TY - JOUR
T1 - Involvement of protein kinase C in the transcriptional regulation of ornithine decarboxylase gene expression by 12-O-tetradecanoylphorbol-13-acetate in T24 human bladder carcinoma cells
AU - Hsieh, J. T.
AU - Verma, Ajit K.
N1 - Funding Information:
‘This work was supported in part by the United States Public Health Service Research Grant CA-35368 from the National Cancer Institute and Institutional Grant IN-35-28-l from the American Cancer Society. A preliminary report of this work was presented at the annual meeting of Federation Association of Biological Chemists (63). J. T. Hsieh was a recipient of a merit scholarship for Ph.D. studies from the Ministry of Education, Republic of China. r To whom correspondence should be addressed. 3 Abbreviations used: TPA, 12-O-tetradecanoyl-
PY - 1988/4
Y1 - 1988/4
N2 - We have analyzed the molecular mechanisms involved in ornithine decarboxylase (ODC) induction by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in T24 cells, an easily manipulable human epithelial cell line. The addition of as low as 10-9m TPA to T24 cells, cultured in a serum-free medium, resulted in ODC induction, with peak ODC activity occurring at about 6 h after TPA treatment. The induction of ODC activity correlates with the steady-state levels of ODC mRNA increased by TPA in T24 cells. TPA treatment did not elicit any change in the size (2.1 kb) of ODC mRNA as determined by Northern blot analysis by hybridization to nick-translated 32P-labeled cDNA prepared from either mouse or human ODC mRNA. Using the DNA-excess filter hybridization technique, we found that increased steady-state levels of ODC mRNA after TPA treatment may be the result of enhanced accumulation of newly synthesized ODC mRNA. The magnitude of the induction of ODC activity was proportional to the amount of ODC mRNA synthesis caused by TPA. In a pulse-chase experiment, we failed to detect any difference in the half-life of ODC mRNA in T24 cells after treatment with either TPA or the vehicle ethanol; the half-life of ODC mRNA was about 6 h in both cases. Furthermore, as determined by the "nuclear runoff transcription assay," the rate of transcription of ODC-gene was increased by treatment of T24 cells with TPA. These results provide direct evidence of the role of transcription-initiation in ODC-gene expression. The examination of the role of protein kinase C in ODC-gene transcription revealed that TPA or diacylglycerol did not induce the synthesis of ODC mRNA in PKC-deficient T24 cells. Taken together these results indicate that the TPA-increased synthesis of steady-state levels of ODC mRNA in T24 cells may be mediated by protein kinase C and is regulated at the transcriptional level.
AB - We have analyzed the molecular mechanisms involved in ornithine decarboxylase (ODC) induction by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in T24 cells, an easily manipulable human epithelial cell line. The addition of as low as 10-9m TPA to T24 cells, cultured in a serum-free medium, resulted in ODC induction, with peak ODC activity occurring at about 6 h after TPA treatment. The induction of ODC activity correlates with the steady-state levels of ODC mRNA increased by TPA in T24 cells. TPA treatment did not elicit any change in the size (2.1 kb) of ODC mRNA as determined by Northern blot analysis by hybridization to nick-translated 32P-labeled cDNA prepared from either mouse or human ODC mRNA. Using the DNA-excess filter hybridization technique, we found that increased steady-state levels of ODC mRNA after TPA treatment may be the result of enhanced accumulation of newly synthesized ODC mRNA. The magnitude of the induction of ODC activity was proportional to the amount of ODC mRNA synthesis caused by TPA. In a pulse-chase experiment, we failed to detect any difference in the half-life of ODC mRNA in T24 cells after treatment with either TPA or the vehicle ethanol; the half-life of ODC mRNA was about 6 h in both cases. Furthermore, as determined by the "nuclear runoff transcription assay," the rate of transcription of ODC-gene was increased by treatment of T24 cells with TPA. These results provide direct evidence of the role of transcription-initiation in ODC-gene expression. The examination of the role of protein kinase C in ODC-gene transcription revealed that TPA or diacylglycerol did not induce the synthesis of ODC mRNA in PKC-deficient T24 cells. Taken together these results indicate that the TPA-increased synthesis of steady-state levels of ODC mRNA in T24 cells may be mediated by protein kinase C and is regulated at the transcriptional level.
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U2 - 10.1016/0003-9861(88)90195-6
DO - 10.1016/0003-9861(88)90195-6
M3 - Article
C2 - 3355171
AN - SCOPUS:0023887311
SN - 0003-9861
VL - 262
SP - 326
EP - 336
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -