Abstract
T47D cells represent an estrogen-responsive human ductal carcinoma cell line which expresses detectable levels of estrogen receptor (ER). We have previously shown that estradiol (E2) treatment of T47D cells causes an increase in the level of p53 and a concomitant phosphorylation of retinoblastoma protein (pRb). In the present study, we have analysed the expression of p53 and phosphorylation state of pRb and compared the effects of E2 and triiodothyronine (T3) on these phenomena. Cells were grown in a medium containing charcoal-treated serum to deplete the levels of endogenous steroids. Upon confluency, the cells were treated with T3 (10−12 to 10−7 M) for 24 h and the presence of p53 and pRb was detected by Western analysis. E2 treatment of cells caused a 2–3-fold increase in the level of p53. Presence of T3 in the medium caused a gradual increase in the level of p53 in a concentration-dependent manner. Under the above conditions, pRb was phosphorylated (detected as an upshift during SDS–PAGE) in the presence of E2 and T3. Supplementation of growth medium with T3 (1 μM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this effect was maintained for up to 12 h. When ICI 164 384 (ICI) (1 μM), an ER antagonist, was combined with E2 (1 nM) or T3 (1 μM), effects of hormones on cell proliferation and hyperphosphorylation of pRb were blocked. Western analysis of p53 was supplemented with its cytolocalization by immuno-labeling using laser scanning confocal fluorescence microscopy, which revealed an ICI-sensitive increase in the abundance of p53 in hormone-treated cells. Steroid binding analysis revealed lack of competition by T3 for the [3H]E2 binding. These results indicate that T3 regulates T47D cell cycle progression and proliferation raising the p53 level and causing hyperphosphorylation of pRb by a common mechanism involving ER and T3 receptor (T3R)-mediated pathways.
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Abbreviations
- ATCC:
-
American type culture collection
- E2:
-
17-β estradiol
- ECL:
-
enhanced chemiluminescence
- ERE:
-
estrogen response element
- FBS:
-
fetal bovine serum
- ER:
-
estrogen receptor
- HBSS:
-
Hanks' balanced salt solution
- HSS:
-
high speed supernatant
- ICI:
-
ICI 164 384
- OHT:
-
4-hydroxy tamoxifen
- p53:
-
53-kDa tumor suppressor protein
- PBS:
-
phosphate-buffered saline
- PMSF:
-
phenylmethylsulfonyl fluoride
- PVDF:
-
polyvinylidene fluoride
- PR:
-
progesterone receptor
- pRb:
-
retinoblastoma protein
- ppRb:
-
phosphorylated pRb
- Rb:
-
retinoblastoma
- SDS–PAGE:
-
sodium dodecyl sulfate polyacrylamide gel electrophoresis
- SSFBS:
-
single stripped fetal bovine serum
- T3:
-
triiodothyronine
- TAM:
-
tamoxifen
- T3R:
-
thyroid hormone receptor
- TRE:
-
thyroid hormone response element.
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Acknowledgements
The studies were supported in part by National Institutes of Health, and the Research Excellence Fund, Center for Biomedical Research, Oakland University.
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Dinda, S., Sanchez, A. & Moudgil, V. Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells. Oncogene 21, 761–768 (2002). https://doi.org/10.1038/sj.onc.1205136
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DOI: https://doi.org/10.1038/sj.onc.1205136
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