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Thyroid hormone-inducible mitochondrial α-glycerophosphate dehydrogenase in the MCF-7 human breast cancer cell line
Eur J Cancer Clan Oncol, Printed in Great Britain.
Vol. 19. No. IO, pp. 1485-1486,
1985.
0
0277-5379/89$3.00+0.00 1985 Pergdmon Press Ltd.
Letter to the Editor Thyroid Hormone-inducible Mitochondrial aGlycerophosphate Dehydrogenase in the MCF-7 Human Breast Cancer Cell Line* DAVID
Division of Clinical Oncology,
P. DRAVES,
IS
Accepted 15 February 1983. *Supported in part by USPHS National Cancer Institute.
Grant CA-17579
J. RUZICKA
and DAVID P. ROSE
experimental flasks, cultured for approximately 12 days and then allocated randomly to 1 of 6 groups, with 5 flasks per group. Five of the 6 experimental groups had one of the following additions: Tr, in concentrations ranging from 1 X 16” to 1 X 1fr6 M; actinomycin D, 15 rg/ml; puromycin, 0.6 mg/ml; T3 plus actinomycin D; and T3 plus puromycin. The sixth group of flasks provided the controls. Incubation was then continued for a further 40 hr, after which the cells were harvested and those from each experimental group pooled and homogenized in chilled 0.25 M sucrose buffer, pH 7.4. The mitochondria were isolated by centrifugation and stored in liquid nitrogen until assayed for GPD activity by the method of Singer [8]. Basal mitochondrial GPD activity of MCF-7 cells ranged from 75 to 135 nmol substrate/min/ mg protein in 6 experiments. The stimulating effect of incubation in the presence of T3, which was maximal at a concentration of 1 X lO_* M, is illustrated in Fig. 1. Puromycin and actinomycin D had no significant effect on basal mitochondrial GPD activity, but hormonal stimulation was completely inhibited when either was added together with T,. We found previously that mitochondria from N-nitrosomethylurea-induced rat mammary carcinomas had an average GPD level of 92.5 3~24.1 nmol substrate/min/mg protein [6], a value which is within the range observed for MCF-7 cells in the present experiments, The observation that a 1 X l@‘-M concentration of T, was optimal for stimulation of GPD activity in MCF-7 cell mitochondria is of some interest. We
generally accepted that the binding of triiodothyronine (T,) to nuclear receptor sites is involved in at least some biological expressions of thyroid hormone action [l], and the kinetics of response to Tr by hepatic cytosol malic enzyme and mitochondrial cY-glycerophosphate dehydrogenase (GPD) suggest that the number of occupied sites determines the maximal thyroid hormone effect [2]. While these receptors have been studied largely in normal tissues, including mammary cells [8], they occur also in some breast carcinomas [4-61. Burke and McGuire [4] demonstrated the presence of T, receptors in the nuclei of MCF-7 human metastatic breast cancer cells, together with a stimulation of tumor cell proliferation when T, was added to the culture medium. In the rat, not only is hepatic mitochondrial GPD activity responsive to thyroid hormone status [I, 71, but mammary carcinomas induced by Nnitrosomethylurea contain nuclear T3 receptors and mitochondrial GPD, the level of which is reduced in hypothyroid animals [6]. We have now demonstrated GPD activity in the mitochondria of MCF-7 cells and a stimulation of the enzyme by T3 which was inhibited in the presence of actinomycin D or puromycin. The cells, a gift from the Michigan Cancer Foundation, were cultured in supplemented Eagle’s Minimal Essential Medium [4]. When they had grown to confluence they were transferred to IT
FRANK
Wisconsin Clinical Cancer Center, Madison, WI 53792, U.S.A.
from the
1485
1486
Letter
to the Editor
250,
5.
I ‘-10
IO
-9
IO
IO-’
T3 Concentration
IO-'
-6 10
CM)
Fig. 1. Stimulation of mitochondrial wglycerophosphate dehydrogenase activity in MCF-7 breast cancer cells by various concentrations of T3 (0). and the inhibitory effects of actinomycin D 15 fig/ml (0), and puromycin 0.6 mg/ml (X).
have assessed MCF-7 cell proliferation during 12 days incubation with various levels of the
hormone, and again found maximal stimulation by 1 X l(r8 M of T,. Also, as was true of GPD (Fig. l), 1 X lO_’M had little or no effect. In a similar experiment, Burke and McGuire [4] obtained maximal stimulation of MCF-7 cell proliferation with 1 X I@’ M of T, and no effect with a concentration of 1 X 1V M. The inhibition of mitochondrial GPD stimulation by T, in the presence of puromycin or actinomycin D is consistent with a true hormonal induction involving synthesis of enzyme and requiring mRNA synthesis. Thus we have shown that the MCF-7 breast cancer cell line, in addition to possessing nuclear T3 receptors, has retained the capacity for responding to thyroid hormone stimulation by both cellular proliferation and de nova synthesis of an enzyme, mitochondrial GPD, which is known to be regulated by T, in normal tissues. The assay, as performed, cannot exclude the possibility that T3 also increased the number of mitochondria per tumor cell.
REFERENCES 1.
OPPENHEIMER JH, SCHWARTZ HL, SURKS MI. Nuclear binding capacity appears to limit the hepatic response to L-triiodothyronine (T,). Endocr Res Commun 1975, 2, 309-325.
2.
3. 4. 5. 6.
7.
8.
OPPENHEIMER JH, SILVA E, SCHWARTZ HL, SURKS MI. Stimulation of hepatic dehydrogenase and malic enzyme by Lmitochondrial a-glycerophosphate triiodothyronine. Characteristics of the response with specific nuclear thyroid hormone binding sites fully saturated. J Clin Invest 1977, 59, 517-527. BHATTACHARYA A, VONDERHAAR BK. Specific binding proteins for 3,5,3’triiodothyronine in mouse mammary epithelium. J Cell Biol 1977, 75, 47a. BURKE RE, MCGUIRE WL. Nuclear thyroid hormone receptors in a human breast cancer cell line. Cancer Res 1978,38, 3769-3773. CERBON M-A, PICHON M-E, MILGROM E. Thyroid hormone receptors in human breast cancer. Cancer Res 1981, 41, 4167-4173. RUZICKA FJ, GUIFFRE L, ROSE DP, DAVISTE. Nuclear thyroid hormone receptors (T,R) and mitochondrial enzymes in N-nitrosomethylurea (NMU)-induced rat mammary carcinomas. Proc Am Assoc Cancer Res 1981,22, 12. LEE Y, LARDY HA. Influence of thyroid hormones on L-cY-glycerophosphate dehydrogenase and other dehydrogenases in various organs of the rat. J Biol Chem 1965, 240, 1427-1436. SINCXR TP. Determination of the activity of succinate, NADH, choline and OLglycerophosphate dehydrogenases. Methods Biochem Anal 1974,22, 123-133.
Vol. 19. No. IO, pp. 1485-1486,
1985.
0
0277-5379/89$3.00+0.00 1985 Pergdmon Press Ltd.
Letter to the Editor Thyroid Hormone-inducible Mitochondrial aGlycerophosphate Dehydrogenase in the MCF-7 Human Breast Cancer Cell Line* DAVID
Division of Clinical Oncology,
P. DRAVES,
IS
Accepted 15 February 1983. *Supported in part by USPHS National Cancer Institute.
Grant CA-17579
J. RUZICKA
and DAVID P. ROSE
experimental flasks, cultured for approximately 12 days and then allocated randomly to 1 of 6 groups, with 5 flasks per group. Five of the 6 experimental groups had one of the following additions: Tr, in concentrations ranging from 1 X 16” to 1 X 1fr6 M; actinomycin D, 15 rg/ml; puromycin, 0.6 mg/ml; T3 plus actinomycin D; and T3 plus puromycin. The sixth group of flasks provided the controls. Incubation was then continued for a further 40 hr, after which the cells were harvested and those from each experimental group pooled and homogenized in chilled 0.25 M sucrose buffer, pH 7.4. The mitochondria were isolated by centrifugation and stored in liquid nitrogen until assayed for GPD activity by the method of Singer [8]. Basal mitochondrial GPD activity of MCF-7 cells ranged from 75 to 135 nmol substrate/min/ mg protein in 6 experiments. The stimulating effect of incubation in the presence of T3, which was maximal at a concentration of 1 X lO_* M, is illustrated in Fig. 1. Puromycin and actinomycin D had no significant effect on basal mitochondrial GPD activity, but hormonal stimulation was completely inhibited when either was added together with T,. We found previously that mitochondria from N-nitrosomethylurea-induced rat mammary carcinomas had an average GPD level of 92.5 3~24.1 nmol substrate/min/mg protein [6], a value which is within the range observed for MCF-7 cells in the present experiments, The observation that a 1 X l@‘-M concentration of T, was optimal for stimulation of GPD activity in MCF-7 cell mitochondria is of some interest. We
generally accepted that the binding of triiodothyronine (T,) to nuclear receptor sites is involved in at least some biological expressions of thyroid hormone action [l], and the kinetics of response to Tr by hepatic cytosol malic enzyme and mitochondrial cY-glycerophosphate dehydrogenase (GPD) suggest that the number of occupied sites determines the maximal thyroid hormone effect [2]. While these receptors have been studied largely in normal tissues, including mammary cells [8], they occur also in some breast carcinomas [4-61. Burke and McGuire [4] demonstrated the presence of T, receptors in the nuclei of MCF-7 human metastatic breast cancer cells, together with a stimulation of tumor cell proliferation when T, was added to the culture medium. In the rat, not only is hepatic mitochondrial GPD activity responsive to thyroid hormone status [I, 71, but mammary carcinomas induced by Nnitrosomethylurea contain nuclear T3 receptors and mitochondrial GPD, the level of which is reduced in hypothyroid animals [6]. We have now demonstrated GPD activity in the mitochondria of MCF-7 cells and a stimulation of the enzyme by T3 which was inhibited in the presence of actinomycin D or puromycin. The cells, a gift from the Michigan Cancer Foundation, were cultured in supplemented Eagle’s Minimal Essential Medium [4]. When they had grown to confluence they were transferred to IT
FRANK
Wisconsin Clinical Cancer Center, Madison, WI 53792, U.S.A.
from the
1485
1486
Letter
to the Editor
250,
5.
I ‘-10
IO
-9
IO
IO-’
T3 Concentration
IO-'
-6 10
CM)
Fig. 1. Stimulation of mitochondrial wglycerophosphate dehydrogenase activity in MCF-7 breast cancer cells by various concentrations of T3 (0). and the inhibitory effects of actinomycin D 15 fig/ml (0), and puromycin 0.6 mg/ml (X).
have assessed MCF-7 cell proliferation during 12 days incubation with various levels of the
hormone, and again found maximal stimulation by 1 X l(r8 M of T,. Also, as was true of GPD (Fig. l), 1 X lO_’M had little or no effect. In a similar experiment, Burke and McGuire [4] obtained maximal stimulation of MCF-7 cell proliferation with 1 X I@’ M of T, and no effect with a concentration of 1 X 1V M. The inhibition of mitochondrial GPD stimulation by T, in the presence of puromycin or actinomycin D is consistent with a true hormonal induction involving synthesis of enzyme and requiring mRNA synthesis. Thus we have shown that the MCF-7 breast cancer cell line, in addition to possessing nuclear T3 receptors, has retained the capacity for responding to thyroid hormone stimulation by both cellular proliferation and de nova synthesis of an enzyme, mitochondrial GPD, which is known to be regulated by T, in normal tissues. The assay, as performed, cannot exclude the possibility that T3 also increased the number of mitochondria per tumor cell.
REFERENCES 1.
OPPENHEIMER JH, SCHWARTZ HL, SURKS MI. Nuclear binding capacity appears to limit the hepatic response to L-triiodothyronine (T,). Endocr Res Commun 1975, 2, 309-325.
2.
3. 4. 5. 6.
7.
8.
OPPENHEIMER JH, SILVA E, SCHWARTZ HL, SURKS MI. Stimulation of hepatic dehydrogenase and malic enzyme by Lmitochondrial a-glycerophosphate triiodothyronine. Characteristics of the response with specific nuclear thyroid hormone binding sites fully saturated. J Clin Invest 1977, 59, 517-527. BHATTACHARYA A, VONDERHAAR BK. Specific binding proteins for 3,5,3’triiodothyronine in mouse mammary epithelium. J Cell Biol 1977, 75, 47a. BURKE RE, MCGUIRE WL. Nuclear thyroid hormone receptors in a human breast cancer cell line. Cancer Res 1978,38, 3769-3773. CERBON M-A, PICHON M-E, MILGROM E. Thyroid hormone receptors in human breast cancer. Cancer Res 1981, 41, 4167-4173. RUZICKA FJ, GUIFFRE L, ROSE DP, DAVISTE. Nuclear thyroid hormone receptors (T,R) and mitochondrial enzymes in N-nitrosomethylurea (NMU)-induced rat mammary carcinomas. Proc Am Assoc Cancer Res 1981,22, 12. LEE Y, LARDY HA. Influence of thyroid hormones on L-cY-glycerophosphate dehydrogenase and other dehydrogenases in various organs of the rat. J Biol Chem 1965, 240, 1427-1436. SINCXR TP. Determination of the activity of succinate, NADH, choline and OLglycerophosphate dehydrogenases. Methods Biochem Anal 1974,22, 123-133.