Progestin stimulation of lactate dehydrogenase in the human breast cancer cell line T-47D

Biochimica et Biophysica Acta 930 (1987) 167-172

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Elsevier BBA 12096

Progestin stimulation of lactate dehydrogenase in the human breast cancer cell line T - 4 7 D Rodney D. Hagley, Judith R. Hissom and Michael R. Moore Department of Biochemistry, Marshall University School of Medicine, Huntington, W V (U.S.A.)

(Received 30 March 1987)

Key words: Progestin; Lactate dehydrogenase; Breast cancer; Cell line T-47D

We have previously reported that physiological levels of progestins alone stimulate lactate dehydrogenase in a dose-responsive manner in the progesterone-receptor-rich human breast cancer cell line T-47D. Using isozyme electrophoresis, we have now found that lactate dehydrogenase isozyme 5 is the only isozyme detectable in these cells, as has been reported for other human breast cancer cells in long-term tissue culture. Upon treatment with progestins, isozyme 5 remains the only isozyme detectable. T-47D cells were plated in charcoal-stripped serum-containing medium and grown for 2 days before treatment with progestin. Lactate dehydrogenase stimulation then plateaued after around 2-3 days of treatment with progestin and was maintained until around day 5, following which a decline in enzyme activity occurred. The effect is specific for progestins, and inhibited by the anti-progestin RU-38486 (17fl-hydroxy-11fl-(4-dimethylaminophenyl-1)-17 a-(prop-l-ynil)-es~a-4,9-dien-3-one). Experiments using actinomycin D and cycloheximide suggest that the effect is dependent on RNA and protein synthesis, respectively. Lactate dehydrogenase stimulation occurs regardless of the presence of the estrogenic pH indicator Phenol red, and of whether it was analyzed per mg DNA or per mg protein.

Introduction This work further characterizes our initial observation that progestins stimulate lactate dehydrogenase in the human breast cancer cell line T-47D [1]. To our knowledge this was the first instance reported of an enzyme whose activity is stimulated by physiological levels of progestins alone in human breast cancer cells in tissue culture. Fournier et al. [2] more recently reported the progestin stimulation of estradiol 17fl-hydroxysteroid dehydrogenase in human breast tumors of

Correspondence: M.R. Moore, Department of Biochemistry, Marshall University School of Medicine, Huntington, WV 25704, U.S.A.

premenopausal and postmenopausal patients, dependent on progesterone receptor presence. Burke et al. [3] have reported elevation of the fifth isozyme of lactate dehydrogenase (LDH-5 or isoo zyme 5) in MCF-7 human breast cancer cells and that isozyme 5 is the only isozyme found in control or estrogen-treated cells. We find the same in T-47D cells with progestin treatment; that is, isozyme 5 is the only detectable isozyme in control or progestin-treated cells. Chalbos and Rochefort [4] have found that progestin induction of a M r 48 000 protein secreted into the medium by T-47D cells reaches a maximum at about 3 - 4 days and then begins to fall off. These same authors [5] have also reported the progestin stimulation of a M r 250000 intracellular protein, which becomes maximal after only 12 h of progestin treatment. Its levels after this time

0167-4889/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (Biomedical Division)

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were not reported. Horwitz and Freidenberg [6] have reported that the progestin R-5020 (17,21-dimethyl- 19-nor-4,9-pregnadiene-3,20-dione) elevates the level Of insulin receptors in T-47Dco, a variant subline of T-47D cells, the effect becoming maximal at around 3 days of treatment with R5020 and then falling off with time. We have found that lactate dehydrogenase stimulation by R-5020 in our T-47D cells plateaus at around 2 - 3 days of treatment, is maintained until around day 5 and then falls off with time, similar to the secreted M r 48 000 protein of Chalbos and Rochefort [4] and the insulin receptor effect of Horwitz and Freidenberg [6]. In addition to the above experiments, we have investigated hormonal specificity and the effect of inhibitors of protein and R N A synthesis on the stimulation of lactate dehydrogenase by progestin. Materials and Methods

Cell culture. T-47D cells, obtained from the American Type Culture Collection, were grown in C o m i n g plastic flasks (75 cm 2) in 5% CO 2 in air at 37 ° C. Growth medium was basal Eagles medium: modified-autoclavable (powdered) plus nonessential amino acids, 2 m M L-glutamine, 10% fetal calf serum (heat-inactivated), 100 u n i t s / m l penicillin, 1 0 0 / ~ g / m l streptomycin (Grand Island Biological Co.) and 6 n g / m l insulin (Sigma). Cells were harvested by replacing the growth medium with Hank's balanced salt solution without calcium and magnesium, but with 1 m M E D T A , incubating for 10 min at 37 ° C and centrifuging. Cells were then washed with Hanks' balanced salt solution at 4 ° C centrifuged and homogenized as below. When cells were going to be used in an experiment, approx. 2.5.10 6 cells were inoculated into each T-75 flask containing the above medium except that the fetal calf serum had been treated with dextran-coated charcoal [7]. Hormones were added in absolute ethanol, control flasks receiving absolute ethanol only, so that the ethanol concentration in experimental and control flasks was 0.1%. Cells were then grown for the times indicated in the figure legends, medium was changed as indicated. Extraction and assay of lactate dehydrogenase. Cell pellets were homogenized in 2 ml of 0.9%

NaC1 using a motor-driven, steel-shafted, teflontipped pestle and a conical ground-glass hand-held homogenizer (Kontes). The mixture was kept in ice except during the actual homogenization (10 up-and-down strokes with 30 s cooling in ice between each series of 10 strokes for a total of 30 strokes). Nuclei, etc. were then centrifuged out using an SW60Ti rotor (Beckman) in a Beckman L5-75 ultracentrifuge at 46 000 x g for 1 h at 2 o C. Lactate dehydrogenase was assayed at 340 nm using a Gilford 250 spectrophotometer and 6050 recorder with the assay mixture described by Wacker [8]. Protein concentrations were determined by the method of Lowry et al. [9]. Enzyme units are defined as micromoles of N A D H formed per minute. D N A concentrations were determined by the method of Burton [10]. Electrophoresis. For lactate dehydrogenase electrophoresis, the Beckman Paragon L D Lactate Dehydrogenase Isoenzyme Electrophoresis kit was used. This kit utilizes prepared agarose slab gels buffered at p H 8.2. Electrophoresis was performed at 100 V for 20 min. After this, the gel was soaked in substrate solution consisting of lactate and N A D to form N A D H , which in the presence of phenazine methosulfate reduces nitroblue tetrazolium chloride, yielding a blue color at the site of enzyme activity. Gels were fixed with 5% acetic acid, dried at 9 0 ° C for 15 min and scanned at 600 nm. Chemicals. Sigma Chemical Co. was the source of progesterone, testosterone, estradiol-17fl, hydrocortisone, L(+)lactic acid (grade L-I), N A D (grade V), cycloheximide and actinomycin D. R5020 (17,21-dimethyl- 19-nor-4,9-pregnadiene-3, 20-dione) was purchased from New England Nuclear. RU-38486 was a gift from Dr. R. Deraedt of Roussel-Uclaf. Results

As shown in Fig. 1, there is only one lactate dehydrogenase isozyme detectable in T-47D cells, isozyme 5, made up of four type M subunits. Under the influence of R-5020, isozyme 5 remains the only isozyme and its specific activity increases, as is also shown in Fig. 1. Fig. 2 shows the results of a time dependency experiment. The effect plateaus after 2 - 3 days of treatment, is maintained until around day 5, and

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Fig. 1. Isozyme 5, the only detectable isozyme in T-47D cells. Using the Beckman Paragon Electrophoresis System, samples of cell extracts from control (C) and 10 -8 M R-5020-treated (R) ceils and a sample of a patient's serum (P) were subjected to lactate dehydrogenase isozyme electrophoresis. 0.3 /~g of protein was applied in the cases of control and R-5020-treated extracts in a vol. of 5/al; 5/tl of patient's serum was applied. The gel was dried with a Beckman gel dryer and scanned using a Beckman Model CDS-2000 gel scanner.

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then falls off with time. The significance of this decline is an yet unclear. While the results in Fig. 2 are analyzed per mg DNA, the same phenomenon occurred when analyzed per mg protein, as in all other experiments in this report. As shown in Fig. 3, the effect is specific for progestins. We did find a similar stimulation by estradiol-17fl using two particular lots of serum. However, the effect occurred only with these two lots and none of the other lots we have tested support any effect of estradiol. All lots of serum we have tried (seven) support the effect of progesterone. In order to determine whether the increase in lactate dehydrogenase upon progestin treatment required new protein and RNA synthesis, respectively, we used cycloheximide and actinomycin D. The data of Fig. 4(A) show that cycloheximide prevents the stimulation of lactate dehydrogenase activity, suggesting that new protein synthesis is required. Cells were seeded in charcoal-stripped serum-containing medium as described in the fig-

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Fig. 2. Time dependency of lactate dehydrogenase stimulation by R-5020. After plating and maintenance in charcoal-stripped serum-containing medium for 2 days, cells were treated with 10 -9 M R-5020 for various time periods, medium was changed every 48 h. For every time point there were triplicate R-5020treated and control flasks. Control flasks received ethanol alone. Activity is in terms of u n i t s / m g DNA and data points refer to mean for triplicate flasks. Data from three separate experiments are presented.

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Fig. 3. Effects of various steroid hormones on lactate dehydrogenase in T-47D cells. After seeding and growth for 3 days in charcoal-stripped serum-containing medium, cells were treated with ethanol alone or 10 -8 M hormone in ethanol for 3 days, medium was changed every day. Data are in terms of units/rag DNA and refer to mean _+S.E. for triplicate flasks. E, estradiol17fl; R, R-5020; Pg, progesterone; T, testosterone; HC, hydrocortisone. This experiment was performed three times with similar results. * indicates statistical difference from control at P < 0.05 by Student-Newman-Keuls' multiple comparison procedure.

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Fig. 4. Effects of cycloheximide and actinomycin D on stimulation of lactate dehydrogenase. (A) CyclohexJmide: cells were seeded and grown for 2 days in charcoal-stripped serum-containing medium. Control cells and progestin-treated cells were then simply medium changed. Cycloheximide at a final concentration of 1 m M was added to the appropriate cells after 24 h of treatment with control or 10 -8 M progestin-containing medium. After 4 h of cycloheximide treatment, the m e d i u m was changed to fresh medium with or without progestin and cells were grown for 20 more hours, when the cells were harvested and assayed for lactate dehydrogenase. Spec. act. in this figure is in units per mg D N A . Data refer to mean + S.E. for triplicate flasks. This experiment was performed three times with similar results. * indicates statistical difference from all others at P < 0.01 by Student-Newman-Keuls' multiple comparison procedure. (B) Actinomycin D: 2 days after plating in charcoal-stripped serum-containing medium, actinomycin D treatment was begun in the indicated cells for 1 h. After this, medium was changed on all flasks with addition of either nothing (control), 10 - 8 M R-5020, 1 / l g / m l (8.10 -7 M) actinomycin D or 1 /~g/ml actinomycin D plus 10 -8 M R-5020. Actinomycin D was dissolved in H 2 0 and R-5020 in ethanol. Control flasks received solvents only. After treatment for 24 h, cells were harvested, extracted and assayed for lactate dehydrogenase. Data refer to m e a n + S . E , for triplicate flasks and spec. act. is in units per mg D N A . This experiment was performed three times with similar results. * indicates statistical difference from all others at P < 0.01 by StudentNewman-Keuls' multiple comparison procedure.

ure legend. Control cells and progestin-treated cells were simply medium changed every 48 h. Cycloheximide was added to the appropriate cells after 24 h of treatment with control or progestin-containing medium. After 4 h of cycloheximide treatment, the medium was changed to fresh medium with or without progestin and cells grown for 20 more hours, when the cells were harvested and assayed for lactate dehydrogenase.

Cell viability was greater than 95% and was unaffected by the cycloheximide. In order to determine whether new RNA synthesis is required for the stimulation of lactate dehydrogenase by progestin, we used actinomycin D. We have determined that RNA synthesis occurs at a level less than 5% of the control under the conditions of our experiments and that cell viability is greater than 95% and unaffected by actinomycin D (data not shown). The data of Fig. 4(B) suggest that new RNA synthesis is required for the effect of progestin on lactate dehydrogenase. We next wanted to determine the effect of the progestin antagonist RU-38486 (17fl-hydroxy-11fl(4-dimethyl-aminophenyl-1)-17a-(prop-l-ynil)-estra-4,9-dien-3-one), which has been reported to mediate its antiprogestin action through the progesterone receptor [11,12], on lactate dehydrogenase stimulation, in part as a probe into whether progestins' stimulation of lactate dehydrogenase is

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R RU RU&R C Fig. 5. Effect of the anti-progestin RU-38486. Cells were seeded in charcoal-stripped serum-containing medium and grown for 2 days. At this time medium was changed to fresh m e d i u m with either 10 - 9 M R-5020 (R), 10 -8 M RU-38486 (RU), R-5020 and RU-38486 ( R U + R), or with solvent alone (C). Solvent was 100% ethanol; final concentration in all flasks was 0.2%. Data refer to mean + S.E. of triplicate flasks. This experiment was performed three times with similar results. * indicates statistical difference from all others at P < 0.01 by Student-Newman-Keuls' multiple comparison procedure.

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occurring through the progesterone receptor. As shown in Fig. 5, RU-38486 prevents the progestin stimulation of lactate dehydrogenase. Finally, in view of the recent finding by Berthois et al. [13] that Phenol red possesses estrogenic activity at the concentrations used in tissue culture, we tested the effect of R-5020 on lactate dehydrogenase, using the same tissue culture medium as in our other experiments except the cells were without Phenol red from 3 days prior to the addition of hormone throughout the experiment. Data from four separate experiments showed that lactate dehydrogenase stimulation still occurs even in the absence of the estrogen Phenol red (not shown). Discussion

Whereas a fair amount of knowledge has been acquired about the estrogen regulation of specific proteins in human breast cancer cells in long term tissue culture, comparatively little is known about progestin effects. We are undertaking a study of the progestin regulation of lactate dehydrogenase, an easily assayable cytoplasmic protein, as a model system for regulation by progestins. This paper represents a continuation of the initial stage of this process. We find that isozyme 5, the isozyme classically found at high levels in skeletal muscle, is the only isozyme detectable in T-47D cells. This is consistent with the report of Burke et al. [3], who found only the isozyme 5 in MCF-7 cells. Lactate dehydrogenase catalyzes the conversion of pyruvate to lactate and as such is an important enzyme in cells at low oxygen tension. Since varying proportions of hypoxic cells exist in a tumor mass, lactate dehydrogenase levels may be important for their survival. A link between lactate dehydrogenase and the survival of malignant cells has been postulated, since Warburg in 1930 showed they contained high levels of lactate [14]. In fact, it is well known that lactate dehydrogenase is elevated in human neoplastic disease [15] and Savlov et al. [16] have suggested the possible use of patient tumor patterns of lactate dehydrogenase in combination with estrogen receptor analysis for selecting therapies for breast cancer patients. Hilf et al [17] showed a doubling of lactate dehydro-

genase activity in breast cancer tissue as compared to benign proliferative disease and normal breast. The isozyme 5 is particularly well suited for a role in anaerobic metabolism as it has an especially low K m for pyruvate and high Vmax for lactate formation. Perhaps progestins help some tumors survive in an environment of low oxygen tension. Stimulation of lactate dehydrogenase by R-5020 plateaus after about 2-3 days of treatment, is maintained through day 5, and then falls off with time. This pattern of elevation of a specific protein by R-5020 in T47D cells, followed by its decline during continuous R-5020 stimulation, has been observed by other authors. Chalbos and Rochefort [4] have reported progestin induction of a M r 48 000 protein which reaches a zenith at 3-4 days and then declines. Insulin receptor stimulation by R-5020 as reported by Horwitz and Freidenberg [6] follows a similar pattern. Horwitz and Freidenberg have speculated that the decline of insulin receptor levels following the rise may be associated with the release of T-47D cells from growth rate inhibitory effects of R-5020. Although we have also observed a slight inhibition of growth of T47D cells by R-5020 (data not shown), we are uncertain as to the significance of the rise/decline pattern of lactate dehydrogenase stimulation. Burke et al. [3] have reported that estrogen stimulates isozyme 5 activity in MCF-7 cells which have high levels of estrogen receptor and low levels of progesterone receptor, and that the effect is specific for estrogens. Although the effect we see in T-47D cells seems to be specific for progestins, we have on occasion, as mentioned earlier, seen an effect with estrogen that may be serum-lot dependent. In view of these facts and the fact that the anti-progestin RU-38486 inhibits the effect of R-5020 on lactate dehydrogenase activity, the data are consistent with the notion that stimulation occurs via the progesterone receptor, although they do not establish this. The cycloheximide data suggest, but do not prove, that new protein synthesis is necessary for stimulation of lactate dehydrogenase by progestins. At this point, it is not known whether the effect is dependent on synthesis of lactate dehydrogenase itself or some other protein(s) necessary for the stimulation of lactate dehydrogenase by progestin.

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The results of experiments with actinomycin D suggest that RNA synthesis is also necessary for the stimulation of lactate dehydrogenase by progestins, suggesting that the regulation is at least partly pretranslational. Although this probably means transcription of the lactate dehydrogenase gene(s) rather than other genes necessary for the lactate dehydrogenase stimulation, this has yet to be determined and these experiments are in progress. Stimulation of lactate dehydrogenase activity is not dependent on DNA synthesis, as 24 h treatment with 10 /~g/ml ara C (cytosine-fl-Darabinofuranoside) still allows progestin stimulation of lactate dehydrogenase (data not shown). The above apparent dependency on RNA and protein synthesis is consistent with the reports of Chalbos and Rochefort [4,5] on the M r 48 000 and M r 250000 proteins in T47D cells, and the report of Prudhomme et al. on 17fl-hydroxysteroid dehydrogenase in primary human breast cell cultures [18]. Finally, we have shown that the stimulation of lactate dehydrogenase occurs in the absence of the pH indicator Phenol red, which has been shown [13] to be estrogenic at the concentrations used in tissue culture. There are several recent reports of progestin-stimulated proteins in human breast cancer cell lines. However, this is, to our knowledge, the first report of this phenomenon in the absence of the estrogen Phenol red.

Acknowledgements This work was supported in part by grants CA29501 and RR05870 from the N.I.H. and by a grant from the Huntington Clinical Foundation. We thank Dr. Howard Quittner of the Marshall University Department of Pathology for use of the

lactate dehydrogenase electrophoresis system, Dr. R. Deraedt of Roussel-Uclaf for supplying us with the RU38486 and Mrs. Dolores Brumfield for typing the manuscript.

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