Abstract
Tumor suppressor p53 induces the cellular response to DNA damage mainly by regulating expression of its downstream target genes. The human securin is an anaphase inhibitor, preventing premature chromosome separation through inhibition of separase activity. It is also known as the product of the human pituitary tumor-transforming gene, pttg, a proto-oncogene. Here we report that the expression of human securin is suppressed in cells treated with the DNA-damaging drugs doxorubicin and bleomycin. This suppression requires functional p53. Analysis of the human securin promoter reveals that DNA-binding sites for Sp1 and NF-Y are both required for activation of securin expression; however, only the NF-Y site is essential for the suppression by p53. Our study indicates that securin is a p53 target gene and may play a role in p53-mediated cellular response to DNA damage.
Highlights
The securin proteins are a family of functional homologues, including the Pimples protein in Drosophila, Cut2 in fission yeast, Pds1 in budding yeast, and vSecurin in vertebrates [1,2,3,4]
To investigate whether vSecurin plays a role in the cellular response to DNA damage as its homologue Pds1 does in yeast, we examined its expression in several human cancer cell lines after treatment with the chemotherapeutic drugs doxorubicin (Dox)1 and bleomycin (Blm), both of which induce strand breaks in DNA [17, 18]
The securin level was significantly lower in the Dox- and Blm-treated U2OS and HCT116 cells, despite that the majority of the treated cells were arrested at G2 (Fig. 1B), indicating that securin expression was suppressed in cells treated with DNA-damaging drugs
Summary
Plasmid Constructs—The wild-type p53 (pCMVp53wt) and dominant negative mutant p53 (pCMVp53mt135) expression vectors were obtained from Clontech Laboratories (Palo Alto, CA). A DNA fragment encoding the influenza hemagglutinin (HA) epitope was fused to the 5Ј-end of the p53mt135 coding region using PCR to generate an HA-tagged p53mt135 expression construct, pCMVHAp53mt135. The control plasmid pCMV expressing -galactosidase was obtained from Clontech. For p-710Luc, a DNA fragment containing the human securin promoter region from Ϫ710 to ϩ45 was isolated from p(Ϫ711/ ϩ201)-Luc and subsequently subcloned into pGL3-basic vector (Promega, Madison, WI). The site-specific mutations in Sp1-binding sites and CCAAT boxes were generated by PCR-based site-directed mutagenesis. For each site-specific mutation, a pair of primers was designed to carry the desired mismatched nucleotides according to the promoter sequences (see below), and the reverse primer was phosphorylated at its 5Ј-end. PCR was performed with p-710Luc as the template using a Takara LA TaqTM Polymerase kit (Panvera, Madison, WI) under the
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