Regulation of growth hormone mRNA synthesis by 3,5,3'-triiodo-L-thyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells)

M. H. Kumara-Siri, Martin I. Surks

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Abstract

3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labelling with [3H]eucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehydetreated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p < 0.005 (from 13,430 ± 609 to 19,150 ± 1160 cpm/106 cells/2 h) and increased GH mRNA, p < 0.001 (from 7.2 ± 1.2 to 14.5 ± 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cells populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61% (counts/min of GH mRNA/106 cells), 49, 59, and 65% (ppm of total RNA), and 64 and 69% (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells. Furthermore, the close relationship between nuclear iodothyronine receptors and GH mRNA synthesis observed by others in asynchronous cells is not apparent in S-phase cells. Our data suggest that position in the cell cycle may mediate the activity of nuclear iodothyronine receptors as expressed by the synthesis of GH mRNA.

Original languageEnglish (US)
Pages (from-to)14529-14537
Number of pages9
JournalJournal of Biological Chemistry
Volume260
Issue number27
StatePublished - 1985

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Thyronines
Pituitary Neoplasms
S Phase
Growth Hormone
Rats
Tumors
Cells
Messenger RNA
Cytoplasmic and Nuclear Receptors
Uridine
Thyroid Hormone Receptors
Cell Cycle
Glucocorticoid Receptors
Complementary DNA
RNA
Collodion
DNA
RNA Stability
Growth
Cell growth

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{6f537c8b61cd4b20bb3f52abb90a1d4e,
title = "Regulation of growth hormone mRNA synthesis by 3,5,3'-triiodo-L-thyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells)",
abstract = "3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labelling with [3H]eucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehydetreated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p < 0.005 (from 13,430 ± 609 to 19,150 ± 1160 cpm/106 cells/2 h) and increased GH mRNA, p < 0.001 (from 7.2 ± 1.2 to 14.5 ± 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cells populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61{\%} (counts/min of GH mRNA/106 cells), 49, 59, and 65{\%} (ppm of total RNA), and 64 and 69{\%} (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells. Furthermore, the close relationship between nuclear iodothyronine receptors and GH mRNA synthesis observed by others in asynchronous cells is not apparent in S-phase cells. Our data suggest that position in the cell cycle may mediate the activity of nuclear iodothyronine receptors as expressed by the synthesis of GH mRNA.",
author = "Kumara-Siri, {M. H.} and Surks, {Martin I.}",
year = "1985",
language = "English (US)",
volume = "260",
pages = "14529--14537",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "27",

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TY - JOUR

T1 - Regulation of growth hormone mRNA synthesis by 3,5,3'-triiodo-L-thyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells)

AU - Kumara-Siri, M. H.

AU - Surks, Martin I.

PY - 1985

Y1 - 1985

N2 - 3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labelling with [3H]eucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehydetreated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p < 0.005 (from 13,430 ± 609 to 19,150 ± 1160 cpm/106 cells/2 h) and increased GH mRNA, p < 0.001 (from 7.2 ± 1.2 to 14.5 ± 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cells populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61% (counts/min of GH mRNA/106 cells), 49, 59, and 65% (ppm of total RNA), and 64 and 69% (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells. Furthermore, the close relationship between nuclear iodothyronine receptors and GH mRNA synthesis observed by others in asynchronous cells is not apparent in S-phase cells. Our data suggest that position in the cell cycle may mediate the activity of nuclear iodothyronine receptors as expressed by the synthesis of GH mRNA.

AB - 3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labelling with [3H]eucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehydetreated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p < 0.005 (from 13,430 ± 609 to 19,150 ± 1160 cpm/106 cells/2 h) and increased GH mRNA, p < 0.001 (from 7.2 ± 1.2 to 14.5 ± 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cells populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61% (counts/min of GH mRNA/106 cells), 49, 59, and 65% (ppm of total RNA), and 64 and 69% (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells. Furthermore, the close relationship between nuclear iodothyronine receptors and GH mRNA synthesis observed by others in asynchronous cells is not apparent in S-phase cells. Our data suggest that position in the cell cycle may mediate the activity of nuclear iodothyronine receptors as expressed by the synthesis of GH mRNA.

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