AN IN VITRO TEST SYSTEM FOR THYROID HORMONE ACTION
Andrea Waltenberger, Sabine Kallenda and Otmar Hohenwarter
Institute of Applied Microbiology, University of Agriculture, Vienna/AUSTRIA
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Introduction
Thyroid hormones are essential for normal growth and development and exert profound effects on cellular metabolism in almost all organs. A number of rat pituitary tumor cell lines respond to physiological concentrations of L-triiodothyronine (T3) by increased cell proliferation und induction of growth hormone secretion (Samuels et al.,1973; Hinkle et al.,1986).
For long time investigations of the mechanism of action of T3 in vitro at physiological concentrations have not been successful because unless one depletes T3 and T4 from serum biological effects of thyroid hormones would always be correlated with hormone concentrations in serum. Using serum obtained from thyroidectomized cows Samuels et al.,(1973) demonstrated the dependence of GH1 pituitary tumor cells on thyroid hormone for growth. Previous studies with GH3 cells, a functional rat pituitary cell line which generates growth hormone as well as prolactin and depends on thyroid hormone for growth (Sato et al., 1976), established that these cells survived passage into serum-free culture only after a period of adaptation and then rapidly progressed to hormone-autonomy (Riss et al.,1989).
This work presents an in vitro test system using GH3 cells in defined serum-free medium, where the addition of L- triiodothyronine stimulates cell division as well as cell activation in a dose dependent manner.
Materials and methods
GH3 cells were obtained from the American Type Culture Collection. The cells were grown at 37oC in a humid atmosphere of 7% (v/v) CO2 in HAM's F12:DMEM supplemented with 10% (v/v) fetal bovine serum and passaged at every fourth day by trypsinizing. From these stock cell cultures serum-free cell growth assays were performed as described in test procedure. The serum-free medium PCM has been described (Sirbasku et al., 1991). Removal of thyroid hormones from fetal bovine serum (stripped serum) was performed by treatment with BIO-RAD AG-1-X8 resin as described by Sirbasku et al.,1991.
MTT-test was performed as described in principle by Mosmann,1983. Determination of cellular DNA content was determined by using a modification of the fluorometric DNA assay described by Rymaszewski et al., 1990. The measurement of lactat dehydrogenase activity present in the cytoplasm of intact cells was performed using the CytoTox 96 TM assay (Promega).
Test procedure
GH3 cells washed three times in serum-free PCM - medium
inoculation: 2500 cells/well into 96-well-plates
addition of different concentrations of T3
incubation at 37oC for 4 days
MTT-test for measuring cell proliferation and activation
Results and discussion
The effect of T3 on GH3 cells at various concentrations is shown in fig. 2a. The minimum T3 concentration which induced a detectabel response was about 10pM. Maximal cell activation is found at a concentration of 1nM T3. The variation of the maximum T3 response, observed in 10 assays performed on different days, was between a 2,5-fold and a 3,5-fold increase of the MTT-signal. The T3 response was also influenced by the plating density of the cells. Using 10000 cells/well instead of 2500 resulted in a lower increase of the MTT signal.
In order to determine if there was an immediate response to T3, MTT-reduction was measured at different timepoints after addition of T3. Within the first 24 hours of incubation no increase in cell activity was detected. A difference in MTT- or LDH-signal between cultures with and without T3 could be obtained only after incubations longer than 24 hours (data not shown).
A comparison between the MTT-signal at different T3 concentrations in serum-free medium and the corresponding LDH- and DNA-measurement is shown in fig. 2b. Enzyme induction was much higher than increase of DNA. Whereas DNA concentration were elevated by about 170% due to effective concentrations of T3, the MTT- and LDH-signals reached a 2,5-fold value suggesting, that cellular dehydrogenases were much more active in T3 stimulated cells.
As previously reported 5,5'-diphenylhydantoin (DPH) inhibits total cellular and specific nuclear T3 binding by cultured GC cells (Gingrich et al., 1985). To determine whether DPH was also effective in our test system MTT- reduction was measured in the presence of DPH. Whereas DPH didn't inhibit GH3 cell growth in PCM medium at all, the stimulatory effect of T3 was decreased dramatically. The addition of 200µM DPH resulted in a 62% decrease in MTT-reduction at 1nM T3 (fig. 2c). To determine whether there is a competitive inhibition of T3 binding by DPH we tried to abolish the DPH-induced decrease in MTT-reduction by increasing T3 concentrations in the media. No augmentation of T3 could make the DPH-induced decrease reversible (data not shown).
In our test system GH3 cells are cultivated in serum containing medium and are then examined under serum-free chemically defined conditions for their specific response to different concentrations of T3 . The use of MTT-reduction for correlating the dose dependent growth promoting effect of T3 on GH3 cells provides a convenient system for studying the regulatory effects of physiological concentrations of thyroid hormone on mammalian cells.
The effect of different media on cell morphology of GH3 cells.
a) DMEM-HAM's F12 supplemented with 10% FCS
b) DMEM-HAM's F12 supplemented with 5% FCS (stripped serum)
c) serum-free PCM-medium without T3
d) serum-free PCM-medium supplemented with 0,1nM T3
GH3 cells obtained from the ATCC were cultivated in medium containing 10% FCS. They could easily be adapted to the serum-free medium PCM when containing 0,1nM T3. The adaptation from serum-containing to serum-free conditions was accompanied by morphological changes and a reduction of attachment (fig. 1). Serum-free cultures didn't need to be trypsinized for splitting any more. When T3 was omitted from the culture medium, either by using stripped serum or PCM without T3, in addition to changes in morphology cell division was reduced dramatically. GH3 cells cultivated in medium containing 10% FCS could be used for T3-testing for about 100 population doublings without any loss in sensitivity as well as cells grown in PCM medium containing 0,1nM T3. GH3 cells didn't show the problem of rapidly progressing to hormone-autonomy after adaptation to serum-free cultivation as it has been observed by others (Riss et al., 1989).
Figure 2
a) Dose-response-relationship of triiodothyronine concentration to MTT-reduction.
GH3 cells were inoculated at a density of 2500 cells/well into 96-well-plates in PCM medium with various amounts of T3. After 4 days of incubation at 37oC the MTT-test for measuring cellproliferation and activation was performed. Data points represent averages of 8 measurements.
b) Effect of triiodothyronine on DNA content and LDH activity of GH3 cells compared to MTT-reduction induced.
c)Influence of different concentrations of 5,5'-diphenylhydantoin on GH3 growth.
References
Gingrich SA, Smith PJ, Shapiro LE, Surks MI (1985) Endocrinology 116, 2306-2313
Hinkle PM, Kinsella PA (1986) Science 234, 1549-1552
Mosmann T (1983) J. Immunol. Methods 65, 55
Riss TL, Ogasawara M, Karey KP, Danielpour D, Stewart BH, Sirbasku DA (1989) In Vitro Cell. Dev. Biol. 25, 127-135
Rymaszewski Z, Abplanalp WA, Cohen RM Chomczynski P (1990) Anal. Biochem. 188, 91-96
Samuels HH, Tsai JS, Cintron R (1973) Science 181, 1253-1256
Sato GH, Hayashi I (1976) Nature 259, 132-134
Sirbasku DA, Pakala R, Sato H, Eby JE (1991) Biochemistry 30, 7466-7477
Acknowledgement
This work was supported by Empire Pharmaceutical & Polymun Scientific Immunbiologische Froschung GmbH
5.95/96 C by IAM