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Induction of heavy metal binding groups by dexamethasone and Zn in cultured Chinese hamster ovary cells
Camp. Biochem. Physiol. Vol. 84C, No. 2, pp. 345-348, Printed
in Great
0306~4492/86 $3.00 + 0.00 Pergamon Journals Ltd
1986
Britain
INDUCTION OF HEAVY METAL BINDING GROUPS BY DEXAMETHASONE AND Zn IN CULTURED CHINESE HAMSTER OVARY CELLS ROLF
A.
and
ANDER&N*
XUE-LAN
WENT
*Department of Toxicology, National Institute of Public Health, Geitmyrsveien 750462 Oslo 4, Norway. Telephone: (02) 35-6020 and tPeking Childrens Hospital, Peking, Peoples Republic of China (Received 4 November
1985)
Abstract-l. Cd binding capacity and pulse polarography were used to study the inducibility of sulfhydryl groups in cultured Chinese hamster ovary cells (wild type and a Cd-resistant mutant) in response to dexamethasone (dex) and Zn. 2. Evidence is presented that both the wild type and the mutant responded to dex and Zn treatment by induction of sulfhydryl groups. 3. In wild type for Zn and dex as well as in the mutant for dex, this induction seems to be in the form of sulfhydryls attached to particulate or membrane fractions in the cells. For Zn in the Cd-resistant mutant the induction was in the form of metallothionein.
INTRODUCTION
Metallothioneins (Mts) are heat stable, low molecular weight (M, = 6000-12,000) proteins that bind heavy metals, belonging to the periodic system transition groups Ia and Ha (Cu+, Ag+, Zn2+ Cd2+ H g 2+ ), through trimercaptide linkages with hi’gh affinity. Mts are characterized by a very high cysteine content and by the absence of aromatic amino acids. Exposure to heavy metals, in particular Zn and Cd, as well as to certain glucocorticoid hormones causes an increase in hepatic and kidney Mt levels. It is believed that Mt plays an important role in metal homeostasis and in protection against heavy metal toxicity. It has been demonstrated that Mt is also induced in cell types other than liver or kidney cells in response to heavy metals and dexamethasone (Karin and Herschman, 1981; Mayo and Palmiter, 1981). More recently Gick and McCarty (1983) showed that high levels of Mt were induced by Zn in a Cdresistant Chinese hamster ovary (CHO) cell mutant. In the present work it is shown that Mt is not found in untreated wild type CHO cells. Neither Zn nor dex were demonstrated to be able to induce Mt in these cells. Evidence is given indicating that CHO cells may respond to Zn and dex by a certain induction of sulfhydryl groups not located on cell Mt. Such groups were assayed by Cd*+ binding affinity and by the electrochemical technique known as pulse polarography. MATERIALS
carrying defects in the proline and hypoxanthine guanine phosphoribosyl transfer&e (HPRT E.C. 2.4.4.8) geies. CHO cells wild tvtx -_ normal (Kl WTN) and a Cd-resistant mutant (458-3) (see Corrigan and Huang, 1983 for selection and preparation of Cd-resistant mutant clones) were allowed to attach and grow in monolayer culture in Corning 75 cm* tissue culture flasks in a-MEM (GIBCO) containing 5% fetal calf serum (KC Biological, Inc) and an antibiotic-antimycotic mixture (GIBCO) at 37°C in a 5% CO, incubator prior to and during incubation with Zn or dex. Zn was added to the cultures in the form of sulfate. Sterile stock solutions of Zn and dex were prepared by autoclaving from which serial dilutions were made. Based on atomic absorption spectrometric analysis (courtesy of Dr. John Frazier), the fetal calf serum used in this study was found to contain approximately I-3pg/ml Zn that when used at 5% in the medium resulted in a concentration of 0.77 PM Zn. The background level of Zn in the medium was further reduced by replacing the commonly used F-10, which contains 6.3 PM Zn, with a-MEM which contains essentially no Zn. Because of the variation in Zn and protein content between batches of sera, fetal calf serum from the same lot was used in all experiments for the present study. The growing solutions were not tested for the possible presence of steroid hormones. After incubation the cells were washed twice with phosphate-buffered saline (pH 7.4) and harvested by the use of 0.1% trvpsin in 0.02% EDTA. The cells were resuspended akd lysed in aq. dest. and then frozen (- 80°C). The supernatant obtained after thawing and centrifugation at 12,OOOg for 1 min was used for pulse polarographic quantitation of sulfbydryl groups or was subjected to gel filtration for further analysis. Electrochemical measurements of suljhydryl groups
AND METHODS
Differential pulse polarography using the BrdiEka procedure was carried out according to PaleEek and Pechan (1971) and Olafson and Sim (1979) by using the Metrohm E502 analyzer and 626 polarecord. Analysis was performed in 10 ml aliquots of supporting electrolyte by scanning from - 1.30 to - 1.60V at 5 mV/sec. Mercury drop time was 0.5 sec. The sensitivity setting was 20 in the PA/mm range. The BrdiEka cobalt electrolyte was used without surface active agents. The electrolyte was purged with high purity nitrogen for 8 min prior to addition of sample and then for
Chemicals Dexamethasone (9a-fluoro-16a-methylprednisolone) was obtained from Sigma Chem. Co. and ZnSO, from Aldrich. The isotope io9Cd was bought from New England Nuclear (sp. act. IL3 Ci/g Cd). Cell cultures and growing conditions The parental cell type was a line of CHO cells (Chinese hamster Cricetulus griseus), originated from Dr. F. T. Kao, 345
346
ROLF A. ANDERSENand XUE-LAN WEN
% +(A) SO0
500 WTN
t
*
100 -
L.
6 12
24
48
72
hrs
% .”
(El
*
1.
:
:
6
12
48
24
72
hrs
Fig. 1. Pulse polarographic responses (calculated to response/cell) of extracts from CHO cells grown for various times in the presence of dexamethasone (IO-’ M) -H-m-mand Zn (10e4M) -_O-_O--_Oas a nercentaee of untreated controls. A: Wild type (WTN), B: bd resist&t mutants (458-3) (Corrigan and-H&g, 1483). Measurements based on cell amounts ranging from IO6 to 3 x 106. For data evaluation normal t-test statistics were used. The curve points given are based on mean values of at least four independent determinations. The level of significance (P) is 0.05. Vertical bars indicate highest +t SEM value observed.
an additional 2 min. In all cases the sample 100 ~1 of the cell supernatant.
size used was
Gel filtration and ion exchange Cell extract samples of 1 ml were run through a Sephadex G-75 column (48.5 x 1.15 cm) at room temperature in elution buffer containing 50 mM Tris (pH 7.4) and 5 mM mercaptoethanol. Eluate fractions of 2ml were collected Each cell extract sample and assayed for ‘09Cd radioactivity. was equilibrated for 20min with 5 pl of the Cd isotope solution containing 500,000 cpm prior to gel filtration. In some experiments cell extracts from Cd-resistant 458-3 cells treated with 10m4 M Zn for 24 hr were run through the column in 5 mM Tris (pH 8.6) and 5 mM mercaptoethanol. Cd isotope solution was added as above. The peak radioactivity fractions were then pooled and applied directly on an ion exchange column (25 x 1.5 cm) composed of DEAE-Sephadex A-50 equilibrated with 5mM Tris (PH 8.6). After washing with 100 ml of the lower gradient buffer, elution was started with a linear Tris gradient (S-500 mM) of total volume of 600 ml (pH 8.6). Eluate fractions of 4 ml were collected and analyzed for “‘%d radioactivity. RESULTS
The polarographic responses obtained from cell extracts prepared from wild type and mutants treated and untreated with lo-‘M dex and 10-4M Zn are
given in Fig. 1. For dex the values ranged from about 12&200% of untreated controls for both cell types. Prior to these experiments cells were tested with dex in different concentrations indicating lo-‘-lOma M to be optimal for induction (not shown).
For Zn, however, wild type and mutants behaved differently. In the wild type an increase in polarographic activity (200% of untreated controls) was seen after 6 hr treatment ( 10e4 M) then a decline was observed reaching a lower value than controls at 72 hr (about 80%). For the mutants, on the other hand, a large increase in polarographic activity was seen for all incubation periods ranging from five to eight times higher values than in untreated controls. For both cell types 10m4 M Zn was found to be the optimal concentration for induction (not shown). In a series of experiments cell extracts, after equiSephadex libration with ‘09Cd, were run through G-75 columns. The time points for treatment of these cells were chosen to be optimal for induced polarographic activity, namely for wild type Zn 6 hr and dex 24 hr and for mutants Zn 24 hr and dex 12 hr (see also Fig. 1 A and B). The elution profiles obtained, when assayed for Cd binding affinity, are given in Fig. 2. Measurements of polarographic activity on individual fractions are also included. Generally one main peak containing most of the Cd binding material as well as the polarographic activity was seen. The peaks, however, were located at different positions for wild type compared to mutant cells. For wild type most of the activity was located in the void (fractions 8-12) while for the mutants it was located at about fraction 16. Some of the profiles also showed a small lo9Cd peak located at about fraction 24 (end of column). This peak corresponded to free lo9Cd or Cd bound to low molecular weight compounds such as glutathione (see also Karin et al., 1980). To show that the lo9Cd binding material from the mutants eluting at about fraction 16 must be characterized as metallothionein (Mt) a standard (MtA) prepared from liver of Cd-treated rats (Rattus noruegicus) was run through the column (procedure given by Ohi et al., 1981). In Fig. 2C it is shown that this Mt also was eluted as one peak at about fraction 16. Another approach for the characterization of the Mt-like material isolated from the mutant cells was performed by running it on ion exchange as already described. When the elution profile from this experiment was compared to the profile obtained for a liver extract from rat given one CdSO, injection of 1 mg/kg and sacrificed after 24 hr, a similarity was found. This is shown in Fig. 3. DISCUSSION
The data in the present work indicate that Zn induces Mt in the Cd-resistant 458-3 CHO cell mutants. This observation is supported by Gick and McCarty (1983) who demonstrated Mt induction by Zn in their Cd-resistant CHO cell mutants. In wild type cells, however, Mt or Mt inducibility could not be demonstrated. This is consistent with the hypothesis that Cd- and Zn-resistant cells might express such resistance as a consequence of a reduction in unbound intracellular metal levels by elevation of Mt synthesis, and that Mt plays an important role in preventing the toxic effects of heavy metals. Polarography, which is supposed to respond to sullhydryls under the conditions used in the present work (PaleEek and Pechan, 1971; Olafson and Sim, 1979), demonstrated an increase in polarographic
347
Induction of heavy metal binding groups WTN
cells
Zn lO-‘M.6
hrs
Fig. 2. Sephadex G-75 profiles of extracts from CHO cells grown in the presence of dexamethasone and Zn for various times as indicated on graphs. Individual fractions assayed for added ‘%d marker -_O-_O--_O---_ The profile of a metallothionein A standard preparation (run in a separate experiment) is given to bottom left *.D..m. .m... Pulse polarographic responses from individual fractions .t fJ..n,,o,.. Curves are not corrected to equal celt numbers. It shonld also be noted that cpm is not a good quantitative measurement of actual metallothionein content.
activity in wild type CHO cells after Zn and dex treatment. It seems reasonable to believe that this increase might be caused by an increase in particleor membrane-bound sulfhydryl groups, because gel filtration of wild type cell extracts showed polarographic activity and Cd binding capacity only in the void volume fraction. Support for the view that membrane bound sullhydryls are inducible is also found in the Cd-resistant CHO cell mutants after dex treatment where Cd binding capacity could be demonstrated in the void volume. Sul~yd~ls could not be detected by pulse polarography in the void volume from untreated mutants. It seems, therefore, difficult to argue that the metal affinity of the Mt demonstrated to be present in the Cd resistant mutants is high enough to strip off radioactive Cd bound to particle sulthydryls, thus being the cause why Cd binding affinity does not show up in the void volume fraction from untreated mutants. The very interesting question of the nature of the sulfhydryls present in the void volume from wild type
I___++_+
.._.._..............
_t
8492KKlmx6E4!92muo65
Fig. 3. Ion exchange profiles (DEAE-Sephadex A-50) of pooled peak radioactivity fractions obtained after gel filtration (SC-75) of a cell extract from Cd-resistant mutant CHO cells (458-3) grown in the presence of 10e4 M Zn for 24 hr 0 0 0 and of metallothionein prepared from rat liver (metallothionein induced by one injection of 1 mg Cd/kg, sacrificed after 24 hr) W n n . See text for procedures. Refractometric index -O-_O--_c1--.
ROLF A. ANDERSENand XUE-LAN WEN
348
CHO cells and the possible role played by Zn and glucocorticoids in their regulation was not further studied
in the present
work.
Acknowledgement-The authors wish to thank Professor P. C. Huang for many fruitful discussions, use of laboratory facilities and for the CHO cells. REFERENCES Corrigan A. J. and Huang P. C. (1983) ~drni~ and zinc flux in wild type and c~mium-resistant CHO cells. Biot. Trace Element Res. 5, 25-33. Gick G. G. and McCarty K. S. (1983) Reduced Cd’+ accumulation and elevated metaliothionein levels in a Cd*+ and Zn*+ resistant clonal CHO-Kl cell line. Toxicology 26, 275-283. Karin M. and Herschman H. R. (1981) Induction of metallothionein in HeLa cells by dexamethasone and zinc. Eur. J. Biochem. 113, 267-272.
Karin M., Herschman H. R. and Weinstein D. (1980) Primary induction of metallothionein by dexamethasone in cultured rat hepatocytes. Biochem. Biophys. Res. Commun. 92, 1052-1059. Karin M., Haslinger A., Holtgreve H., Richards R. I., Krauter P., Westphal H. W. and Beato M. (1984) Characterization of DNA sequences through which cadmium and induce glucocorticoid hormones human metallothionein-II, gene. Nature, Land. 308, 513-519. Mayo K. E. and Palmiter R. D. (1981) Glucocorticoid regulation of metallothionein-1mRNA synthesis in cuhured mouse ceils. J. biol. Chem. 256, 2621-2624. Ohi S., Cardenosa G., Pine R. and Huang P. C. (1981) Cadmium-induced accumulation of metalkothionein messenger RNA in rat liver. .I. biol. Chem. 256. 2180-2184. Olaf& R. W. and Sim R. G. (1979) An electrochemical approach to quantitation and characterization of metallothioneins. Analyt. Biochem. 100, 343-351. PaleEek E. and Pechan 2. (1971) Estimation of nanogram quantities of proteins by pulse-polarographic technique. Analyt. Biochem. 42, 59-71.
in Great
0306~4492/86 $3.00 + 0.00 Pergamon Journals Ltd
1986
Britain
INDUCTION OF HEAVY METAL BINDING GROUPS BY DEXAMETHASONE AND Zn IN CULTURED CHINESE HAMSTER OVARY CELLS ROLF
A.
and
ANDER&N*
XUE-LAN
WENT
*Department of Toxicology, National Institute of Public Health, Geitmyrsveien 750462 Oslo 4, Norway. Telephone: (02) 35-6020 and tPeking Childrens Hospital, Peking, Peoples Republic of China (Received 4 November
1985)
Abstract-l. Cd binding capacity and pulse polarography were used to study the inducibility of sulfhydryl groups in cultured Chinese hamster ovary cells (wild type and a Cd-resistant mutant) in response to dexamethasone (dex) and Zn. 2. Evidence is presented that both the wild type and the mutant responded to dex and Zn treatment by induction of sulfhydryl groups. 3. In wild type for Zn and dex as well as in the mutant for dex, this induction seems to be in the form of sulfhydryls attached to particulate or membrane fractions in the cells. For Zn in the Cd-resistant mutant the induction was in the form of metallothionein.
INTRODUCTION
Metallothioneins (Mts) are heat stable, low molecular weight (M, = 6000-12,000) proteins that bind heavy metals, belonging to the periodic system transition groups Ia and Ha (Cu+, Ag+, Zn2+ Cd2+ H g 2+ ), through trimercaptide linkages with hi’gh affinity. Mts are characterized by a very high cysteine content and by the absence of aromatic amino acids. Exposure to heavy metals, in particular Zn and Cd, as well as to certain glucocorticoid hormones causes an increase in hepatic and kidney Mt levels. It is believed that Mt plays an important role in metal homeostasis and in protection against heavy metal toxicity. It has been demonstrated that Mt is also induced in cell types other than liver or kidney cells in response to heavy metals and dexamethasone (Karin and Herschman, 1981; Mayo and Palmiter, 1981). More recently Gick and McCarty (1983) showed that high levels of Mt were induced by Zn in a Cdresistant Chinese hamster ovary (CHO) cell mutant. In the present work it is shown that Mt is not found in untreated wild type CHO cells. Neither Zn nor dex were demonstrated to be able to induce Mt in these cells. Evidence is given indicating that CHO cells may respond to Zn and dex by a certain induction of sulfhydryl groups not located on cell Mt. Such groups were assayed by Cd*+ binding affinity and by the electrochemical technique known as pulse polarography. MATERIALS
carrying defects in the proline and hypoxanthine guanine phosphoribosyl transfer&e (HPRT E.C. 2.4.4.8) geies. CHO cells wild tvtx -_ normal (Kl WTN) and a Cd-resistant mutant (458-3) (see Corrigan and Huang, 1983 for selection and preparation of Cd-resistant mutant clones) were allowed to attach and grow in monolayer culture in Corning 75 cm* tissue culture flasks in a-MEM (GIBCO) containing 5% fetal calf serum (KC Biological, Inc) and an antibiotic-antimycotic mixture (GIBCO) at 37°C in a 5% CO, incubator prior to and during incubation with Zn or dex. Zn was added to the cultures in the form of sulfate. Sterile stock solutions of Zn and dex were prepared by autoclaving from which serial dilutions were made. Based on atomic absorption spectrometric analysis (courtesy of Dr. John Frazier), the fetal calf serum used in this study was found to contain approximately I-3pg/ml Zn that when used at 5% in the medium resulted in a concentration of 0.77 PM Zn. The background level of Zn in the medium was further reduced by replacing the commonly used F-10, which contains 6.3 PM Zn, with a-MEM which contains essentially no Zn. Because of the variation in Zn and protein content between batches of sera, fetal calf serum from the same lot was used in all experiments for the present study. The growing solutions were not tested for the possible presence of steroid hormones. After incubation the cells were washed twice with phosphate-buffered saline (pH 7.4) and harvested by the use of 0.1% trvpsin in 0.02% EDTA. The cells were resuspended akd lysed in aq. dest. and then frozen (- 80°C). The supernatant obtained after thawing and centrifugation at 12,OOOg for 1 min was used for pulse polarographic quantitation of sulfbydryl groups or was subjected to gel filtration for further analysis. Electrochemical measurements of suljhydryl groups
AND METHODS
Differential pulse polarography using the BrdiEka procedure was carried out according to PaleEek and Pechan (1971) and Olafson and Sim (1979) by using the Metrohm E502 analyzer and 626 polarecord. Analysis was performed in 10 ml aliquots of supporting electrolyte by scanning from - 1.30 to - 1.60V at 5 mV/sec. Mercury drop time was 0.5 sec. The sensitivity setting was 20 in the PA/mm range. The BrdiEka cobalt electrolyte was used without surface active agents. The electrolyte was purged with high purity nitrogen for 8 min prior to addition of sample and then for
Chemicals Dexamethasone (9a-fluoro-16a-methylprednisolone) was obtained from Sigma Chem. Co. and ZnSO, from Aldrich. The isotope io9Cd was bought from New England Nuclear (sp. act. IL3 Ci/g Cd). Cell cultures and growing conditions The parental cell type was a line of CHO cells (Chinese hamster Cricetulus griseus), originated from Dr. F. T. Kao, 345
346
ROLF A. ANDERSENand XUE-LAN WEN
% +(A) SO0
500 WTN
t
*
100 -
L.
6 12
24
48
72
hrs
% .”
(El
*
1.
:
:
6
12
48
24
72
hrs
Fig. 1. Pulse polarographic responses (calculated to response/cell) of extracts from CHO cells grown for various times in the presence of dexamethasone (IO-’ M) -H-m-mand Zn (10e4M) -_O-_O--_Oas a nercentaee of untreated controls. A: Wild type (WTN), B: bd resist&t mutants (458-3) (Corrigan and-H&g, 1483). Measurements based on cell amounts ranging from IO6 to 3 x 106. For data evaluation normal t-test statistics were used. The curve points given are based on mean values of at least four independent determinations. The level of significance (P) is 0.05. Vertical bars indicate highest +t SEM value observed.
an additional 2 min. In all cases the sample 100 ~1 of the cell supernatant.
size used was
Gel filtration and ion exchange Cell extract samples of 1 ml were run through a Sephadex G-75 column (48.5 x 1.15 cm) at room temperature in elution buffer containing 50 mM Tris (pH 7.4) and 5 mM mercaptoethanol. Eluate fractions of 2ml were collected Each cell extract sample and assayed for ‘09Cd radioactivity. was equilibrated for 20min with 5 pl of the Cd isotope solution containing 500,000 cpm prior to gel filtration. In some experiments cell extracts from Cd-resistant 458-3 cells treated with 10m4 M Zn for 24 hr were run through the column in 5 mM Tris (pH 8.6) and 5 mM mercaptoethanol. Cd isotope solution was added as above. The peak radioactivity fractions were then pooled and applied directly on an ion exchange column (25 x 1.5 cm) composed of DEAE-Sephadex A-50 equilibrated with 5mM Tris (PH 8.6). After washing with 100 ml of the lower gradient buffer, elution was started with a linear Tris gradient (S-500 mM) of total volume of 600 ml (pH 8.6). Eluate fractions of 4 ml were collected and analyzed for “‘%d radioactivity. RESULTS
The polarographic responses obtained from cell extracts prepared from wild type and mutants treated and untreated with lo-‘M dex and 10-4M Zn are
given in Fig. 1. For dex the values ranged from about 12&200% of untreated controls for both cell types. Prior to these experiments cells were tested with dex in different concentrations indicating lo-‘-lOma M to be optimal for induction (not shown).
For Zn, however, wild type and mutants behaved differently. In the wild type an increase in polarographic activity (200% of untreated controls) was seen after 6 hr treatment ( 10e4 M) then a decline was observed reaching a lower value than controls at 72 hr (about 80%). For the mutants, on the other hand, a large increase in polarographic activity was seen for all incubation periods ranging from five to eight times higher values than in untreated controls. For both cell types 10m4 M Zn was found to be the optimal concentration for induction (not shown). In a series of experiments cell extracts, after equiSephadex libration with ‘09Cd, were run through G-75 columns. The time points for treatment of these cells were chosen to be optimal for induced polarographic activity, namely for wild type Zn 6 hr and dex 24 hr and for mutants Zn 24 hr and dex 12 hr (see also Fig. 1 A and B). The elution profiles obtained, when assayed for Cd binding affinity, are given in Fig. 2. Measurements of polarographic activity on individual fractions are also included. Generally one main peak containing most of the Cd binding material as well as the polarographic activity was seen. The peaks, however, were located at different positions for wild type compared to mutant cells. For wild type most of the activity was located in the void (fractions 8-12) while for the mutants it was located at about fraction 16. Some of the profiles also showed a small lo9Cd peak located at about fraction 24 (end of column). This peak corresponded to free lo9Cd or Cd bound to low molecular weight compounds such as glutathione (see also Karin et al., 1980). To show that the lo9Cd binding material from the mutants eluting at about fraction 16 must be characterized as metallothionein (Mt) a standard (MtA) prepared from liver of Cd-treated rats (Rattus noruegicus) was run through the column (procedure given by Ohi et al., 1981). In Fig. 2C it is shown that this Mt also was eluted as one peak at about fraction 16. Another approach for the characterization of the Mt-like material isolated from the mutant cells was performed by running it on ion exchange as already described. When the elution profile from this experiment was compared to the profile obtained for a liver extract from rat given one CdSO, injection of 1 mg/kg and sacrificed after 24 hr, a similarity was found. This is shown in Fig. 3. DISCUSSION
The data in the present work indicate that Zn induces Mt in the Cd-resistant 458-3 CHO cell mutants. This observation is supported by Gick and McCarty (1983) who demonstrated Mt induction by Zn in their Cd-resistant CHO cell mutants. In wild type cells, however, Mt or Mt inducibility could not be demonstrated. This is consistent with the hypothesis that Cd- and Zn-resistant cells might express such resistance as a consequence of a reduction in unbound intracellular metal levels by elevation of Mt synthesis, and that Mt plays an important role in preventing the toxic effects of heavy metals. Polarography, which is supposed to respond to sullhydryls under the conditions used in the present work (PaleEek and Pechan, 1971; Olafson and Sim, 1979), demonstrated an increase in polarographic
347
Induction of heavy metal binding groups WTN
cells
Zn lO-‘M.6
hrs
Fig. 2. Sephadex G-75 profiles of extracts from CHO cells grown in the presence of dexamethasone and Zn for various times as indicated on graphs. Individual fractions assayed for added ‘%d marker -_O-_O--_O---_ The profile of a metallothionein A standard preparation (run in a separate experiment) is given to bottom left *.D..m. .m... Pulse polarographic responses from individual fractions .t fJ..n,,o,.. Curves are not corrected to equal celt numbers. It shonld also be noted that cpm is not a good quantitative measurement of actual metallothionein content.
activity in wild type CHO cells after Zn and dex treatment. It seems reasonable to believe that this increase might be caused by an increase in particleor membrane-bound sulfhydryl groups, because gel filtration of wild type cell extracts showed polarographic activity and Cd binding capacity only in the void volume fraction. Support for the view that membrane bound sullhydryls are inducible is also found in the Cd-resistant CHO cell mutants after dex treatment where Cd binding capacity could be demonstrated in the void volume. Sul~yd~ls could not be detected by pulse polarography in the void volume from untreated mutants. It seems, therefore, difficult to argue that the metal affinity of the Mt demonstrated to be present in the Cd resistant mutants is high enough to strip off radioactive Cd bound to particle sulthydryls, thus being the cause why Cd binding affinity does not show up in the void volume fraction from untreated mutants. The very interesting question of the nature of the sulfhydryls present in the void volume from wild type
I___++_+
.._.._..............
_t
8492KKlmx6E4!92muo65
Fig. 3. Ion exchange profiles (DEAE-Sephadex A-50) of pooled peak radioactivity fractions obtained after gel filtration (SC-75) of a cell extract from Cd-resistant mutant CHO cells (458-3) grown in the presence of 10e4 M Zn for 24 hr 0 0 0 and of metallothionein prepared from rat liver (metallothionein induced by one injection of 1 mg Cd/kg, sacrificed after 24 hr) W n n . See text for procedures. Refractometric index -O-_O--_c1--.
ROLF A. ANDERSENand XUE-LAN WEN
348
CHO cells and the possible role played by Zn and glucocorticoids in their regulation was not further studied
in the present
work.
Acknowledgement-The authors wish to thank Professor P. C. Huang for many fruitful discussions, use of laboratory facilities and for the CHO cells. REFERENCES Corrigan A. J. and Huang P. C. (1983) ~drni~ and zinc flux in wild type and c~mium-resistant CHO cells. Biot. Trace Element Res. 5, 25-33. Gick G. G. and McCarty K. S. (1983) Reduced Cd’+ accumulation and elevated metaliothionein levels in a Cd*+ and Zn*+ resistant clonal CHO-Kl cell line. Toxicology 26, 275-283. Karin M. and Herschman H. R. (1981) Induction of metallothionein in HeLa cells by dexamethasone and zinc. Eur. J. Biochem. 113, 267-272.
Karin M., Herschman H. R. and Weinstein D. (1980) Primary induction of metallothionein by dexamethasone in cultured rat hepatocytes. Biochem. Biophys. Res. Commun. 92, 1052-1059. Karin M., Haslinger A., Holtgreve H., Richards R. I., Krauter P., Westphal H. W. and Beato M. (1984) Characterization of DNA sequences through which cadmium and induce glucocorticoid hormones human metallothionein-II, gene. Nature, Land. 308, 513-519. Mayo K. E. and Palmiter R. D. (1981) Glucocorticoid regulation of metallothionein-1mRNA synthesis in cuhured mouse ceils. J. biol. Chem. 256, 2621-2624. Ohi S., Cardenosa G., Pine R. and Huang P. C. (1981) Cadmium-induced accumulation of metalkothionein messenger RNA in rat liver. .I. biol. Chem. 256. 2180-2184. Olaf& R. W. and Sim R. G. (1979) An electrochemical approach to quantitation and characterization of metallothioneins. Analyt. Biochem. 100, 343-351. PaleEek E. and Pechan 2. (1971) Estimation of nanogram quantities of proteins by pulse-polarographic technique. Analyt. Biochem. 42, 59-71.