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IS Audo, SR Darjatmoko, CL Schlamp, DM Albert, RW Nickells; Increase of P53 and P21 Expression by Vitamin D Analogs on Human Retinoblastoma Xenograft in Athymic Mice . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2601.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Treatment with vitamin D analogs increases tumor cell death by apoptosis in human retinoblastoma xenografts grown in athymic mice. The mechanism by which vitamin D activates apoptosis in these cells is not well understood. Previously (ARVO Abstract 2000), we used immunohistochemistry to show an increase in p53 and p21 but no change in Bax expression in tumors treated with vitamin D analogs. To compliment these qualitative observations, we have been quantifying changes in the mRNA levels of p53, p21 and Bax. Methods: Athymic mice were injected subcutaneously with Y79 human retinoblastoma cells and, after five days, treated intraperitoneally 5 times a week for 5 weeks with either mineral oil (control) or vitamin D analogs [calcitriol 0.05µg and 1,25-(OH)2-16-ene-23yne vitamin D3 (16,23-D3) 0.5µg] before euthanasia. The tumors were then harvested and frozen for total RNA extraction by the phenol/chloroform method. Short cDNA clones corresponding to human actin (control), Bax, p53, and p21 were first cloned from tumor samples by PCR. Changes in mRNA levels were initially examined by RT-PCR. Template first strand cDNA was obtained for each tumor by reverse transcription. The amount of template cDNA for PCR was normalized by measuring the incorporation of a-32P-dCTP in the RT step. Equal amounts of cDNA were then used for the PCR reaction with specific primers. Changes in levels of PCR generated products were quantified by Southern blotting using labeled cDNA probes to the target sequence. The level of p53, p21, and Bax were normalized to actin levels. Data from the RT-PCR experiments are being corroborated using Northern blotting and RNase protection analysis. Results: The results from the RT-PCR study show an increase of p53 mRNA after treatment with both vitamin D analogs. This increase appeared very early in the course of the treatment (after 3 weeks of treatment) and is more pronounced with 16-23 D3. The expression of p21 was also increased by vitamin D analogs. This increase was detected by 3 weeks in tumors treated with16-23 D3, and after 5 weeks in tumors treated with calcitriol. No change was detected in the expression of Bax with either treatment. Conclusion: The analysis of mRNA expression from human retinoblastoma cells xenograft in athymic mice show that p53 and p21 are good candidates to explain the antitumoral effect of vitamin D analogs.
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