(CANCER RESEARCH 56. 4407-4412. Occobcr I, 1996]

Selenite and Selenate Inhibit Human Lymphocyte Growth viaDifferent Mechanisms1

Giannis Spyrou,2 Mikael Björnstedt, Sven Skog, and Arne Holmgren

The Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry und Biophysics, Karolinska Inslilutet, S-I7Ì77 Stockholm [G. S., M. B., A. H.¡:Department ofBioscience, Center for Biotechnology, Karolinska ¡nstilutet, Novum. S-I4I 57 Huddinge [G. S.}: and Department of Medical Radiobiology, Karolinska Institute!, S-I7Õ 76

Stockholm ¡S.S.], Sweden

ABSTRACT

Selenium compounds like selenite and selenate have strong inhibitoryeffects, particularly on mammalian tumor cell growth by unknown mechanisms. We found that the addition of sodium selenite and sodium selenateinhibited the growth of human 3B6 and BL41 lymphocytes. Selenite wasmore potent because 10 fiM selenite produced a growth inhibitory effectsimilar to that of 250 // Mselenate. The mechanism of action of selenite andselenate appears to be different. 3B6 and BL41 cells treated with seleniteaccumulated in the S-phase; however, selenate caused an accumulation ofcells in G2. Selenite-mediated growth inhibition was irreversible, although

the effects of selenate could be reversed. Selenite, in contrast to selenate,is efficiently reduced by the thioredoxin system (thioredoxin, thioredoxinreductase, and NADPH). At concentrations required to observe a similareffect on cell growth, the activity of thioredoxin reductase, recently shownto be a selenoprotein, increased in selenite-treated cells and decreased inselenate-treated cells. Ribonucleotide reductase activity was inhibited in

an in vitro assay by selenite and selenodiglutathione but not by selenate.These results show that selenite and selenate use different mechanisms toinhibit cell growth.

INTRODUCTION

Selenium is an essential trace element with a number of biologicalroles ( 1). The selenium atom is incorporated as a Secys3 residue in

important redox-active enzymes, including glutathione peroxidase andtype I iodothyronine 5'-deiodinase (2, 3). Higher doses of inorganic

and organic selenium compounds have strong inhibitory effects on thegrowth of cultured mammalian cells (4-6). The inhibitory effects inparticular have been extensively studied in animal models with trans-

plantable tumors as well as chemically or virally induced tumors(7-9). On the other hand, selenium compounds in physiological doses

exert preventive effects to chemical carcinogenesis, and inhibition oftumor development is suggested to occur in the initiation and postinitiation phases (10).

Trx (12 kDa) contains a redox-active disulfide/dithiol in the active

site in its oxidized and reduced form. The first described function ofTrx was to be a hydrogen donor for ribonucleotide reductase (11), akey enzyme for DNA synthesis which reduces ribonucleotides todeoxyribonucleotides (12). Recent results show that Trx is present inhuman plasma (13) and is secreted by normal and neoplastic cellsthrough a leaderless secretory pathway (14, 15). It synergizes withinterleukin 1 and interleukin 2 and has been suggested to be animportant modulator of the immune system and in the regulation ofcell growth (15-18). Trx operates in conjunction with the FAD-

Received 2/12/96; accepted 8/1/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked ailvenisemenl in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by grants from the Swedish Medical Research Council

(Projects 13X-10370. 13P-10636, and I3X-3529). the Swedish Cancer Society (961). andthe Karolinska Institute.

- To whom requests for reprints should be addressed, at Department of Bioscience.

Center for Biotechnology. Karolinska Institute!. Novum, S-141 57 Huddinge. Sweden.Phone: 46-8-608-91-62; Fax: 46-8-74-55-38; E-mail: Giannis.Spyrou@mbb.ki.se.

' The abbreviations used are: Secys, selenocysteine; Trx, thioredoxin; GS-Se-SG.

selenodiglutathione; TR, thioredoxin reductase.

containing enzyme, TR, and NADPH (the Trx system) as a generalprotein disulfide reductase (11, 19).

Selenite (ScOj ) is a rather strong oxidizing agent and is reducedby an excess of thiols. It may react with reduced glutathione, the mostabundant thiol in mammalian cells, to form the covalent adduciGS-Se-SG (20) or undergo reduction by the Trx system (21, 22).GS-Se-SG, considered to be a central metabolite of inorganic sele

nium compounds, is further efficiently reduced by glutathione reductase (23) or the Trx system (22, 24, 25). Reduction of GS-Se-SG leadsto formation of selenide (HSe~/Se2^), a highly reactive form of

selenium. Selenide may redox cycle with oxygen, thus leading to anonstoichiometric oxidation of thiols and NADPH.

In selenate (SeO42 ), selenium exists in its highest oxidation state

(+VI). Selenate is much less reactive with thiols, and the metabolismof this selenium compound is largely unknown. One proposed mechanism is the formation of "active selenate" by ATP sulfurylase. ATPsulfurylase would catalyze the formation of adenosine-5'-selenophos-

phate, which then would be further reduced nonenzymatically byglutathione (26).

In this article, we describe the effects of selenite and selenate ontwo human B-lymphocyte cell lines either EBV-containing (3B6) orEBV-negative (BL-41). Specifically, we have examined proliferation

and cell cycle distribution as well as the effects on the Trx system andribonucleotide reductase. Our results indicate that selenite and selenate act via different mechanisms.

MATERIALS AND METHODS

Chemicals. Sodium selenite (Na2SeO,), sodium selenate (Na,SeO4), andglutathione were purchased from Sigma. Bovine insulin was obtained fromNordisk Insulin A/S (Geritone, Denmark). GS-Se-SG was prepared as de

scribed previously (22). RPMI 1640 was obtained from Flow Laboratories(Ayshire, G.B.) and PCS, glutamine, and antibiotics were obtained from LifeTechnologies, Inc., G.B. All other chemicals were obtained from Sigma.

Growth and Incubation of Cells. The EBV-transtbrmed lymphoblastoidcell line 3B6 was kindly provided by Professor T. Tursz (Institute Gustave-

Roussy, Paris, France). The cells were maintained suspended in RPMI 1640supplemented with 10% heat-inactivated PCS, glutamine, and antibiotics in a

5% CO2 atmosphere. For experiments, viable cells were counted after trypanblue staining (final concentration, 0.4%), and the cell density was adjusted to2.8 X IO5 cells/ml in RPMI 1640-10% FCS.

Enzyme Preparations. Recombinant human Trx was prepared as described previously (25). Human TR was prepared from placenta essentially asdescribed by Holmgren and Björnstedt (27). Mouse ribonucleotide reductase.subunits Rl and R2, was a kind gift from Professor L. Thelander (UmeâUniversity, Urnea, Sweden). Protein concentrations were determined using theBradford assay (Bio-Rad).

Enzyme Assays. Trx and TR activities were determined in cell extracts bythe ability to reduce insulin disulfides according to the method described byHolmgren and Björnstedt (27). Briefly, for TR activity measurements, 50 ju,gof each sample cell extract were incubated at 37°Cfor 20 min in a total volume

of 120 ¡uwith 80 DIMHEPES (pH 8.5). 0.7 mg/ml NADPH, 3.4 mM EDTA,2 mg/ml insulin, and 5 /J.M human Trx. The reaction was stopped by theaddition of 500 /nl of 0.4 mg/ml DTNB/6 M guanidine hydrochloride in 0.2 MTris-HCl (pH 8.0), and the absorbance at 412 nm was recorded. A standard

curve with purified TR was used to calculate the amount of TR in the cellextracts.

4407

on April 14, 2020. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MECHANISM OF GROWTH INHIBITION BY SELENIUM COMPOUNDS

Ribonucleotide reducíase activity was determined as described previouslyby measuring the formation of [3H]dCDP from [3H]CDP (28, 29). Purified

recombinant mouse ribonucleotide reducíase (7 /j.g Rl and 1.5 /xg R2/assay)was incubated in the presence of 4 juM human Trx, 25 nM human placenta TR.0.8 mM NADPH. and selenite or selenate. The R2 subunit was reactivatedbefore each experiment following the anaerobic procedure described by Mannet al. (30).

Elutriation and Cell Cycle Composition. Cells from the various cell cyclestages were isolated by using an elutriator rotor (JE-G; Beckman Instruments,

Inc., Fullerton, CA) as described previously (31). The separation was carriedout at +4°Cin Tris buffer (0.1 M Tris, 0.07 M NaCl, and 5 mM EDTA, pH 7.5)

at a rotor speed of 2800 rpm. The cell suspension was loaded at a flow rate of30 ml/min, and the volume of each fraction was 100 ml.

The distribution of cells in the cell cycle was determined using flowcytofluorometry as described previously (32). Cells were fixed in 4% formaldehyde in phosphate buffer. After removal of formaldehyde by washing inethanol and rehydration with water, the cells were incubated with Pronase(protease XXIV; Sigma), stained directly with 4',6-diamidino-2-phenylindole,

and analyzed using a PAS II flow cytometer (Partee, Münster.Germany). Therelative numbers of cells in G,, S-phase, and G2 + M were calculated by

computer analysis.

RESULTS

Effect of Selenite and Selenate on Human 3B6 LymphocyteGrowth. The relative sensitivities of human 3B6 cells to differentconcentrations of selenium compounds are shown in Fig. 1. In eachexperiment, the cells were collected at 24, 48, 72, and 96 h afteraddition of the selenium compounds to the medium, and the numberof viable cells was determined using trypan blue exclusion. Bothselenite and selenate caused a dose-dependent decrease in the number

of viable cells. However, much higher concentrations of selenate (250/U.M)were required to obtain the same degree of growth inhibition aswith selenite (10 /J.M).

Effect of Selenite and Selenate Withdrawal on Cell Growth andViability. From the experiment described in Fig. 1, it was not possible to conclude whether selenite or selenate affected cell viability orwhether these compounds inhibited proliferation of viable cells. Toinvestigate this, cells were grown for 3 days in the presence of 10 /J.Mselenite (Fig. 2A) or 250 JUMselenate (Fig. IB}. At the end of the thirdday, the cells were collected by centrifugation, washed with freshmedium, and each culture was divided in two parts: one part wasreplated in fresh medium with no addition of selenite or selenate, andthe other part in medium containing selenite or selenate. The cultureswere allowed to grow for an additional 4 days, and aliquots wereanalyzed each day. Cells originally exposed to selenate resumedgrowth after removal of selenate from the medium. In contrast, cellsexposed to selenite could not recover. Similar to selenate, selenitetreatment decreased the number of viable cells; however, selenite alsoinhibited the proliferation of cells previously exposed to selenite,indicating more severe damage.

Flow Cytofluorometric Analysis of Selenite- and Selenate-

treated Cells. The effect of selenite and selenate on the cell cycleprogression of 3B6 cells was examined by using cytofluorometry.Cells treated with either 10 JU.Mselenite or 250 fj.M selenate werecollected at 24, 48, or 72 h and analyzed. The results are shown in Fig.3 and Table 1. The control culture before addition of selenite orselenate showed a typical profile of DNA content distribution withpeaks at 60-90, cells in G, and at 140-160, cells at G2-M, and as abroad band between the two peaks (S-phase). Treatment with seleniteseemed to block cells in the S-phase with more exaggerated results

after 48 and 72 h. In contrast, selenate gave another profile with cellsaccumulated in G2-M at the expense of cells in the S-phase. These

results show that selenite and selenate affect the cell cycle distributiondifferently, indicating different mechanisms of action. To ensure that

0.8-

I=5 0.6-

D control

•¿�2.5uM selenite

A 5uM selenite

•¿�lOuM selenite

o

X 0.4-

0.2-

1.0

.-, 0.8-

I•¿�3

0.6-

0.4-

= 0.2-

D contro!A lOjaM selenate

o 50 |iM selenate

•¿�100 (J.Mselenate

À250 |iM selenate

B

Days

Fig. 1. Growth inhibition effects of selenite and selenate. 3B6 lymphocytes at a densityof 0.28 X If/1 cells/ml were incubated with medium containing selenite (A) or selenate (ß)

at the concentrations indicated for each curve. At defined times, the number of viable cellswas determined by trypan blue exclusion. Each experiment was repeated three times. Eachtime point for each experiment was duplicated. Similar results were obtained betweenexperiments.

the effect of selenite and selenate on the growth and cell cycledistribution was not restricted to 3B6 cells, which are EBV-infectedand known to have a high content of Trx, we used an EBV-negative

B cell line, BL41. Selenite and selenate inhibited the growth of BL41in a similar way as in 3B6 cells (Fig. 4A). Furthermore, flow cyto-metric analysis showed a dose-dependent accumulation of cellstreated with selenite in the S-phase and accumulation in G-,-M after

treatment with selenate (Fig. 4ß).Effect of Selenite and Selenate on Cellular TR Activity. Studies

in our laboratory have previously shown that selenite is a goodsubstrate for mammalian TR in vitro, whereas in the same assayselenate was no substrate (21). Therefore, we analyzed the activity ofTR in cells treated with selenite or selenate. We prepared total extractsof cells treated with different doses of selenite and selenate for 24 hand measured TR activity (Fig. 5). Selenite at concentrations up to 10P.Minduced TR activity (Fig. 5A), whereas selenate had the oppositeeffect (Fig. 5B).

Since the level of TR during the cell cycle is not known, we usedelutriation to obtain fractions with cells enriched at various phases ofthe cell cycle (Fig. 6A). Cell fractions which eluted early wereenriched for G, cells, with subsequent fractions becoming enrichedfor cells at progressively more advanced phases. The levels of TRwere detectable at all phases of the cell cycle and parallel the per-

4408

on April 14, 2020. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MECHANISM OF GROWTH INHIBITION BY SELENIUM COMPOUNDS

0.5

A 0.4-

ç" 0.3-exS °-2^

-o 0.1

10.0

250 uM selenate

medium change

B

0 1

Days

Fig. 2. Ability of 3B6 cells to resume growth after exposure to selenite or selenate. 3B6cells were grown in the presence of 10 /¿Mselenite or 250 |UMselenate. Following a 3-day

treatment, the medium was changed, and the cultures were divided into two parts with (O.A), or without (•.A) the selenium compounds present. Growth was determined as in Fig.1. Each experiment was repeated twice. Each time point for each experiment wasduplicated. Similar results were obtained between experiments.

centage of cells in the S-phase in each fraction (Fig. 65). Thus, TRexpression is higher in the S-phase.

Effect of Selenite and Selenate on CDP Reduction by Ribonu-

cleotide Reductase. Ribonucleotide reducíaserequires the dithiolform of Trx to reduce a ribonucleotide to a deoxyribonucleotide. Weanalyzed the effects of selenite, selenate, and GS-Se-SG on ribonu

cleotide reduction in an assay using purified ribonucleotide reducíaseand the Trx system. The results (Fig. 7) show that 10 JLIMselenite orGS-Se-SG reduced levels of dCDP formation to 15% of the control

value, whereas 250 /UMselenate only had a slight effect. Selenite andGS-Se-SG but not selenate inhibit the ribonucleotide reducíaseactiv

ity as well as the Trx system (24), suggesting that ribonucleotidereducíaseis inhibited because ils hydrogen donor system is inhibited.A direct effect of selenite and GS-Se-SG on the redox-aclive SH

groups in the active site of ribonucleotide reducíaseor struclural SHgroups causing formation of activated disulfides appears less likely.The results point to a direct effect on deoxyribonucleotide produclionresulting in S-phase inhibilion by selenite.

DISCUSSION

Selenium compounds like selenite and selenate in higher doses areknown to inhibit the growth of mammalian cells (4-6). The mecha

nisms of aclion of the different selenium compounds in this processare largely unknown. The metabolism of selenium compounds and thereaclions with cellular sulfhydryls have been suggested to be important for the growth inhibitory effects. Proposed inhibitory mechanisms

include interaclion wilh reduced glulalhione (33), inhibition of DNAand RNA synlhesis (34, 35), protein synlhesis (36), or inhibilion of thetranscriplion factor AP-1 DNA-binding activity (37).

In our experiments, the addition of selenate or selenite inhibited thegrowth of the human B cell line 3B6. Selenite was much more potent,because 10 ¿IMselenite showed an inhibitory effect similar lo lhal of250 /UMselenate. Furthermore, inhibilion by selenate was reversible,and cellular growth was obtained after removal of the medium containing selenate after a lag period. On Ihe contrary, 3 days after iheaddilion of selenite, cell growih had ceased irreversibly. At this time

1600-O.e£

1200-gB=

800-u400-n.acontrol•

A

O 40 80 120 160 200 240 280

Relative DNA content

2000

40 80 120 160 200 240 280

Relative DNA content

2000'

1600-

v"I 1200-

9e=5 800H

250 uM Selenate

O 40 80 120 160 200 240 280

Relative DNA content

Fig. 3. Flow cytofluorometric analyses of 3B6 cells following a 3-day exposure to 10

/¿Mselenite or 250 U.M selenate. The effect of selenite and selenate in cell cycleprogression was determined by measuring the DNA content distribution using flowcytofluorometry after treatment for 72 h. The lower DNA content (60-90) represents cellsin G,. the intermediate DNA content (90-140) cells in the S-phase. and the higher DNAcontent (140-160) represents G2-M.

4409

on April 14, 2020. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MECHANISM OP GROWTH INHIBITION BY SHLHNIUM COMPOUNDS

Table 1 Fltnv cylofluommetric analysis of 3B6 cells treated with .selenite and selenale

Distribution of cells at différentlime points

Cell cyclephaseControlGÃŒSG,-MSelenite

(lüfia}G,SG,-MSelenate

(250;IM)G,SG2-M24

h523995636g66191548h5636g4944765191672h65287414g10552(125

1

selenite seténate

donor for ribonucleotide reducíase.Inhibition of de novo synthesis ofdeoxyribonucleotides prevents cell cycle progression and causes accumulation of cells in the S-phase. The results of the in vitro assay for

ribonucleotide reducíase activity are in agreement with the resultsfrom the flow cytofluorometric experiments where selenite inhibitedthe cells at the S-phase.

Selenite is known to efficiently react with thiols (20, 21, 23).Selenodiglutathione (GS-Se-SG) is further reduced to the highly reactive aniónselenide. Selenide can undergo auto-oxidation and redoxcycling with thiols (21, 24, 38). Selenite and GS-Se-SG are highly

efficient substrates for TR but they can also inhibit the Trx system byoxidation of structural cysteine residues in human Trx or by NADPHdepletion (25). Inhibition of the Trx system by competition could leadto impaired reduction of the active site of ribonucleotide reducta.se.Conversely, selenate is not reactive with thiols and does not affectsignificantly the CDP reduction in the in vitro assay. This low reactivity with thiols may explain why selenate is 25 times less potent ininhibiting cell growth compared to selenite. The suggested mechanisms of selenite toxicity include interaction with thiols (39, 40). Aredox mechanism for the selenium-dependent inhibition of cellgrowth is also supported by previous reports showing that redox-active selenium compounds are far more potent than redox-inactive

compounds like selenate and selenomethionine (41, 42).Recently, Tamura and Stadtman (43) have discovered that TR from

human adenocarcinoma cells contains Secys. Also, TR from human Tcells is a selenoprotein containing at least 0.74 selenium/subunit (44).The Secys was present in an unusual C-terminal Cys-Secys-Gly

sequence. The presence of Secys in human TR may account for itscatalytic activity with selenite and other various low molecular weightcompounds (27), including lipid hydroperoxides (45). In our experiments, TR in extracts from cells treated with selenite was increased by

3.C

selenite selenate

Fig. 4. Growth and flow cytofluorometric analyses of BL41 cells after a 72-h exposureto selenite or selenate. BL4I cells at a density of 0.22 X IO6 cells/ml were incubated with

medium containing selenite (5 and 10 fxM)or selenate (50 and 250 ^IM). After 72 h. thenumber of viable cells (A) and the DNA content distribution (ß)were determined.

point, most cells were in the S-phase, indicating that impairment of

DNA synthesis was making progress in the cell cycle impossible.Medina and Oborn (4) have reported that the inhibitory effect ofselenite in mouse mammary epithelial YN-4 cells was reversible. This

result could be due to a number of factors including cell type, thiolcontent, and selenium resistance.

The cell cycle analysis by cytofluorometry showed that cells treatedwith selenite were inhibited in the S-phase. Similarly, YN-4 cellsaccumulate at S-phase-G2-M following selenite treatment (9). Selenate treatment caused accumulation in G2-M as determined by flowcytometric analysis. Selenite and its suggested main metabolite GS-Se-SG, at 10 /XMconcentrations, both effectively inhibited the reduc

tion of CDP to dCDP when the Trx system was used as a hydrogen

10 20 30 40

Selenite, uM

100 200 300

Selenate, uM

400 500

Fig. 5. Effect of selenite and selenale concentrations on the levels of TR. lixtracls fromcells treated with different concentrations of selenite or selenate for 24 h were analy/.edfor TR activity. The results are expressed as ng TR/mg total protein. ß«rv.SD.

4410

on April 14, 2020. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MECHANISM OF GROWTH INHIBITION BY SELENIUM COMPOUNDS

40% as measured in an assay for enzyme activity. Thus, the additionof selenite in moderate concentrations could increase the levels of TRby elevating the levels of selenide essential for the synthesis andincorporation of Secys. Furthermore, this induction by selenite mayact as a possible defense mechanism to prevent oxidative damage oran attempt to metabolize selenite to more inert forms such as elemental selenium via TR. On the other hand, selenate reduced the TRactivity by 50%. The levels of TR activity after selenite and selenatetreatment mirror the cell cycle analysis and the regulation of TRduring the cell cycle. Cells treated with selenite accumulate in theS-phase, and TR activity peaks in the S-phase. Cells treated withselenate accumulate in G,-M, and TR activity is down-regulated atG-.-M. The protein levels of Trx in cells treated with selenite or

selenate were not affected (data not shown). However, the assay forTrx did not discriminate between Trx inactivated through oxidation ofstructural cysteines and active Trx; thus, the effect of selenite andselenate on the activity of Trx remains to be determined.

Selenium is effective in inhibiting both virally induced [dietary Sesupplementation inhibited mammary tumorigenesis in BALB/cfC3Hmice infected with the murine mammary tumor virus MuMTV-S (46)]

and transplantable tumors [growth of Ehrlich ascites was retarded byselenium treatment (7)]. Tumor cells appear to be more sensitive thannormal cells to the effects of selenium compounds on cellular functions (35, 47). The strong irreversible growth inhibitory effect ofselenite is of great interest for the inhibition of tumor growth. Tumorcells are dependent on efficient synthesis of DNA, i.e., a highlyefficient ribonucleotide reduction. Our data show efficient inhibitionof ribonucleotide reducíaseby selenite and the metabolite GS-Se-SG.

The clinical potential should be important since selenite inhibited thegrowth of lymphoblastoid cells at rather moderate concentrations.

ig•¿�§.«

ll¿s•¿�3

23456

Fractions

Btî1

1I-i'o-o

—¿�'S-a2.e.2n-BA^

"\l

1234567

Fractions

Fig. 6. Analysis of cell cycle regulation of TR activity. Fractions containing cellpopulations enriched for cells in different phases of the cell cycle were obtained byelutriation (A). Total cell extracts of each fraction were assayed for TR activity (B) usingTrx-dependent insulin reduction. Btirs. SD.

control selenate selenite GS-Se-SG

Fig. 7. Ribonucleotide reducíase activity in the presence of selenite, selenate. andGS-Se-SG. The effect of selenite (10 JIM), selenate (250 JIM), and GS-Se-SG (10 fiM) onmouse ribonucleotide reducíasein the presence of NADPH/Trx and TR as hydrogen donorsystem was assayed as described in "Materials and Methods." The activity of ribonucleotide reducíasewas determined as the reduction of ?[H]CDP to '[H]dCDP. The results are

presented as percentage of control (100% equals the production of 2.8 nmol dCDP/30min). All assays were done in triplicate. Bars, SD.

Our results may provide mechanistic explanations to the inhibitoryeffects of some selenium compounds on cell growth.

ACKNOWLEDGMENTS

We are grateful to Carina Svensson for help with the cell experiments.

REFERENCES

1.Stadtman. T. C. Some selenium dependent biochemical processes. Adv. Enzymol.,48: 1-28, 1979.

Behne. D.. Kyriakopoulos. A.. Meinhold. H., and Köhrle.J. Identification of type Iiodothyronine 5'-deiodinase as a selenoenzyme. Biochcm. Biophys. Res. Commun..

173: 1143-1149. 1990.Flohe. L. The selenoprotein glutathione peroxidase. In: D. Dolphin, O. Avramovic.and R. Poulson (eds.). Glutathione: Chemical. Biochemical and Medical Aspects, pp.643-731. New York: John Wiley & Sons, 1988.Medina, D., and Oborn, C. J. Selenium inhibition of DNA synthesis in mousemammary epithelial cell line YN-4. Cancer Res.. 44: 4361-4365. 1984.

Nano. J. L., Czeruka. D.. Menguy, F.. and Rampal. P. Effect of selenium on thegrowth of three human colon cancer cell lines. Biol. Trace Elem. Res., 20: 31-43,

1989.Kuchan, M. J.. and Milner. J. A. Influence of intracellular glutathione on selenite-mediated growth inhibition of canine mammary tumor cells. Cancer Res.. 52: 1091-

1095, 1992.Greeder. G. A., and Milner, J. A. Factors influencing the inhibitory effect of seleniumon mice inoculated with Ehrlich ascites tumor cells. Science (Washington DC), 209:825-827. 1980.Milner. J. A. Effect of selenium on virally induced and transplantable tumor models.Fed. Proc., 44: 2568-2572, 1985.Medina. D., Lane, H. W., and Tracey, C. M. Selenium and mouse mammarylumorigenesis: an investigation of possible mechanisms. Cancer Res., 43: 2460-2464, 1983.Ip, C. The chemopreventive role of selenium in carcinogenesis. J. Am. Coll. Toxico!..5: 7-20. 1986.Holmgren, A. Thioredoxin. Annu. Rev. Biochem., 54: 237-271. 1985.

Thelander. L.. and Reichard. P. Reduction of ribonucleotides. Annu. Rev. Biochem.,48: 133-158, 1979.

Nakamura, H., DeRosa, S.. Roederer, M.. Anderson, M. T., Dubs. J. G.. Yodoi. J.,Holmgren. A., Herzenberg, L. A., and Herzenberg. L. A. Elevation of plasmathioredoxin levels in HIV infected individuals. Int. Immunol.. 8: 603-611. 1996.Rubarteli!. A.. Bajetto, A.. Allavena, G., Wollman, E., and Sitia, R. Secretion ofthioredoxin by normal and neoplastic cells through a leaderless secretory pathway.J. Biol. Chem., 267: 24161-24164, 1992.Ericson. M. L., Hörling.J., Wendel. H. V., Holmgren. A., and Rosen. A. Secretion ofthioredoxin after in vitro activation of human B cells. Lymphokine Cytokine Res., II:201-207, 1992.

Tagaya. Y., Maeda, Y.. Mitsui, A.. Kondo, N.. Matsui, H., Hamuro, J., Brown, N..16. Tagay:Arai, K.. Yokota. T.. Wakasugi. H.. el al. ATL-derived factor (ADE), an IL-2receptor/Tac inducer homologous to thioredoxin; possible involvement of dithiol-reduction in the IL-2 receptor induction. EMBO J., S: 757-764. 1989.

Wakasugi. N.. Tagaya. Y., Wakasugi. H., Mitsui. A., Maeda. M.. Yodoi, J.. andTursz. T. Adult T-cell leukemia-derived factor/thioredoxin, produced by both humanT-lymphotropic virus type I- and Epstein-Barr virus-transformed lymphocytes, acts asan autocrine growth factor and synergizes with interleukin 1 and interleukin 2. Proc.Nati. Acad. Sci. USA, 87: 8282-8286. 1990.

Rosen, A., Lundman, P., Carlsson, M.. Bhavani, K., Srinivasa. B. R., Kjellstrom, G..

4411

on April 14, 2020. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

MECHANISM OF GROWTH INHIBITION BY SELI-NIl'M COMPOUNDS

Musson. K., and Holmgren, A. A cd4( + ) T cell line-secreted factor, growth promoting for normal and leukemic a cells, identified as thioredoxin. Int. Immunol., 7:625-633, 1995.

19. Holmgren, A. Thioredoxin and glutaredoxin systems. J. Biol. Chem.. 264: 13963-13966. 1989.

20. Ganther, H. E. Selenotrisulfides. Formation by the reaction of thiols with seleniousacid. Biochemistry, 7: 2898-2905, 1968.

21. Kumar. S.. Bjornstedt, M.. and Holmgren, A. Selenite is a substrate for calf thymusthioredoxin reducíaseand thioredoxin and elicits a large non-stoichiometric oxidationof NADPH in the presence of oxygen. Eur. J. Biochem., 207: 435-439, 1992.

22. Bjornstedt, M., Kumar, S.. and Holmgren. A. Selenite and selenodiglutathione:reactions with thioredoxin systems. Methods Enzymol., 252: 209-219, 1995.

23. Ganther, H. E. Reduction of the selenotrisulfide derivative of glutathione lo apersulfide analog by glutathione reducíase. Biochemistry. 10: 4089-4098, 1971.

24. Bjornstedt, M., Kumar, S., and Holmgren, A. Selenodiglutathione is a highly efficientoxidant of reduced thioredoxin and a substrate for mammalian thioredoxin reducíase.J. Biol. Chem., 267: 8030-8034, 1992.

25. Ren. X., Bjornstedt, M.. Shen, B., Ericson, M. L., and Holmgren, A. Mutagenesis ofstructural half-cystine residues in human thioredoxin and effects on the regulation ofactivity by selenodiglutathione. Biochemistry, 32: 9701-9708, 1993.

26. Dilworth. G. L., and Bandurski, R. S. Activation of selenate by adenosine 5'-

triphosphate sulphurylase from Sacchuromvces cerevisiae. Biochem. J., 163: 521-

529, 1977.27. Holmgren, A., and Bjornstedt. M. Thioredoxin and thioredoxin reducíase.Methods

Enzymol., 252: 199-208, 1995.

28. Engström,Y., Eriksson, S., Thelander, L., and Àkerman, M. Ribonucleotide reducíasefrom calf Ihymus. Purification and properties. Biochemistry, 18: 2941-2948, 1979.

29. Ingemarson, R.. Graslund. A.. Darling. A., and Thelander, L. Herpes simplex virusribonucleotide reducíase:expression in Escherichia colt and purification to homoge-neily of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodallon subunil. J. Virol., 63: 3769-3776, 1989.

30. Mann, G. J., Graslund. A., Ochiai, E-I., Ingemarson, R., and Thelander, L. Purification and characterization of recombinant mouse and herpes simplex virus ribonucle-olide reducíaseR2 subunit. Biochemistry. 30: 1939-1947. 1991.

31. Skog, S., Tribukait, B.. and Sundius, G. Energy metabolism and ATP turnover duringthe cell cycle of Ehrlich ascites lumor cells. Exp. Cell Res., 141: 23-29, 1982.

32. Caslro, J., Heiden, T., Wang, N., and Tribukail. B. Preparalion of cell nuclei fromfresh tissues for high quality DNA flow cytometry. Cylomelry, 14: 793-804, 1993.

33. Baiisi, G., Katki, A. G., Klecker. R. J., and Myers. C. E. Selenium-induced cytotox-icily of human leukemia cells: inleraclion wilh reduced gluiaihione. Cancer Res., 46:5482-5485, 1986.

34. Jiang, X. R.. Macey, M. G., Lin, H. X., and Newland, A. C. The anti-leukaemiceffects and Ihe mechanism of sodium selenite. Leuk. Res., 16: 347-352. 1992.

35. Abdullaev, F. I., MacVicar, C.. and Frenkel, G. D. Inhibition by selenium of DNA andRNA synthesis in normal and malignant human cells in ritro. Cancer Leu., 65:43-49, 1992.

36. Vemie, L. N.. Collard. J. G., Eker, A. P. M., De Wildt, A., and Wilders, I. T. Studieson the inhibition of protein synthesis by selenodiglutathione. Biochem. J., 180:213-218, 1979.

37. Spyrou, G., Bjornstedl, M.. Kumar. S., and Holmgren, A. AP-I DNA-binding activityis inhibited by selenite and selenodiglutathione. FEBS Lett.. 368: 59-63. 1995.

38. Shamberger. R. J. The genotoxicity of selenium. Mutât.Res., 154: 29-48, 1985.

39. Garberg. P., and Högberg,J. The role of hypoxia in selenium metabolism. Biochem.Pharmacol., 36: 1377-1379, 1987.

40. Spallholz, J. E. On the nature of selenium toxicity and carcinostatic activity. J. FreeRadicals Biol. & Med., 17: 45-64, 1994.

41. Siwek, B., Bahbouth. E., Serra, M-A.. Sabbioni, E., de Pauw-Gillet. M-C., and

Bassleer, R. Effect of selenium compounds on murine B16 melanoma cells andpigmented cloned pB16 cells. Arch. Toxicol., 68: 246-254, 1994.

42. Yan, L., Yee, J. A.. Boylan. L. M.. and Spallholz. J. E. Effect of selenium compoundsand thiols on human mammary tumor cells. Biol. Trace Elem. Res., 30: 145-162,1991.

43. Tamura. T., and Stadtman, T. C. A new selenoprotein from human lung adenocarci-noma cells. Purification, properties and thioredoxin reducíase activity. Proc. Nail.Acad. Sci. USA, 93: 1006-1011, 1996.

44. Gladyshev, V. N., Jeang, K-T.. and Stadtman, T. C. Selenocysteine. identified as thepenultimate C-terminal residue in human T-cell thioredoxin reducíase,corresponds toTGA in the human piacental gene. Proc. Nati. Acad. Sci. USA, 93: 6146-6151, 1996.

45. Bjornstedt, M., Hamberg. M.. Kumar. S., Xue. J., and Holmgren, A. Human thioredoxin reducíasedirectly reduces lipid hydroperoxides by NADPH and selenocystinestrongly stimulates the reaction via catalytically generated selenols. J. Biol. Chem.,270: 11761-11764. 1995.

46. Medina, D.. and Shepherd, F. Selenium mediated inhibition of mouse mammarytumorigenesis. Cancer Lett., 8: 241-245, 1980.

47. Medina. D.. and Oborn, C. J. Differential effects of selenium on the growth of mousemammary cells in vitro. Cancer Lett., 13: 333-344, 1981.

4412

on April 14, 2020. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1996;56:4407-4412. Cancer Res   Giannis Spyrou, Mikael Björnstedt, Sven Skog, et al.   Different MechanismsSelenite and Selenate Inhibit Human Lymphocyte Growth via

  Updated version

  http://cancerres.aacrjournals.org/content/56/19/4407

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/56/19/4407To request permission to re-use all or part of this article, use this link

on April 14, 2020. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from