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NB1011 induces Ser15 phosphorylation of p53 and activates the G2/M checkpoint
Ryan W Dellinger1, Patricia L Karjian, Saskia T C Neuteboom
1NewBiotics, Inc., San Diego, CA 92121, USA.
Abstract:
NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, is a novel anti-cancer agent that selectively targets tumor cells expressing high levels of thymidylate synthase (TS), an enzyme required for DNA biosynthesis. NB1011 treatment of high-TS-expressing breast carcinoma cells (MCF7TDX) results in the induction of p53 and p21 protein levels, whereas no p53 or p21 induction is observed in the low-TS-expressing MCF7 tumor cells. Furthermore, MCF7TDX cells accumulate in the G(2)/M phase of the cell cycle in response to NB1011. In this study, the effect of NB1011 on the phosphorylation status of p53 was analyzed. We demonstrate that NB1011 treatment of various tumor cell lines expressing high TS results in the phosphorylation of p53 on Ser15, whereas this p53 phosphorylation is not observed in low-TS-expressing tumor cells. Also, we examined the role of several key cell cycle regulators in the growth inhibition observed in response to NB1011. Our results show that the mRNA and protein levels of the G(2)/M regulators cdc2, cyclin B1 and cdc25C are down-regulated in MCF7TDX cells, while unaffected in MCF7 cells. The mRNA and protein levels of 14-3-3sigma, also a direct transcriptional target of p53, are up-regulated in MCF7TDX cells following NB1011 treatment, while unchanged in MCF7 cells. Taken together, our data indicate that the growth inhibition caused by NB1011 in MCF7TDX cells is mediated through phosphorylation of p53 and activation of the G(2)/M checkpoint.
Insights
NB1011, an anti-cancer agent, selectively targets thymidylate synthase (TS)-high tumor cells. It induces p53 phosphorylation and G(2)/M cell cycle arrest, leading to growth inhibition in these cells.
Area of Science:
- Oncology
- Molecular Biology
- Cell Cycle Regulation
Background:
- NB1011 is a novel phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine.
- It acts as an anti-cancer agent targeting thymidylate synthase (TS)-expressing tumor cells.
- TS is crucial for DNA biosynthesis, making it a target for cancer therapy.
Purpose of the Study:
- To investigate the effect of NB1011 on p53 phosphorylation in tumor cells.
- To elucidate the role of cell cycle regulators in NB1011-induced growth inhibition.
- To determine the mechanism of NB1011's anti-cancer activity in high-TS-expressing cells.
Main Methods:
- Treatment of high-TS-expressing (MCF7TDX) and low-TS-expressing (MCF7) tumor cell lines with NB1011.
- Analysis of p53 and p21 protein levels.
- Cell cycle analysis to assess G(2)/M phase accumulation.
- Western blotting and RT-PCR to examine the expression of cell cycle regulators (cdc2, cyclin B1, cdc25C, 14-3-3sigma).
Main Results:
- NB1011 induced p53 and p21 protein levels and G(2)/M cell cycle arrest specifically in high-TS-expressing MCF7TDX cells.
- NB1011 treatment resulted in p53 phosphorylation at Ser15 in high-TS-expressing cells, but not in low-TS-expressing cells.
- Down-regulation of cdc2, cyclin B1, and cdc25C mRNA and protein levels, and up-regulation of 14-3-3sigma, were observed in MCF7TDX cells.
Conclusions:
- NB1011 selectively inhibits the growth of high-TS-expressing tumor cells.
- The anti-cancer effect of NB1011 is mediated by p53 phosphorylation and subsequent activation of the G(2)/M cell cycle checkpoint.
- NB1011 represents a promising therapeutic agent for cancers with high thymidylate synthase expression.