p53 regulates PAPP-A levels by binding to the PAPP-A promoter.

<p>A) The mammary glands from wild type, pregnant (wild type) MMTV-Skp2B and tumors were analyzed for the mRNA levels of PAPP-A by quantitative RT-PCR. B) The level of IGFBP-4 in the mammary gland of wild type mice that are either virgin, pregnant, lactating or involuting was determined by western blot. C) The estrogen receptor null breast cancer cells MDA-MB-157 were transfected with 3 ug of estrogen receptor plasmid and D) after 24 hours cells were harvested for analyzing the expression of the estrogen receptor and mRNA levels of PAPP-A by quantitative RT-PCR. E) The p53 null MDA-MB157 cells were transfected with 2 µg of wild type p53 and PAPP-A mRNA levels determined. F) The level of expression of transfected wild type p53 was determined by western blot. G) MDA-MB 157 cells were transfected with 2 µg of different mutants of p53 (p53–143 and p53–248), 24 hours after, cells were harvested for analysis of PAPP-A mRNA. H) Western blot showing the expression of mutant p53. Tubulin was used as a loading control. I) p53 binding site on PAPP-A promoter. MDA-MB157 cells transfected with 2 µg of wild type and mutant p53 constructs. After 24 hours of transfection cells were fixed with formaldehyde and collected for chromatin immunoprecipitation (ChIP) using the p53 (DO-1) antibody. Normal mouse IgG served as a negative control.