A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity

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Experimental

Cell Research 124 (1979) 237-246

A CADMIUM-RESISTANT VARIANT OF THE CHINESE HAMSTER (CHO) CELL WITH INCREASED METALLOTHIONEIN INDUCTION CAPACITY C. E. HILDEBRAND,

R. A. TOBEY, E. W. CAMPBELL

and M. D. ENGER

Cellular and Molecular Biology Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, NM 87545, USA

SUMMARY The toxic trace metal Cd2+ has been used to select a variant (designated Cd3 of the Chinese hamster cell (line CHO) resistant to the growth-inhibitory and cytotoxic effects of Cd*+. Resistance of the Cd’ cell to Cd*+-mediated cytotoxicity is not due to a decreased capability of the Cd’ cell to accumulate Cd2+since CdZ+uptake in the Cd’ cell is indistinguishable from that in the CHO cell at both toxic and subtoxic CdZ+ exposures. Comparison of the relative capacities of these two cell types to induce specific low molecular weight Cd2+-binding proteins (metallothioneins) reveals that the Cd’ cell has an increased capacity to induce metallothionein and to sequester intracellular Cd’+ in metallothioneins. These results suggest that the greater competence of the Cd’ cell to induce metallothionein is a major factor in the Cd*+-resistant phenotype of the variant.

Studies on the metabolism of cadmium (Cd) are important in areas of trace metal toxicology and trace metal nutrition [l-4]. Although Cd is recognized as a highly toxic environmental contaminant which accumulates in animals, little is known about the cellular mechanism(s) which underlie its toxicologic actions [l-4]. In animals, Cd2+ is concentrated primarily in the liver and kidneys where it is stably bound to low molecular weight cysteine-rich, metal-binding protein(s), called metallothionein(s) [ 1, 2, 5-71. Metallothionein synthesis is induced both in animals [8-121 and in cultured cells [13-191 in response to Cd2+ or Zn2+ exposure [8-211 and is believed to provide a protective function against the toxic actions of intracellular Cd2+ [16, 22-281. The fact that metallothioneins isolated from animal tissues contain considerable amounts of ZrP, in addition to Cd2+ [29, 301, to16-791815

gether with the observation that metallothionein synthesis also is induced by Znz+ has led to the hypothesis that metallothioneins are an important component in the regulation of cellular Zn2+ levels. Since the induction of metallothionein synthesis is specific [31] and appears to be regulated at the level of transcription [ll, 17, 21, 311, this system has great potential for studies on the regulation of induced gene expression. The problem of cellular metabolism of Cd2+ including (a) mechanism of transport and accumulation; (b) regulation of metallothionein induction; and (c) definition of targets for the primary cytotoxic actions of Cd2+ can be approached by applying the techniques of somatic cell genetics for isolating stable variants which display altered Cd2+ metabolism. Further, since Cd2+ behaves in several respects as a toxic anaExp Cd/ Res 124 (1979)

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100 \. I \

logue of the essential trace metal, ZP, . l \* especially as an inducer of metallothionein synthesis, biochemical characterization of 80 these variants will provide new clues to essential trace metal homeostasis. Finally, the 60 . . derivation of variants with different capac\ ities to induce metallothionein synthesis offers new tools for delineating the steps involved in regulation of gene expression and for probing the organization of the thio0 20 40 60 80 100 nein gene within the cellular genome. The present study demonstrates the use- Fig. 1. Abscissa: time (hours); ordinate: % survival. Effects of 0.2 uM Cd*+ on survival of CHO cells in fulness of Cd2+ as a selective agent for iso- monolayer culture. In this experiment cell survival was lating a variant mammalian cell line with in- determined as follows: CHO cells from a suspension were counted and plated into 60 mm tissue culcreased resistance to the cytotoxic effects culture ture dishes. After incubation at 37°C for 1.5 h, during of Cdz+. The Chinese hamster cell (line which time the cells attached and began to spread out on the plastic matrix, CdCl, solution was added (0.2 CHO) is particularly suited to this study be- PM, final cont.). The plates were incubated for difcause it is a well-characterized mammalian ferent oeriods of time. after which the cadmium-conwas removed and replaced with fresh, cell line and has been of great value in the taining-medium Cd-free medium. Following incubation for 7 days, the isolation of numerous nutritional (condi- plates were washed, fixedystained and the number of (250 cells) was determined and expressed as tional-lethal) variants ([32, 33, 341 and ref- colonies the percent of colonies formed relative to a Cd-free erences therein). A Cd2+-resistant popula- control culture. Each data point represents the results tion was obtained by long-term culturing of obtained from six replicate plates. Chinese hamster cells (line CHO) in low, marginally toxic, levels of Cd2+. A stable, Cd2+-resistant variant, designated Cd’, was MATERIALS AND METHODS obtained by cloning a single cell isolate from the Cd2+-resistant population. The Cell culture and selection and isolation Cd2+-sensitive parent CHO cell and the Cd’ of the Cd2+-resistant variant cell were compared during exposure to both Chinese hamster (line CHO) cells were maintained in or suspension culture in calcium-deficient high and low levels of Cd2+ for (a) growth monolayer Ham’s F-10 medium supplemented with 15% newborn rate effects; (6) cytotoxic effects as meas- calf serum (Biocell) plus penicillin and streptomycin. Stock solutions of CdCl, (Mallinkrodt, analytical ured by cell survival; (c) cellular Cd2+ up- grade; or Apache, 99.999%) were prepared in 0.1 M HCl, and dilutions of the stock solutions were made take; and (d) induction of metallothionein into sterile, deionized distilled water. The diluted and extent of Cd2+-binding to metallothiostock CdCl, solutions were stored frozen at -20°C in nein. These studies show that the Cd’ vari- polyethylene tubes to minimize adsorptive loss. Cadconcentrations in stock solutions were verified ant has a markedly increased capacity, rela- mium by atomic absorption analysis (Perkin Elmer Atomic tive to the Cd2+-sensitive parental cell, for Absorption Spectrophotometer, Model 303). The Cd*+ in medium without added Cd*+ deterinducing synthesis of metallothionein which concentration mined by atomic absorption analysis was below limits strongly suggests that metallothionein syn- of detection (<0.09 L&Q. Further, rosCd2+isotope dilution experiments indicated that the endogenous Cd” thesis is a major factor in the cellular de- concentration in the growth medium was CO.01 PM. toxification of Cd2+. Preliminary results of The zinc concentration in Ham’s F-10 medium is 0.1 However, this does not account for the zinc conthis work have been presented elsewhere PM. tributed bv the inclusion of 15% serum. CuSO, was I

[311.

I

I

I

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present in-F-10 medium at 0.01 PM, but as with’zinc,

Metallothionein

induction

the final concentration of copper in the growth medium depends upon the level in the added serum. The concentrations of zinc and copper, as well as other trace metals, in complete growth medium are currently being measured and will be reported elsewhere. To obtain a Cd*+-resistant population, mass selection was initiated by adding Cd2+ to a monolayer culture in a T-75 flask to a-final concentration of 0.2 WM. This level of Cd*+ is marginally toxic to the parent CHO cell as shown in fig. 1.-The Cd*+-treated monolayer was confluent within 3 days, at which time it was subcultured (1 : 6) with fresh medium containing 0.2 WM Cd*+. Thereafter, subculturing was performed at weekly intervals with dilutions of-1 : 50 to 1 : 60. The growth medium was changed every 2 days to maintain a steady state intracellular level of Cd2+ [ 191.After 11 weeks of selective pressure by exposure to 0.2 PM CdCl*, the monolayer was harvested and stored frozen at -70°C in 10% glycerol-saline in F-10 medium containing 0.2 PM CdCl,. Simultaneously, an aliquot of the Cd*+-treated monolayer cells was transferred to suspension culture and tested for resistance by exposure to 2 WM CdCl,, a concentration which stops CHO cell division within 24 h. The 11 week Cd2+-treated population grew in 2 PM Cd2+-containing medium with a doubling time indistinguishable from the 16-17 h doubling time characteristic of both an unexposed CHO cell and the 11 week Cd*+-treated cell in the absence of Cd*+. Upon establishing the Cd*+-resistant character of the population of cells obtained by the 11-week mass selection, a monolayer of the resistant population was washed with Cd2+-free F-10 medium. Following trypsinization, a 0.2 ml aliquot of an appropriate dilution was placed into each well of a 96well Micro Test II tissue culture plate (Falcon) under conditions in which aonrox. 30 of the 96 wells contained a single cell. Afier allowing sufficient time in a 5 % CO, incubator for cells to attach. wells containing single cells were identified. Each of the single cell clones was allowed to become confluent, whereupon each clone was subcultured into a 24-well dish, again in Cd2+-free medium. Upon reaching confluency these clones were subcultured into T-25 flasks also in Cdl+-free medium. Twenty-one clones were selected and stored frozen as described above. The fastest growing clone, designated Cd’, was subcultured both in monolayer and in suspension culture. All subsequent comparative studies of the CHO and the Cd’ cells described herein were performed with cells in suspension culture. The doubling times for cultures of CHO and Cd’ cells maintained in cadmiumfree medium were the same (16-17 h) whether grown in suspension or in monolayer culture.

Determination

of cell survival

Cells exposed to CdCl, in suspension culture for indicated intervals were pelleted by centrifugation, resuspended in Cdz+-free medium, counted, diluted and plated into 60x 15-mm tissue culture dishes. The plates (6 replicates/data point) were incubated for 7 days at 3PC in a CO* incubator, then washed, fixed, and stained with 1% crystal violet prior to determination of the number of colonies with ~50 cells.

in a Cd-resistant

CHO variant

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Measurement of Cd2+ uptake and subcellular distribution Tracer amounts of carrier-free “YdCl, (obtained from either New England Nuclear Corporation or Amersham) were added to calibrated stock solutions of CdCl,. losCd activity was measured by liquid scintillation counting, utilizing either PCS (Nuclear-Chicago Corporation) or Aquasol (New England Nuclear Corporation) scintillation solvent. Cd*+ incorporation into cells was determined in samples of cells harvested from culture by centrifugation and washed in 0.25 M sucrose by resuspension and centrifugation. The cell pellet was resuspended in buffer A (0.01 M Tris-Cl (oH 7.4). 0.01 M KCl, 0.0015 M MgCl,, 0.1 mM dithioi’hreitol): Cells were lysed by adding one-tenth volume of a 10% solution of the non-ionic detergent NP-40 (Shell Oil Co.) at W’C. The suspension was agitated by vortex mixing, allowed to stand at 4°C for 15 min, and then agitated again by vortex mixing for 30 sec. After centrifugation of the cell lysate at 500 g for 5 min, the supernat& cytoplasm was transferred to polystyrene tubes, frozen in dry ice and ethanol, and stored at -20°C. The nuclear pellet was resuspended in 1.0 ml of buffer A. ‘Aliquots of total cell suspension, cytoplasm, and nuclear suspension, were utilized for determination of losCd activities. ‘OgCdactivities were converted to pg Cd2+/10gcell equivalents. This number (pg CdZ+/109cells) is nearly equivalent to ppm CdZ+ since lo9 cells weigh - 1 g 1191.

Measurement of metallothionein synthesis rates and levels of metallothionein-bound Cd2+ in cells exposed to CdC12 The relative rate of thionein synthesis was determined by measuring the kinetics of incorporation of [3sS]cysteine (New England Nuclear Corp. >260 Cilmmol) prior to and at various times during CdZ+ exposure. Cultures exposed to CdCll (no ‘Wd) were ‘pulselabeled’ with 0.350 pCi/ml [35S]cysteine for 30 min prior to the times indicated in the figures. The ‘pulselabeling’ was terminated by pouring the cultures (100 ml) over frozen 0.25 M sucrose cubes and stirring the suspension for 30 sec. Cell harvesting and fractionation were performed as described above. The amount of [Wlcysteine incorporated into nascent metallothionein was determined by separating the low molecular weight metallothioneins from the other, much larger cytoplasmic components using molecular sieve chromatoeraohv. The use of Sephadex G-75 or G-50 to accom&h this separation represents a standard procedure [9-21,24-291. Sephadex G-75 columns (1.2X85 cm) were equilibrated with 0.05 M T&Cl (pH 8.4) at 20°C and eluted with the same buffer at 20°C. The elution profile of Y3 activity from these columns showed three distinct peaks: the first peak contained material excluded from the column (70000 D), the second peak eluting was identified to be metallothionein and the peak eluting last contained unincorporated [Yjlcysteine. The Em Cell RPS 124 (1979)

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identity of the metallothionein neak was confirmed in separate experiments showing-that (a) its chromatographic behavior on Sephadex G-75 is indistinguishable from that reported for well characterized metallothioneins from other sources [18, 25, 27, 281; (b) it preferentially binds cytoplasmic Cd; (c) it is induced in response to Cd2+ or Zn*+ exposure: and (d) it disnlavs relative cysteine-richness and leucme deficiencyA([iS] and unpublished data). The relative rate of metallothionein synthesis was determined by comparing the amount of [YS]cysteine incorporated into the metallothionein peak with the amount of incorporation into proteins eluting with the excluded peak. The practice of utilizing the ratio of [?S]metallothionein to %-labeled non-metallothionein proteins as a relative measure of thionein synthesis rate depends upon the constancy of the non-metallothionein protein synthesis rate. Measurements of the specific activity of incorporation into non-metallothionein proteins (those eluting in the excluded peak on Sephadex G-75 columns) were found to be approximately constant during the Cd2+exposure intervals described in this report. This procedure for calculating the relative thionein (the apoprotein of metallothionein) synthesis rate is therefore valid and, also, avoids requirements for maintaining constant cell concentrations from one pulse labeling period to the next. The amount of intracellular Cd2+ bound to metallothionein was determined by column chromatography as described above, using the cytoplasm from cells exposed to ‘09CdCl,.

growth rate characteristic of the parental cell. The uncloned subpopulation gradually exhibited a loss of resistance to Cd2+ toxicity when cultivated for prolonged periods in Cd2+-free medium [36]; however, a Cd2+resistant clone derived from this subpopulation was found to be extremely stable during continued growth in Cd2+-free medium. This Cd’ variant was utilized in comparative studies with the sensitive CHO parental cell. Comparison of growth, survival, and Cd2f uptake characteristics in parental and Cdzt-resistant cell types

Initial studies of the growth of CHO cells in suspension culture in medium containing 0.2 PM CdCl, revealed a growth rate comparable to that obtained in cadmium-free medium (data not shown); however, in medium containing 1 PM CdCl, the cells stopped dividing within 48 h following addition of Cd2+ (fig. 2A) [19]. At 1 PM Cd2+ RESULTS there was no reduction in the growth rate Selection and isolation of Cd2+of Cd’ cells relative to that of the unexresistant variants posed Cd’ or CHO cell. Prolonged cultivaInitial attempts to obtain Cd2+-resistant tion of the Cd’ cell for up to 10 days in 1 variants directly by mass selection of sur- PM CdCl, had no effect on growth rate. It viving colonies from cultures of CHO cells should be noted that the growth kinetics of exposed continuously to high levels of Cd2+ CHO cells in 1 PM CdCl, were dependent (> 1 PM) were unsuccessful due to the ex- upon cell concentration at the time of additremely slow growth rates obtained for cells tion of CdCl, to the growth medium. Speisolated in this fashion. However, it was cifically, when Cd2+ is added to a culture at possible to obtain a resistant subpopulation low cell concentration (e.g. 80000 cells/ml), of CHO cells by maintaining monolayer cul- the decrease in growth rate was more rapid tures continuously for prolonged periods than that observed at a higher cell concen(11 weeks in this case) in medium contain- tration (e.g. 200000 cells/ml). This differing 0.2 PM CdC12.Monolayer cultures were ence in growth rate most likely arises employed during the selection process pri- through differences in the rate of uptake of marily because it is not experimentally Cd2+ by the high and low density cultures; feasible to maintain CHO cells in suspen- further studies will be required to test this sion culture for such extended periods. possibility. In any event, to ensure experiThis selection procedure produced a mental reproducibility, exposure conditions population of Cd2+-resistant cells with a were standardized by utilization of cultures E.rp Cd Res 124 (IY7Y)

Metallothionein induction in a Cd-resistant CHO variant

b” ‘0

, 12

I 24

I 36

I 48

1 6C

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Fig. 2. Abscissa: time (hours); ordinate: (A) cells/ml; (B) % survival, (C) fig Cd2+/10gcells. Growth (A), survival (B), and Cd2+ uptake (C) characteristics of CHO (solid symbols) and Cd’ (open symbols) cells during continuous exposure to 1.O PM Cd*+. (A) Growth kinetics of CHO cells plus 1 PM CdZ+ (O-O); CHO cells, no Cd*+ (Cm); Cd’ cells plus 1 PM Cd*+ (A-A); Cd’ cells, no Cd*+ (O-O). (B) Cell survival measurements: CHO cells (O-O); Cd’ cells cultured in Cdz+-free medium for 1 week (O-O); 4 weeks (0-O); or 8 weeks (0-O) prior to challenge with 1 /IM Cd*+. (C) Cellular Cd*+ accumulation in CHO cells (CO) or Cd’ cells (O-O, A-A). In all cases Cd2+ exposure was continuous beginning at 0 h.

of uniform cell concentration (approx. lo5 cells/ml in the experiments described in this report) at the time of addition of cadmium. Continuous exposure of parental CHO cells to 1 PM CdCl, resulted in an exponential decrease in viable cells after a 4to 6-h lag (fig. 2B). By 60 h, less than 1%

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of the cells were capable of giving rise to colony formation. Under the same conditions of challenge, Cd’ cells retained better than 90% viability for 72 h of continuous exposure, and this property of the Cd’ cells was stable through 2 months of continuous growth in the absence of Cd*+ (fig. 2B). Recent experiments have extended the stability period of the Cd’ variant subline to at least 6 months. The kinetics of incorporation of Cd*+ into both CHO and Cd’ cells are compared in fig. 2 C. In this experiment, cells were exposed continuously to 1 PM CdCl, and at various times aliquots of the culture were removed for the determination of Cd*” levels. These data indicate that, during the first 36 h of exposure, when the effects of Cd*+ on CHO cell growth and viability are maximal (fig. 2A, B), the accumulation of Cd*+ into CHO cells was indistinguishable from that of Cd’ cells. Therefore, a difference in rate of accumulation of Cd*+ in Cd’ and CHO cells cannot account for their differential sensitivities to Cd*+ exposure. An alternative explanation for the differential Cd*+ sensitivities of the CHO and Cd’ cells could reside in the manner in which these two cell types accommodate intracellular Cd*+. Since metallothioneins sequester intracellular Cd*+ and are believed to provide protection against the cytotoxic actions of Cd*+ ions, measurements were made to compare the CHO and Cd’ cell types for (a) the efficiency of induction of metallothionein synthesis; and (b) the extent to which Cd2+ was bound by metallothioneins during exposure to both toxic (1 .OPM) and subtoxic (0.2 PM) levels of Cd2+. The relative rates of metallothionein synthesis in both sensitive and resistant cell types prior to Cd2+ exposure are low and are only marginally detectable when assayed by pulse-labeling with [35S]Exp Cell Res 124 ( 1979)

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IO 6 6 4 2 0 LJLJ’L

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14 12 IO * 6 4 2 0 LLL 14 12 IO 8 6 4 2 0 LL 0

6

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A

time (hours): ordinate: rel. rate of metallothionein synthesis. Kinetics of metallothionein induction in Cd’ (0) and CHO (0) cells exposed to (A) 1.0 PM Cd*+ or (B) 0.2 UM Cd*+. The relative rates of metallothionein svnihesis are determined as described in Materials &d Methods. The incorporation of [V]cysteine into metallothionein at 8 h in the Cd* cell in frame (A) was 8715 cpm/lO’ cells. [35S]Cysteine incorporated into non-metallothionein proteins was 50 321 cpm/lO’ cells.

Fia. 4. Abscissa: C

20

40

60

60

Fig. 3. Abscissa: fraction no.; ordinate:

35Scpm X 10m3. Sephadex G-75 column chromatographs of cytoplasm from cells pulse-labeled with [%]cysteine. (A) Cd’ cells cultured in Cd*+-free medium; (B) Cd’ cells exposed continuously for 7 h to 1.0 PM Cd*+ and pulse-labeled from 6.5 to 7.0 h; (C) CHO cells exposed continuously for 7 h to 1.OPM Cd*+ and pulse-labeled from 6.5 to 7.0 h. Large cellular components labeled with [%]cysteine during the 30 min pulse elute from the column in fractions E-20. Metallothionein elutes in fractions 36-44, and it is seen clearly in the cytoplasm from the Cd”+-induced Cd’ cell in (B). The large peak of % radioactivity eluting last (fractions N-65) contains unincorporated [Vlcysteine.

cysteine followed by separation of metallothionein from other cytoplasmic proteins by Sephadex G-75 column chromatography (fig. 3A). After 7 h of continous exposure to 1 PM Cd2+ the rate of metallothionein synthesis in the Cd’ cell increases at least 30-fold (fig. 3B) over the low basal level (fig. 3A). In contrast, over the same time period, metallothionein synthesis in CHO cells increased only slightly (fig. 3C). Detailed induction kinetics at 1.0 (fig. 4A) and 0.2 PM Cd2+ (fig. 4B) show that in Cd’ cells, metallothionein synthesis begins rapidly and reaches a maximal rate by 8 h. In contrast, CHO cells respond less draExp Cell Res 124(1979)

matically, reaching a maximal synthesis rate later (11 h) than Cd’ cells which is approximately one-fifth as great as that found in Cd’ cells. Since metallothionein induction is low in CHO cells grown in the presence of both toxic (1.0 PM) and subtoxic (0.2 PM) levels of Cd, toxicity per se does not interfere with the induction process. The increased induction of metallothionein synthesis observed in the Cd’ cell compared with the CHO cell suggests that the mass of Cd-thionein should also increase faster and to a higher level in the Cd’ cell than in the CHO cell. To ascertain that this is the case, determinations of Cdthionein levels were made coincident with measurements of Cd2+ accumulation by the two cell types (fig. 2C, table 1). Cells were fractionated and the amount of Cd2+ bound to Cd-thionein in the cytoplasmic fraction was determined. Elution profiles of the cytoplasm from CHO and Cd’ cell types exposed to 1.0 ,uM Cd2+ for 24 h are shown in fig. 5A andB, respectively. In these studies

Metallothionein

induction

A

in a Cd-resistant

Table 1. Accumulation

and intracellular distribution of Cd2+ in CHO and Cd’ cells exposed to 0.2 or I .O PM CdZ+ for 24 h FFCX;] 1.0 0.2

Cell type

pg Cd’+1 lo9 cells

CHO Cd’ CHO Cd’

45.9 42.6 15.5 14.3

pg Cd2+ in metallothionein/ lo9 cells

IO

metallothionein is found only in the cytoplasm in agreement with reports by others ([ 121, unpublished results). The accumulation of Cd2+ in both CHO and Cd’ cell types and the amount of Cd2+ bound to Cd-thionein are summarized in table 1 for exposure to toxic and subtoxic Cd2+ levels. Although the two cell types incorporate approximately the same total amounts of Cd2+ at a given exposure level, the amount of Cd2f in Cdthionein in the Cd’ cell is significantly greater than that in the CHO cell. This result is expected on the basis of the greatly increased metallothionein synthesis rates in the Cd’ cell compared with the CHO cell.

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6 6 4 2

15.9 37.6 3.3 11.7

CHO variant

0

L T

00

20

40

60

fraction no.; ordinate: ‘Td cpm x10-3. Sephadex G-75 chromatographs of cytoplasm from (A) CHO cells; or (B) Cd’ cells exposed to 1 /AM CdCl, (plus tracer levels of 10gCdC1z)for 24 h. Large cytoplasmic Cdl+-binding components elute in the excluded peak (fractions 15-20). Metallothionein (Cdthionein) elutes in fractions 32-42. No free Cd*+ is found in cytoplasm from either CHO or Cd’ cells.

Fig. 5. Abscissa:

attributable to increased metallothionein synthesis [27, 281. However, their studies did not provide data to establish the stability of the Cd2+ resistance of adapted cells to long-term culture in Cd2f-free medium nor DISCUSSION did they distinguish between adaptation to The isolation of a stable Cd2+-resistant var- Cd2+ due to reduced Cd2+ uptake or to the iant of the CHO cell reported here is, to our constitutive or induced synthesis of metalknowledge, the first account of the isolation lothionein [27, 281. In the present study the experimental of a Cd2+-resistant cell line having welldefined alterations in the regulation of me- evidence shows that the resistance of the tallothionein induction. In previous reports Cd’ cell to Cd2+-mediated growth inhibition [27, 281 Rugstad & Norseth showed that and cytotoxicity is stable for up to 6 months cultured human skin epithelial cells and of continuous culture in Cd2+-free medium mouse L cell tibroblasts could be adapted and that Cd2+ resistance cannot be attrito grow in high levels (100 PM) of Cd2+. buted to decreased Cd2+ uptake by the Cd’ Further, they found that the adapted cells cell compared with the CHO cell under had increased levels of Cd2+ in the cyto- identical Cd2+ exposure conditions. Furplasmic metallothionein and concluded that ther, the measurements of metallothionein the Cd2f resistance to growth inhibition was induction and accumulation of thioneinExp Cell Res 124 (1979)

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bound Cd*+ in the two cell types demonstrate that the Cd* cell possesses the ability to induce metallothionein synthesis more rapidly and to synthesize a greater level of metallothionein than the CHO cell. The latter findings imply that the increased capacity to induce metallothionein in the Cd’ cell is a major factor in its resistance to Cd2+-mediated cytotoxicity by virtue of the capacity of metallothionein to sequester the toxic Cd2+ ion, thus rendering it inert with respect to its ability to interfere with normal biological function (e.g. activities of a number of enzymes, especially Zn2+-requiring enzymes, are altered by Cd2+ [l]). This observation supports and strengthens the hypothesis that intracellular metallothionein participates in protection against the toxic actions of Cd2+[8]. Although the Cd’ variant displays an increased capacity to synthesize metallothionein compared with the CHO cell, the level of metallothionein synthesized in the Cd’ cell (-200 ,ug Cd2+ in metallothioneinlg of cells, wet weight, following exposure for 24 h to 2 ,uM Cd2+) is comparable (within a factor of 2) to levels of metallothioneinbound Cd2+ obtained in cell lines derived from pig kidney [ 141,rat liver [ 181or in liver tissue from animals exposed chronically to Cd2+ [2, 141. This indicates that the Cd’ cell behaves like. liver cells in vivo or cell lines isolated from liver, suggesting that the parent CHO cell may have attained a Cd2+sensitive state (i.e. a decreased metallothionein induction capability) due to maintenance of a constant, acceptable level of Zn2+ in the culture medium, thus reducing requirements for the cell to regulate intracellular Zn2+. Hence, it is necessary to qualify the nomenclature of the Cd’ cell, recognizing that it is resistant to Cd2+ only with respect to the parent CHO cell. These considerations are not intended to imply Exl, Cell RPS 124 (1979)

that a differentiated function (i.e., inducibility of metallothionein synthesis) has been ‘repressed in the CHO cell. However, this possibility deserves consideration in future studies. While the present study demonstrates that the Cd’ cell has a heritable, Cd2+-resistant phenotype, the genetic nature of this stable change to Cd2+ resistance remains to be elucidated. Is this phenotypic variation a result of a stable genetic change, or is the increased Cd2+ resistance a consequence of an epigenetic change, e.g. a post-transcription alteration which restores the metallothionein-synthesizing capacity of the CHO cell to a higher, more ‘normal’ (i.e. with respect to the in vivo situation) rate? Both somatic cell genetics and biochemical approaches are currently being employed to answer these questions. One further question which arises is whether mutagenesis would increase the frequency of appearance of Cd2+-resistant cells. Although this experiment has not been done using established mutagens, it should be mentioned that a preliminary report has indicated that Cd2+ is mutagenic in the CHO cell system [37]. While these unanswered questions provide avenues for further investigation, they do not detract from the utility of the CHO and Cd’ cell types (a) to define mechanisms involved in the regulation of cellular metabolism of not only the toxic trace metal Cd2+but also the essential trace metal Zn2+; (b) to study the interplay between toxic and essential trace metals in cellular metabolism; (c) to identify cellular targets involved in the Cd2+-mediated perturbation of normal cellular function; and (d) to elucidate mechanisms in the regulation of transcription, processing and translation of thionein mRNA. Following the notion that variants selected using Cd2+ as a toxic analogue of

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Zn2+ may have altered cellular mechanisms The authors wish to thank J. L. Hanners, M. Jones, R. J. Kissane for their capable assistance in these for regulating Zn2+ levels [point (a) above], and studies and P. Stein for performing the atomic absorpongoing experiments include studies on the tion analyses. This work was performed under the of the US Department of Energy and the comparative metabolism of Zn2+ in Cd’ and auspices Environmental Protection Agency under interagency CHO cells. Further, with regard to point agreement EPA-IAG-DS-E6. (d), studies of metabolism of thionein REFERENCES mRNA both in animals [38-40] and in cul1. Vallee, B L & Ulmer, D D, Ann rev biochem 41 tured cells [41, 421 have appeared. The (1972) 91. techniques described in those investigations 2. &berg, L, Piscator, M, Nordberg, G F & Kjellstrom, T, Cadmium in the environment. CRC [41, 421 are being used to define the level at Press, Cleveland, Ohio (1974). which regulation (e.g. transcription vs 3. Fassett. D W. Ann rev nharmacol 15 (1975) 425. 4. Prasad,‘A S & Oberleas, 0 (ed), Trace elements in translation) of induction of metallothionein human health and disease vol. 2. DD. 401-444. synthesis is controlled in the Cd’ and CHO Academic Press, New York 1976. - cell types. 5. K&i, J H R & Vallee. B L. J biol them 235 (1960) Finally, since the Cd’ cell has the capa- 6. -346%): Ibid 236 (1961) 2435. city to induce synthesis of an easily assayed I. Biihler, R H 0, Leuthardt, L & Kagi, J H R, Fed oroc 37 (1978) 1814. specific gene product rapidly and in the 8. Piscator; M, Nord hyg tidskr 45 (1964) 76. 9. Webb, M, Biochem pharmacol21 (1972) 2751. absence of any comparable increase in gen10. Winge, D R, Premakumar, R & Ragogopalan, K V, eral RNA or protein synthesis [3 11,this cell Arch biochem bionhvs 170(1975) 242. will be valuable in studies of regulation of 11. Shaikh, Z A & &pin-Smith, ‘J, Chem-biol interactions 15 (1976) 327. inducible gene expression. Continuing ef- 12. Bryan, S E & Hidalgo, H A, Biochem biophys res commun 68 (1976) 858. forts to isolate more variants with a broader 13. Lucis, 0 J, Shaikh, Z A & Embil, Jr, J A, Exrange of sensitivities to Cd2+ resistance will perientia 26 (1970) 1109. make it possible to determine, e.g., whether 14. Webb, M & Daniel, M, Chem-biol interactions 10 (1975) 269. increased Cd2+ resistance can be achieved 15. Daniel, M R, Webb, M & Cempel, M, Chem-biol interactions 16 (1977) 101. by alterations in the pathways regulating M D, Hildebrand, C E, Tobey, R A, thionein mRNA transcription and transla- 16. Enger, Camnbell. E W. Jones, M & Hanners, J L, Fed proc37 (1978) 1349. tion or by amplification of the gene(s) codH A, Koppa, V &Bryan, S E, Biochem j ing for the metallothioneins since gene am- 17. Hidalgo, 170 (1978) 219. plification has been demonstrated to confer 18. Rudd, C J & Herschman, H R, Toxic01 appl pharmacol27 (1978) 647. drug resistance in a number of cell lines 19. Enger, M D, Hildebrand, C E, Jones, M & Barrington, H L, Proc 17th Hanford biology sym[431. oosium. Battelle Northwest Laboratories. RichWith regard to the latter possibility, gene iand, Wash. (Oct. 17-19,1977). US Department of Energy Symposium Series, US Technical inforamplification appears to be an event which mation service (1978) 37. occurs with low frequency and which deWebb, M, Biochem sot trans 3 (1975) 632. pends upon variant (mutant) selection pro- E: Richards, M P & Cousins, R J, Biochem biophys res commun 64 (1975) 1215. cedures using highly specific substrate anaYoshikawa, H, Ind health 8 (1970) 184. biochem 2 logues. Since Cd2+ behaves as an analogue ;:: Nordbera. G F. Environ nhvsiol _ _ (1972) 7.’ of Zn2+ both in a variety of Zn-metallopro24. Webb, M & Magos, L, Chem-biol interactions 14 (1976) 357. teins [l], as well as in the induction of me25. Webb, M & Verschoyle, R D, Biochem pharrnacol tallothionein, the DNA sequences involved 25 (1976) 673. in thionein synthesis may be good candi- 26. Squibb, K S, Cousins, R J, Silbon, B L & Levitt, S, Exp mol path01 25 (1976) 163. dates for amplification under appropriate 27. Rugstad, H E & Norseth, T, Nature 257 (1975) 136. selective conditions. Exp Cell Res 124 (1979)

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37. Hsie, A W, O’Neill, J P, San Sebastian, J R, Couch, D B, Fuscoe, J C, Sun, W N C, Brimer, P A, Machanoff, R, Riddle, J C, Forbes, N L & Hsie, M H, Fed proc 37 (1978) 1384. 38. Squibb, K S & Cousins, R J, Biochem biophys res commun 75 (1977) 806. 39. Shapiro, S G, Squibb, K S, Markowitz, L A & Cousins, R J, Biochem j 175(1978) 833. 40. Anderson, R D & Weser, U, Biochem j 175 (1978) 841. 41. Rail, L B, Enger, M D & Hildebrand, C E, Fed proc 38 (1979) 399. 42. Hildebrand, C E, Rail, L B & Enger, M D, Fed proc 38 (1979) 398. 43. Ah, F W, Kellems, R E, Bertino, J R & Schimke, R T, J biol them 253 (1978) 1357. Received February 6, 1979 Revised version received June 15, 1979 Accepted June 18, 1979