampton, C.C. Winterbourne, Proteomic de-tection of hydrogen peroxide-sensitive thiol proteins in Jurkatcells, Biochem. J. (2005), in press.

[34] H.H. Jang, K.O. Lee, Y.H. Chi, B.G. Jung, S.K. Park, J.H. Park,J.R. Lee, S.S. Lee, J.C. Moon, J.W. Yun, Y.O. Choi, W.Y. Kim,J.S. Kang, G.-W. Cheong, D.-J. Yun, S.G. Rhee, M.J. Cho, S.Y.Lee, Two enzymes in one: two yeast peroxiredoxins displayoxidative stress-dependent switching from a peroxidase to amolecular chaperone function, Cell 117 (2004) 625–635.

[35] T.-C. Meng, T. Fukuda, N.K. Tonks, Reversible oxidation andinactivation of protein tyrosine phosphatases in vivo, Mol. Cell9 (2002) 387–399.

[36] L.M. Del Razo, B. Quintanilla-Vega, B.-C.E.E.S. Calderon-Aranda, M. Manno, A. Albores, Stress proteins induced by ar-senic, Toxicol. Appl. Pharmacol. 177 (2001) 132–148.

[37] B. Shi, R.R. Isseroff, Thapsigargin induces phosphorylation ofthe 27-kDa heat shock protein in human keratinocytes, J. Invest.Dermatol. 107 (1996) 749–754.

[38] J. Liu, M.B. Kadiiska, Y. Liu, T. Lu, W. Qu, M.P. Waalkes,Stress-related gene expression in mice treated with inorganicarsenicals, Toxicol. Sci. 61 (2001) 314–320.

[39] M.A. Deaton, P.D. Bowman, G.P. Jones, M.C. Powanda, Stressprotein synthesis in human keratinocytes treated with sodium ar-senite, phenyldichloroarsine, and nitrogen mustard, Fund. Appl.Toxicol. 14 (1990) 471–476.

[40] L. Ellgard, A. Helenius, Quality control in the endoplasmicreticulum, Nat. Rev. Mol. Cell. Biol. 4 (2003) 181–191.

[41] J. Lynch, M. Michalak, Calreticulin is an upstream regulatorof calcineurin, Biochem. Biophys. Res. Commun. 311 (2003)1173–1179.

[42] H. Hennings, D. Michaels, C. Cheng, P.M. Steinert, K. Hol-brook, S.H. Yuspa, Calcium regulation of growth and differ-entiation of mouse epidermal cells in culture, Cell 19 (1980)245–254.

[43] B. Wilkinson, H.F. Gilbert, Protein disulfide isomerase,Biochim. Biophys. Acta 1699 (2004) 35–44.

[44] R. Chapman, C. Sidrauski, P. Walter, Intracellular signallingfrom the endoplasmic reticulum to the nucleus, Ann. Rev. CellDev. Biol. 14 (1998) 459–485.

[45] C.M. Haynes, E.A. Titus, A.A. Cooper, Degradation of mis-folded proteins prevents ER-derived oxidative stress and death,Mol. Cell 15 (2004) 767–776.

[46] N.A. Robinson, S. Lapic, J.F. Welter, R.L. Eckert, S100A11,S100A10, annexin I, desmosomal proteins, small proline-rich proteins, plasminogen activator inhibitor-2, and involucrinare components of the cornified envelope of cultured humanepidermal keratinocytes, J. Biol. Chem. 272 (1997) 12035–12046.

[47] B.C. Bastian, U. van der Piepen, J. Romisch, E.P. Paques, E.B.Brocker, Localization of annexins in normal and diseased hu-man skin, J. Dermatol. Sci. 6 (1993) 225–234.

[48] M. Fauchon, G. Lagnile, J.-C. Aude, L. Lombardia, P. Soularue,C. Petat, G. Marguerie, A. Sentenac, M. Werner, J. Labarre, Sul-fur sparing in the yeast proteome in response to sulfur demand,Mol. Cell 9 (2002) 713–723.

[49] K.A. Webster, Evolution of the coordinate regulation ofglycolytic enzymes by hypoxia, J. Exp. Biol. 206 (2003)2911–2922.

[50] D.G. Hardie, Metabolic control: a new solution to an old prob-lem, Curr. Biol. 10 (2000) R757–R759.

[51] C.A. Raguso, S.L. Guinot, J.-P. Janssens, B. Kayser, C. Pichard,Chronic hypoxia: common traits between chronic obstructivepulmonary disease and altitude, Curr. Opin. Clin. Nutr. Metab.Care 7 (2004) 411–417.

[52] D.S. Kirkpatrick, K.V. Dale, J.M. Catania, A.J. Gandolfi, Low-level arsenite causes accumulation of ubiquitinated proteins inrabbit renal cortical slices and HEK293 cells, Toxicol. Appl.Pharmacol. 186 (2003) 101–109.

human epidermal cell protein responses to arsenite treatment in culture

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