[CANCER RESEARCH 46, 3384-3388, July 1986]

Differential Effects of Selenium on Normal and Neoplastic CanineMammary Cells1'2

M. E. Fico, K. A. Poirier,2 A. M. Watrach, M. A. Watrach, and J. A. Milner

Department of Food Science, Division of Nutritional Sciences [M. E. F., K. A. P., J. A. MJ, and Department of Pathobiology [A. M. W., M. A. WJ, University of Illinois,Urbana, Illinois 61801

ABSTRACT

Four different canine mammary tumor (CMT) cell lines and a nonneoplastic primary culture of mammary cells were examined for their invitro responsiveness to selenium supplementation. These cell lines werefound to vary in their metabolic response to increasing concentrations ofselenium. Sensitivity to selenium, as sodium selenite, increased withincreasing concentrations of this trace element in all of the neoplasticlines. These data also suggest that increasing the plating density of tumorcells further increases the sensitivity to selenium. A relatively selenium-sensitive cell line (CMT-13) and relatively insensitive cell line ((Mill) were characterized on the basis of reduced growth resulting fromselenium supplementation. Increasing the concentration of selenium to0.75 »ig/mldepressed the growth of CMT-13 and CMT-11 cells by 75%and 11%, respectively, while no inhibition was observed in nonneoplasticcells. These cell lines also varied in their sensitivity to different forms ofselenium. Selenodiglutathione was the most effective form of seleniumexamined that inhibited tumor cell growth. The sensitivity of the neoplastic lines was selenodiglutathione »sodium selenite »selenocystine> selenomethionine. None of the forms of selenium examined inhibitedthe growth of the nonneoplastic mammary cells in culture. Supplementation with sodium selenite (l »igSe per ml) for 60 min resulted in adramatic depression in RNA biosynthesis in CMT-13, but not CMT-11or nonneoplastic cells.

INTRODUCTION

Dietary factors constitute important variables that may eitherenhance or suppress tumor development. One factor, implicatedas a potential inhibitor of tumor development, is the traceelement selenium. Regional and national variation in dietaryselenium intake is inversely correlated with mortality from avariety of cancer sites including breast, colon, rectum, prostate,lung, and blood (1,2). Considerable evidence also indicates thatdietary selenium supplementation can reduce the incidence ofvirally and chemically induced tumors (3-14).

Weisberger and Suhrland (15) observed that selenocystinesignificantly retarded the growth of Murphy's lymphosarcoma

implanted into rats. Our laboratory has shown that selenium isalso capable of retarding the in vitro growth of Ehrlich ascitestumor cells (16-18), LI210 leukemic cells (19), SV40-3T3tumor cells (20), canine mammary tumor cells (21), and humanmammary tumor cells (22). The inhibition of tumor development was dependent upon the form and dose of seleniumadministered. Data obtained with solid transplantable tumorssuggest the antitumorigenic properties of selenium are not onlydue to direct cytotoxicity, but are associated with a decrease inthe proliferation of neoplastic cells (20-23).

The present studies were designed to examine the effect ofselenium on the growth of nonneoplastic and neoplastic canine

Received 1/22/85; revised 2/24/86; accepted 3/28/86.1Supported in part by USPHS Grant 5T32 CA 090607 and United States

Department of Agriculture, Science, and Education Administration Grant 5901-0410-9-0243; American Cancer Society, Illinois Division, Inc., Grant 82-2; andUSPHS Grant CA33699 awarded by the National Cancer Institute, Departmentof Health and Human Services. Presented in part at the Third InternationalSymposium on Selenium in Biology and Medicine, Beijing, China (42).

2 Present address: University of Medicine and Dentistry of New Jersey, Schoolof OstéopathieMedicine, Medical Ans Building, 300 Broadway, Camden, NJ08103.

mammary cells in culture. Differences in sensitivity to seleniumamong cell types, as influenced by different forms of selenium,were also examined. In addition, the effect of selenium onmacromolecular biosynthesis was examined.

MATERIALS AND METHODS

Tumor Cells. Four CMT3 cell lines designated CMT 11, 13, 14A,

and 14B were used in the following studies. (Mill was isolated froma primary neoplasm of a mammary gland of a 9-yr-old Airedale. Thistumor had the histológica!characteristics of a spindle-cell carcinoma.Electron microscopic examination revealed numerous desmosomaljunctional complexes. This line was purified and has been maintainedin the laboratory of A. Watrach for over 105 passages. Its morphologicalproperties, karyotypic configuration, hormone receptors, and tumori-genicity were defined in athymic nude mice. These cells are essentiallyepithelial in type and have continued to maintain a modal chromosomenumber of 78. The CMT-13 cell line was developed from a primarytumor of the mammary gland of an 1l-yr-old mixed terrier. This tumorhad morphological properties of a cystic adenocarcinoma. This cell linewas morphologically stabilized by cultivation and further purified byisolation of colonies arising from single cells in soft agar. Cultures havebeen continuously propagated through 95 passages in the laboratory ofA. Watrach. These cells were determined to be of myoepithelial derivation and have a modal chromosome number of 68. In nude micethese cells consistently produced neoplastic growths with the samemorphological characteristics as the original tumor.

CMT-14A cells were isolated from a primary neoplasm of the rightfifth mammary gland of a 9-yr-old female dachshund. The originaltumor had the structural characteristics of a carcinoma of the "clearcell" type (24). These cells are of a distinctly epithelial type with

numerous desmosomal junctional complexes and a modal chromsomenumber of 86. The cells have been continuously propagated through115 passages. In nude mice, CMT-14A cells grow slowly and produceneoplastic growths of the same morphological characteristics as theoriginal tumor. CMT-14B was developed from the same neoplasm asCMT-14A. The CMT-14A and B cell lines were separated early duringisolation and developed different cultural and morphological properties.CMT-14B is essentially epithelial like in its structural characteristicsand has a strong clonogenic potential. This cell line has been maintainedover 104 passages in the laboratory of A. Watrach and has a modelchromosomal number of 86. This cell is very aggressive upon transplantation in nude mice and readily gives rise to large tumors, eventuallykilling the host.

Isolation of Nonneoplastic Cells. A short-term cell line was developedfrom a normal mammary gland of a 4-yr-old lactating pointer. Thegland was surgically removed and processed within 1 h. Small specimens of mammary tissue were trimmed of fat and excess connectivetissue and minced with scissors. The minced tissue was then transferredto an Erlenmeyer flask containing sufficient 0.25% trypsin in phosphate-buffered saline to form a suspension. The resulting suspensionwas constantly stirred at 37°C,utilizing a magnetic stirrer. At intervals

the supernatant was harvested and centrifuged, the trypsin was decanted, and each cell pellet was washed once in Earle's diploid basal

medium (25), containing 10% fetal bovine serum. The cells were thenplated in plastic flasks, using the same medium supplemented withantibiotics. A majority of cells in the primary cultures had morphological characteristics suggestive of epithelial-like cells. Primary culturesused in the present studies were grown in Earle's diploid basal medium

3The abbreviation used is: CMT, canine mammary tumor.

3384

on June 26, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

RESPONSIVENESS OF CMT CELL LINES TO SELENIUM

supplemented with 10% fetal bovine serum and antibiotics.Selenium Sensitivity. The CMT cell lines and the primary nonneo-

plastic primary culture were examined for their sensitivity to seleniumaddition in vitro. Neoplastic and nonneoplastic cultures were maintained with chemically defined Ringer's inclusive phosphate medium

and 10% fetal bovine serum. Neoplastic and nonneoplastic cells weremaintained at 37'C in an environment of 5% CO2:95% air and a

relative humidity of 95%. All subsequent cultures were maintained inthis manner.

In Experiment 1, each cell culture, at the time of confluency, wasreplenished with fresh medium supplemented with selenium as Na2SeOj

molecule. Radioactivity in each of the macromolecular fractions wasdetermined with a Beckman LS9000 liquid scintillation counter (Beck-man Instrument Co., Palo Alto, CA). Statistical analysis was performedby the Student t test. Mean differences with P < 0.05 were consideredstatistically significan!.

RESULTS

Growth Response to Selenium. In Experiment 1, two of thefour canine tumor cell lines, CMT-13 and CMT-14B, were

(Sigma Chemicals, St. Louis, MO) at concentrations of 0, 0.2 0.4, 0.6, sensitive to the addition of 0.2 /¿gof selenium per ml (Fig. 1).0.8, or 1.0 fig,Se per ml. Cells were harvested by trypsinization at 24-hintervals for 72 h following medium repletion for determination ofselenium toxicity and total cell number. Viabilities were determined bythe trypan blue dye viable exclusion technique (26, 27) on a Bright-

Line hemocytometer (American Optical Corporation, Buffalo, NY).Cell survival is expressed as a percentage of cells excluding trypan blue,in relation to control cultures at the same time interval. Duplicateassays were performed on each concentration of selenium for each cellline at each of the time points.

In Experiment 2, the effect of selenium on the growth of CMT-11,CMT-13, and nonneoplastic canine mammary cell cultures was examined during a 4-day experiment. In this study all lines were plated at 1x 10s cells per flask. After 24 h, selenium, as sodium selenite at

concentrations ranging from 0.0 to 0.75 jig/ml, was added to each cellline. Attached cells were harvested by trypsinization and counted at 24,48, and 72 h after the addition of selenium. Cells were counted with aCoulter Counter (Coulter Electronics, Hialeah, FL). Four flasks perconcentration of selenium were examined at each time point.

Influence of Different Forms of Selenium on Cell Growth. In Experiment 3, the relatively selenium-insensitive (CMT-11), selenium-sensitive (CMT-13), and the nonneoplastic mammary cell cultures wereexposed to different forms of selenium to determine their efficacy inreducing growth. Nonneoplastic, CMT-11, and CMT-13 cell lines wereplated at 1 x 10s cells per flask and allowed to attach for 24 h. After

24 h the medium was replenished, and the flasks were divided intogroups and treated with 0.75 PKof selenium per ml as selenomethionine,selenocystine, sodium selenite, and selenodiglutathione. All seleniumcompounds were purchased from Sigma Chemical Co., St. Louis, MO.Selenodiglutathione was prepared and separated on a series of Dowex50Wx4 (200-400 mesh hydrogen form) columns according to themethod of Ganther (28) as modified by Vernie et al. (29). Cells wereharvested by trypsinization and counted at 24 and 48 h after the additionof selenium. Four flasks per experimental treatment were counted ateach time period. All data were analyzed by analysis of variance with aleast significance test applied for mean comparison.

Biosynthesis of Macromolecules. In Experiment 3, CMT-11, CMT-13, and NCM cells were grown to subconfluency before repletion withmedium containing 0 or l ¿igof Se per ml. To each group of flasks wasadded either 2.5 /iCi of |5'-3H]thymidine (17 Ci/mmol), 5.0 //Ci of (5'-3H]uridine (29 Ci/mmol), or 25 MCiof L-[4,5-3H]leucine (36 Ci/mmol)

(Amersham Corporation, Northbrook, IL). All flasks were incubatedfor an additional 60 min before the medium was decanted. The cellswere washed twice with phosphate-buffered saline and removed byscraping. DNA, RNA, and protein were fractionated by centrifugationusing a neutral cesium chloride density gradient. For these samples thecell pellet was lysed with sodium dodecyl sulfate (0.5% in 0.02 M TrisHCl, pH 7) and mixed with 150% CsCl (1.5:1.0 CsCl:0.05 M Tris-HCl,pH. 7.0). The gradient was spun to equilibrium at 105,000 x g in aBeckman Ti 70.1 rotor for 48 h. Total RNA was determined by themethod of Ceriotti (31). An adaptation of the method of Taylor andMilthorpe (32) was used to estimate DNA spectrofluorometrically usingthe DNA-specific fluorochrome 4,6-diamidino-2-phenylindole (Boeh-ringer Manheim, West Germany). A standard curve was generated andfound to be linear between 0.5 and 10 ¿tgof DNA per ml. Protein wasdetermined by the method of Lowry et al. (33). The purity of eachfraction was assessed for contamination by the other macromoleculesand found to be minimal. Any cross-contamination of the fractions wascorrected for in the calculation of radioactivity per unit of the macro-

CMT-14A cells were not sensitive to the addition of this quantity of selenium, and they had viabilities reduced only by about20% when the medium was supplemented with 0.6 ng of selenium per ml. Greater quantities of selenium were effective insuppressing the viability of these cells. The CMT-11 cell linewas also extremely resistant to selenium supplementation. Concentrations of selenium of 0.8 ¿¿g/mlor more were required tocause a modest, less than 20%, decrease in the viability of thiscell line. Incubation of CMT-13, CMT-14A, and CMT-14Bwith this quantity of selenium resulted in a 97, 51, and 40%decrease in viabilities within 48 h. Although the CMT-11 cellline was relatively insensitive to selenium supplementation, itwas approximately 4 times more sensitive to l /ig of seleniumper ml than was the primary culture of nonneoplastic caninemammary cells. Viabilities of nonneoplastic canine mammary

100 1001

0 24 48 72

Duration of Treatment (hours)0 24 48 72

Duration of Treatment (hours)Fig. 1. The influence of increasing concentrations of selenium on (he viability

of CMT-13, CMT-11, CMT-14A, and CMT-14B and a primary culture ofnonneoplastic mammary cells in culture. Selenium was added to cultures atconfluency. The concentrations of selenium were 0.2 fig/ml (•).0.4 ¿ig/ml(•),0.6 iig/ml (A), 0.8 (ig/ml (D), and 1.0 Mg/ml (O), atoe refer to CMT-13, CMT-14B, CMT-14A, nonneoplastic mammary cells, and CMT-11, respectively.

3385

on June 26, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

RESPONSIVENESS OF CMT CELL LINES TO SELENIUM

cells were not altered by selenium supplementation up to 0.8ííg/ml.Addition of 1.0 ng of Se per ml decreased cell numberless than 10% in these nonneoplastic canine mammary cells.

In Experiment 2, selenium, as sodium selenite, inhibited thegrowth of both the CMT-11 and 13 cell lines but not the growthof the primary culture of the nonneoplastic cells (Figs. 2 and 3;Table 1). Growth of the CMT-13 cells was more sensitive toselenium supplementation than was CMT-11, as observed inthe viability studies of Experiment 1. Sodium selenite supplementation caused a greater depression in the growth rate inCMT-13 cells in the first 24 h than after longer incubations.Within 24 h 0.1 ng of Se per ml resulted in a significantinhibition of growth of CMT-13 cells (Fig. 2). Addition of 0.05ng of Se per ml actually stimulated the growth of the CMT-11cell line (Fig. 3). Increasing the concentration of selenium to0.1 ng/m\ in the CMT-11 cells reduced growth compared tothat observed with no selenium supplementation. Concentrations of selenium greater than 0.5 /¿g/mlwere required to inducea significant inhibition of growth in CMT-11 cells. Even thisconcentration required approximately 48 h of incubation todetect a depression in growth. The percentage of growth inhibition caused by selenium supplementation of cultures of CMT-11 was reduced after 3 days of incubation, indicating someadaptation occurs in these cells.

Selenium supplementation up to 0.1 ng/m\ had no influenceon the growth of nonneoplastic mammary cells (Table 1). Thegrowth of these cells was stimulated by greater than 0.5 ng ofSe per ml by Day 3 of incubation (Table 1). Increasing theconcentration of Se to 0.75 ng/m\ reduced the time required todetect a growth stimulation in the nonneoplastic cells.

Growth Response to Different Forms of Se. CMT-13 wasmore sensitive than CMT-11 to all forms of selenium examined.Selenocystine inhibited the growth of CMT-13 by 29.1% buthad no effect on the growth of CMT-11 cells. Selenomethioninehad minimal effects on cellular growth in both cell lines when

-1

Fig. 2. Influence of increasing concentrations of selenium on the growth ofCMT-13 cells in culture. Selenium was added 24 h after plating. Points, mean of4 observations per concentration of selenium per day of incubation. Seleniumsignificantly inhibited growth (/' < O.OS)when present in the medium at concentrations of >0.1 UKml (Days 1 and 2) and aO.05 ¿ig/ml(Day 3).

Control— 0.05

-•—0.10-O 0.5-o 0.75

-1

Fig. 3. Influence of increasing concentrations of selenium on the growth ofCMT-11 cells in culture. Selenium was added 24 h after plating. Points, mean of4 observations per concentration of selenium per day of incubation. Seleniumsignificantly inhibited growth (P < O.OS)when present in the medium at concentrations of >O.S pg/ml (Day 2) and stimulated growth at - .0.05 pg/ml (Days 2and 3).

Table 1 Effect of selenium as NatSeO, on the growth of nonneoplastic caninemammary cells

SeleniumG/g/ml)0.0

0.050.100.500.75No.

of cells x 10 ' on the followingdaysI2.17

±0.15"2.05 ±0.23°2.12 ±0.15"2. 10 ±0.23"2.23 ±0.18"23.09

±0.11"2.90 ±0.14"3.05 ±0.24«2.89 ±0.15°3.46 ±0.09*34.66

±0.24'5.11 ±0.32°5.52 ±0.22"6.44 ±0.25*6.55 ±0.21*

Vertical means ±SE with unlike superscripts differing by P < 0.05.

present 48 h (Table 2). In other studies longer incubation withthis concentration has been found to retard the growth of CMT-13 cells but not CMT-11 cells (data not presented). Selenium,as selenodiglutathione and sodium selenite, markedly inhibitedthe growth of both the CMT-13 and CMT-11 cell lines but hadno adverse effects on nonneoplastic canine mammary cells(Table 2). Selenodiglutathione completely inhibited the growthand resulted in cell killing of CMT-13 cells in less than 24 h.Only a 39% decrease in growth occurred after exposure toselenodiglutathione in the CMT-11 cell line during this sametime of incubation. The percentage of growth inhibition increased to 76% after 2 days of exposure to selenodiglutathionein CMT-11 cells. Sodium selenite supplementation at 0.75 fig/ml inhibited the growth of CMT-13 by 21 % at 24 h but had nodetectable effect on the growth of the CMT-11 cell line. However, after 2 days of treatment with selenite the inhibition ofCMT-13 was 51% and CMT-11 was 42%.

Macromolecular Biosynthesis. Addition of sodium selenitefor 60 min at a concentration of 1 ng of Se per ml significantlyincreased the incorporation of thymidine into DNA in both theCMT-11 and nonneoplastic cells. However, incorporation of

3386

on June 26, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

RESPONSIVENESS OF CMT CELL LINES TO SELENIUM

label into the DNA of CMT-13 cell was not influenced byselenium supplementation during this incubation time. Proteinsynthesis was actually depressed by approximately 15% in onlythe nonneoplastic mammary cells. RNA biosynthesis was decreased approximately 50% in CMT-13 but was not significantly altered in either the CMT-11 or nonneoplastic culturesexposed to selenium for 60 min (Table 3).

DISCUSSION

Increasing information strongly suggests that selenium hasan inhibitory effect on the proliferation of neoplastic tissue.However, little information exists to explain the mechanism bywhich selenium exerts its antiproliferative effect. Broghamer etal. (34) reported that increased concentrations of blood selenium in some cancer patients are associated with tumors remaining confined to the region of origin, fewer distant metastasis, reduced numbers of primary neoplasms, and decreasedfrequency of recurrences. Neoplastic tissue, regardless of cellular origin, is known to concentrate a variety of seleniumcompounds at levels higher than that of normal differentiatingtissue (35). Human and animal experiments suggest the incorporation of selenium in neoplastic tissue is negatively correlatedwith the degree of differentiation of the tissue (34-38). Theability of selenium to inhibit the growth of neoplastic but notnonneoplastic cells in the present experiments is consistentwith this hypothesis.

The use of established cell lines provides a good model tostudy the mechanism of selenium-induced inhibition of tumorgrowth. In vitro differences in the growth of cultured normal,preneoplastic, and neoplastic murine mammary cells treatedwith selenium have been reported (39). This differential response is also reflected in the various canine mammary celllines used in the present studies. The range of response toselenium in the present studies was from relatively sensitive torelatively resistant. The inhibiting effects of selenium on these

Table 2 Effect of different forms of selenium on the growth of CMT-13, CMT-11,and nonneoplastic canine mammary cells

No. of cells x10-*Forms

ofselenium"Day

1*ControlSelenomethionineSelenocystineSodium

seleniteSelenodiglutathioneDay

2ControlSelenomethionineSelenocystineSodium

seleniteSelenodiglutathioneCMT-135.77

±0.42'5.87±0.75'5.79±0.59e4.57±0.38'0'9.30

±0.62C8.94±0.74'6.59±LIO**4.55±0.60*ofCMT-115.09

±0.54C4.78±0.20e5.33±0.51'4.94±0.22'3.09±0.34''15.21

±0.51'13.26±0.74'14.25

±0.51'8.78±1.00a3.60±1.06'Non

neoplastic1.

72 ±0.27e1.81±0.23'2.15±0.44'1.84±0.12'1.68±0.18'4.40

±0.26'4.46±0.22'4.56

±0.12'4.28±0.16'4.46±0.27'

" Selenium was added to the medium in each form at 0.75 im/mi.* Days 1 and 2 indicate 24 and 48 h after selenium treatment, respectively.'"•^Verticalmeans ±SE with unlike superscripts differing by P< 0.05.

cells were dependent on the total concentration and form, thelength of exposure, cell type and growth state, or plating density.

Comparison of confluent and growing cultures indicates thatcell density modifies the sensitivity to selenium (Figs. 1-3).These data are consistent with the data of Medina et al. (40)showing that the inhibition of COMMA-D cells caused byselenium increased with cell density. The reason for this increased sensitivity is unknown.

The present studies are consistent with previous observationsfrom our laboratory showing that the form of selenium is animportant factor determining the ability of this trace elementto inhibit tumor growth (16, 19, 23). Vernie et al. (29) reportedthat Selenodiglutathione was a potent inhibitor of protein biosynthesis in cells in culture. This inhibition is thought to occuras a result of a depression in the synthesis of the elongationfactor eIF-2 (29). Selenodiglutathione was the most potent formof selenium examined in these studies. However, not all cellsresponded identically to this form of selenium. Whether thedifferences in efficacy of the forms of selenium examined in ourstudies are related to cellular uptake or metabolism of seleniumwithin the cell is unknown.

The quantity and form of selenium accumulated by thesecells and their ability to metabolize selenium may explain theirdifferences in sensitivity to this trace element. Whether theform of selenium present in the CMT-11 cell differs from thatfound in the CMT-13 or nonneoplastic cell line is unknown.Our data suggest that the CMT-11 cell line may be moreefficient in acclimating to higher concentrations of selenium asa function of time (Fig. 3). The ability to acclimate suggeststhat the rate of selenium detoxification may be a factor in thesensitivity of cells to this trace element.

The inhibitory effects of selenium on the growth of tumorcells are not completely understood. Gruenwedel and Cruik-shank (41) reported that, after incubating Hoi.a cells with 2, 5,and 10 /IM sodium selenite, the synthetic activity of RNA,DNA, and protein was significantly decreased. However, theinhibition of one of these marcomolecules may lead to alterations in the synthesis or degradation rates of the other macro-molecules. Medina and Oborn (39) observed that the growthresponse of a selenium-responsive tumor cell as measured by[3H]thymidine incorporation into DNA was altered 72 h, fol

lowing the addition of selenium to the medium. Lewko et al.(42) have also reported a progressive depression in the growthof human breast cancer lines after the addition of sodiumselenite from 0.1 to 1.0 \i%of Se per ml. Accompanying thisdepression in growth was a depression in protein biosynthesisin cells cultured for 24 h. In the present studies, only nonneoplastic cells exposed to selenium were found to have depressedincorporation of leucine into protein. These cells are less sensitive to selenium since they are able to withstand relativelyhigh selenium concentrations without an inhibition of growth.At present, it is unknown if protein biosynthesis remains depressed in these cells or if they are able to compensate for

Table 3 Effect of selenium treatment on macromolecular biosynthesis in canine mammary tumorsCMT-13"RNA

biosynthesis (dpm [3H]uridine/pg RNA)DNA biosynthesis (dpm [3H)thymidine/Mg DNA)Protein biosynthesis (dpm [3H]leucine/>ig protein)Se

treated2955±154*

22.9 ±I.I*724±21*Control6295

±176'23.8 ±0.3*767 ±23*CMT-1

1°Se

treated8891±558*

10.0 ±0.2*992 ±9i»Control8471

±39*6.6 ±1.2'

1005 ±36*NCMC°Se

treated2246±206*

4.2 + 0.1*

482 ±yControl2247

±159*3.2 ±0.1'

541 ±13*

* CMT-13 is selenium-responsive, and CMT-11 is selenium-nonresponsive canine mammary tumor cell line. NCMC area is nonneoplastic, nonresponsive primary

cell line.b~cHorizontal paired means ±SE with unlike superscripts differing, /*< 0.05.

3387

on June 26, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

RESPONSIVENESS OF CMT CELL LINES TO SELENIUM

exposure to this concentration of this trace element. Nevertheless, an inhibition of protein biosynthesis does not appear to bethe principal factor accounting for the enhanced sensitivity ofCMT-13 cells to selenium.

In the present studies, the incorporation of uridine into RNAwas modified only in the selenium-sensitive cell line (CMT-13).These data suggest RNA may be the first crucial macromoleculeto change after selenium supplementation. This early detectionof depressed RNA synthesis in the selenium-sensitive cell linemay indicate a specific site of action for selenium in tumor cellmetabolism. Abdullaev et al. (30) reported that selenite significantly inhibited the incorporation of [3H]uridine into RNA of

loach (Misgurnus fossilis) embryos at the gastrula stage. Theeffect was first detected with incubation medium supplementedwith 10 HIMsodium selenite after 60 min of incubation. Suchinformation suggests that selenium, in addition to inhibitingprotein biosynthesis, may alter RNA transcription leading tospecific alterations in RNA translation and ultimately depressed protein biosynthesis. Increased time of exposure toselenium would likely have modified the incorporation of precursors into DNA and protein in the neoplastic cells examinedin the present studies. Our growth data suggest that concentration, form, and time of exposure to selenium are factors thatdetermine when macromolecule biosynthesis will be altered.Support for this hypothesis comes from the delay in cell deathof confluent cultures of canine mammary tumors as the selenium concentration is decreased (Fig. 1) and the delay in thesensitivity of CMT-11 cells to selenodiglutathione.

In conclusion, the use of these neoplastic and nonneoplasticcells for comparative purposes may yield a better understandingof the biological effects of selenium. Such information is neededto evaluate the true efficacy of selenium as a potential antitu-

morigenic agent.

REFERENCES

1. Schnauzer, G. N., White, D. A., and Schneider, C. J. Cancer mortalitycorrelation studies. III. Statistical association with dietary selenium intakes.Bioinorg. Chem., 7: 23-34, 1977.

2. Schnauzer, G. N., White, D. A., and Schneider, C. J. Cancer mortalitycorrelation studies. IV. Associations with dietary intakes and blood levels ofcertain trace elements, notably selenium-antagonists. Bioinorg. Chem., 7:35-56, 1977.

3. Schnauzer, G. N., and Ishmael, D. Effects of selenium and of arsenic on thegenesis of spontaneous mammary tumors in inbred (Ml mice. Ann. Clin.Lab. Sci., 4: 441-447, 1974.

4. Witting, C., Witting, U., and Krieg, V. The tumor-protective effect ofselenium on an experimental model. J. Cancer Res. Clin. Oncol., 104: 109-113, 1982.

5. Harr, J. R., Exon, J. H., Whanger, P. D.. and Weswig, P. H. Effect of dietaryselenium on /V-2-fluorenyl-acetamide (FFA)-induced cancer in vitamin Isupplemented, selenium depleted rat. Clin. Toxicol., 5: 187-194, 1972.

6. Ip, C., and Ip, M. M. Chemoprevention of mammary tumorigenesis by acombined regimen of selenium and vitamin A. Carcinogenesis (Lond.), 2:915-918, 1981.

7. Ip, C., and Sinha, D. Anticarcinogenic effect of selenium in rats treated withdimethylbenz(a)anthracene and fed different levels and types of fat. Carcinogenesis (Lond.), 2:435-438, 1981.

8. Jacobs. M. M., Janssen, B., and Griffin, A. C. Inhibitory effects of seleniumon 1,2-dimethylhydrazine and methylazoxymethanol acetate induction ofcolon tumors. Cancer Lett., 2: 133-138, 1977.

9. Medina, D., and Shepard, F. Selenium-mediated inhibition of mouse mammary tumorigenesis. Carcinogenesis (Lond.), 2: 451-455, 1981.

10. Clayton, C. C., and Baumann, C. A. Diet and azodye tumors: effect of dietduring a period when the dye is not fed. Cancer Res., 9: 575-582, 1949.

11. Schrauzer, G. N., White, D. A., and Schneider, C. J. Inhibition of the genesisof spontaneous mammary tumors in C3H mice: effects of selenium and ofselenium-antagonistic elements and their possible role in human breastcancer. Bioinorg. Chem., 6:265-270, 1976.

12. Shamberger, R. J. Relationship of selenium to cancer. I. Inhibitory effect ofselenium on Carcinogenesis. J. Nati. Cancer Inst., 44:931-936, 1970.

13. Soullier, B. K., Wilson, P. S., and Nigro, N. D. Effect of selenium onazoxymethane-induced intestinal cancer in rats fed high fat diet. CancerLett., 72:343-348, 1981.

14. Daoud, A. H., and GrifTm, A. C. Effects of selenium and retinoic acid on themetabolism of Ar-acetylaminofluorene and JV-hydroxyacetylaminofluorene.Cancer Lett., 5: 231-237, 1978.

15. Weisberger, A. S., and Suhrland, L. G. Studies on analogues of L-cysteineand L-cystine. II. The effect of selenium cystine on Murphy lymphosarcomatumor cells in the rat. Blood, //: 11-18, 1956.

16. Greeder, G. A., and Milner, J. A. Factors influencing the inhibitory effect ofselenium on mice inoculated with Ehrlich ascites tumor cells. Science (Wash.DC), 209:825-827, 1980.

17. Poirier, K. A., and Milner, J. A. The effect of various selenocompounds onEhrlich ascites tumor cells. Biol. Trace Elem. Res., 1:25-34, 1979.

18. Milner, J. A., Greeder, G. A., and Poirier, K. A. Selenium and transplantabletumors. In: J. E. Spallimi/, J. L. Martin, and H. E. Ganther (eds.). Seleniumin Biology and Medicine, Second International Symposium, pp. 146-159.Westport, CT: AVI Publishing Company, Inc., 1980.

19. Milner, J. A., and Hsu, C. Y. Inhibitory effects of selenium on the growth ofL1210 leukemic cells. Cancer Res., 41:1652-1656, 1981.

20. Milner, J. A. Selenium and the transplantable tumor. J. Ag. Food. Chem.,52,436-442, 1984.

21. Watrach, A. M., Milner, J. A., and Watrach, M. A. Effect of selenium ongrowth rate of canine mammary carcinoma cells in athymic nude mice.Cancer Lett., 15, 137-143, 1982.

22. Watrach, A. M., Milner, J. A., Watrach, M. A., and Poirier, K. A. Inhibitionof human breast cancer cells by selenium. Cancer Lett., 25:41-47, 1984.

23. Poirier, K. A., and Milner, J. A. Factors influencing the antitumorigenicproperties of selenium. J. Nutr., 113: 2147-2154, 1983.

24. Watrach, A. M., Hager, J. C., Wong, P. K. Y., Watrach, M. A., and MacLeod,R. Induction of oncornavirus like particles in cell line of canine mammarycarcinoma. Br. J. Cancer, 38:639-642, 1978.

25. Hayflick, L. Fetal human diploid cells. In: P. K. Kruse and J. R. Patterson(eds.). Tissue Culture, pp. 43-45. Academic Press, New York: 1973.

26. Holmberg, B. On the permeability to lissamine green and other dyes in thecourse of cell injury and cell death. Exp. Cell. Res., 22:106-111, 1960.

27. Tennant, J. A. Evaluation of the trypan blue technique for determination ofcell viability. Transplantation (Baltimore), 2: 685-693, 1964.

28. Ganther, H. E. Reduction of the selenotrisulflde derivative of glutathione toa persulfide analog by glutathione reducíase.Biochemistry, 22: 4089-4098,1971.

29. Vernie, L. N., Hamburg, C. J., and Bont, W. S. Inhibition of the growth ofmalignant mouse lymphoid cells by selenodiglutathione and selenodicystine.Cancer Lett., 14: 303-308, 1981.

30. Abdullaev, G. B., Mekjtiyev, N. K., Abdullaev, P. I., and Kafani, K. A. Effectof sodium selenite on RNA synthesis in loach embryo. Biochimie (Paris), 45:98-102, 1980.

31. Ceriotti, G. The determination of nucleic acid in animal tissue. J. Biol.Chem., 214: 59-70, 1955.

32. Taylor, I. W., and Milthorpe, B. K. An evaluation of DNA fluorochromesstaining techniques, and analysis for flow cytometry. J. Histochem. Cyto-chem., 28:1224-1232, 1980.

33. Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J. Proteinmeasurement with the Polin phenol reagent. J. Biol. Chem., 193: 265-275,1951.

34. Broghamer, W. L., Jr., McConnell, K. P., and Blotcky, A. L. Relationshipbetween serum selenium levels and patients with carcinoma. Cancer (Phila.),57: 1384-1388, 1976.

35. Cavalieri, R. R., Scott, K. C., and Sairene, E. Selenite as a tumor-localizingagent in man. J. Nucí.Med., 7:197-208, 1966.

36. Herrera, N. E., Gonzales, R., Schwartz, R. D., Diggs, A. M., and Belsky, O.Seleno-methionine as a diagnostic agent in malignant lynphoma. J. Nucí.Med., 6: 792-804, 1965.

37. McConnell, K. P., Broghamer, W. L., Jr., Blotcky, A. J., and Hurt, O. J.Selenium levels in human blood and tissues in health and disease. J. Nutr.,IOS: 1026-1031,1975.

38. Spencer, R. P., Montana, G., Scanion, G. F., and Evans, O. R. Uptake ofselenomethionine by mouse and in human lymphomas, with observations onselenite and selenate. J. Nucí.Med., 8: 197-208, 1967.

39. Medina, D., and Oborn, C. J. Differential effects of selenium on the growthof mouse mammary cells in vitro. Cancer Lett., 13: 333-344, 1981.

40. Medina, D., Morrison, D. G., and Oborn, C. J. Selenium retention andinhibition of cell growth in mouse mammary epithelial cell lines in vitro.Biol. Trace Element Res., in press, 1986.

41. Gruenwedel, D. W., and Cruikshank, M. K. The influence of sodium seleniteon the viability and intracellular synthetic activity (DNA, RNA, and proteinsynthesis) of HeLaS3 cells. Toxicol. Appi. Pharmacol., 50:1-7, 1979.

42. Lewko, W. M., Winn, D., and McConnell, K. A. Effect of sodium seleniteon growth and protein synthesis in cultured human breast cancer cells. Fed.Proc., 42:669, 1983.

3388

on June 26, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1986;46:3384-3388. Cancer Res   M. E. Fico, K. A. Poirier, A. M. Watrach, et al.   Canine Mammary CellsDifferential Effects of Selenium on Normal and Neoplastic

  Updated version

  http://cancerres.aacrjournals.org/content/46/7/3384

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/46/7/3384To request permission to re-use all or part of this article, use this link

on June 26, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from