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Downregulation of VPAC1R expression in breast cancer cell lines
B Madsen1, B Georg, M W Madsen
1Department of Clinical Biochemistry, Bispebjerg University Hospital, DK-2400 Copenhagen, NV, Denmark.
Abstract:
The breast carcinoma cell line T47D was tested for 17 beta-estradiol (E2) mediated regulation of vasoactive intestinal polypeptide receptor type-1 (VPAC1) expression. E2 was found to downregulate the mRNA level. The number of VIP binding sites was reduced 66% on treatment with E2 for 72 h. Experiments with cycloheximide suggested that the effect was independent (at least partly so) of protein synthesis. Experiments with the transcriptional inhibitor, actinomycin D, showed that E2 did not influence the VPAC1 mRNA halflife. Both of two antiestrogens, ICI 182,780 and 4-hydroxy-tamoxifen, mediated a concentration dependent inhibition of the effect of E2 on the mRNA level. Transient transfection with reporter-gene constructs containing various portions of the VPAC1 5'-flanking sequence revealed the most proximal 100 bp to be essential for the basal transcriptional activity. However, E2 did not influence the expression of the reporter gene using up to 3,250 bp of the VPAC1 5'-flanking region.
Insights
17 beta-estradiol (E2) downregulates vasoactive intestinal polypeptide receptor type-1 (VPAC1) mRNA in breast cancer cells. This effect appears independent of protein synthesis and does not alter mRNA stability.
Area of Science:
- Endocrinology
- Molecular Biology
- Cancer Research
Background:
- Vasoactive intestinal polypeptide receptor type-1 (VPAC1) plays a role in various cellular functions.
- The regulation of VPAC1 expression by 17 beta-estradiol (E2) in breast cancer cells is not fully understood.
- T47D cells, a human breast carcinoma line, are often used to study estrogenic effects.
Purpose of the Study:
- To investigate the effect of 17 beta-estradiol (E2) on VPAC1 expression in T47D breast carcinoma cells.
- To elucidate the mechanisms underlying E2-mediated regulation of VPAC1.
- To assess the role of protein synthesis and mRNA stability in this regulatory process.
Main Methods:
- Quantitative analysis of VPAC1 mRNA levels following E2 treatment.
- Measurement of VIP binding sites to determine receptor expression.
- Use of cycloheximide to assess protein synthesis involvement.
- Application of actinomycin D to evaluate mRNA halflife.
- Reporter-gene assays with VPAC1 5'-flanking sequences to study transcriptional regulation.
- Treatment with antiestrogens (ICI 182,780 and 4-hydroxy-tamoxifen) to investigate estrogen receptor-mediated effects.
Main Results:
- E2 significantly downregulated VPAC1 mRNA levels in T47D cells.
- A 66% reduction in VIP binding sites was observed after 72 hours of E2 treatment.
- The E2-induced downregulation was partly independent of new protein synthesis.
- E2 did not affect the halflife of VPAC1 mRNA.
- Antiestrogens inhibited the E2-mediated downregulation in a concentration-dependent manner.
- Reporter-gene assays indicated that the proximal 100 bp of the VPAC1 5'-flanking region are crucial for basal transcription, but E2 did not modulate reporter gene expression within this region.
Conclusions:
- 17 beta-estradiol negatively regulates VPAC1 expression in T47D breast cancer cells at the mRNA level.
- The downregulation mechanism appears to involve post-transcriptional or translational events rather than direct transcriptional activation or mRNA stabilization.
- Estrogen receptor antagonists can counteract the effect of E2 on VPAC1 expression, suggesting an estrogen receptor-dependent pathway.