Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...

Interstitial Cystitis Antiproliferative Factor Modulates MDM2,
Leading to P53 Stability
Nichole Jones
Mentor: Jayoung Kim, PhD
Children’s Hospital Boston
Objectives: Interstitial cystitis (IC) Antiproliferative Factor (APF) has been reported
as a sialoglycopeptide urinary biomarker of IC, a chronic syndrome of unknown
etiology with variable symptoms including pelvic and or perineal pain, urinary frequency,
and urgency. We previously reported that APF suppressed the proliferation
rate of normal bladder epithelial cells through a mechanism that involves increased
levels of p53 and activated signal pathways (Kim et al. FEBS Lett. 581:3795, 2007;
Kim et al. BJU Int. 103(4):514, 2009). The goal of present study is to understand
the process whereby p53 expression level elicits cell growth arrest in response to APF.
Methods: We performed MTT-based proliferation assay to measure APF effects on
growth of T24 human bladder cells. We engineered the expression levels of p53 and
the murine double minute 2 E3 ubiquitin ligase (MDM2) by transient transfection
with silencing RNAs or by the use of overexpression constructs. Various biochemical
experiments including Immunofluorescence Staining, western blot analysis and immunoprecipitation
were used. Results: APF treatment significantly decreased protein
level of MDM2 within 2 hours. Time course experiments showed that p53 level was
enhanced when MDM2 expression was suppressed. Inhibition of proteasomal activity
enhanced the APF-induced p53 increase. MDM2 bound to p53 and triggered
ubiquitination and degradation of p53. Furthermore, APF attenuated the association
of p53/MDM2 and delayed p53 degradation. Conclusions: We demonstrate that
MDM2, a negative regulator of p53 expression, is functionally attenuated by APF
in bladder epithelial cells. These data suggest that targeting MDM2 or p53/MDM2
complex formation may be relevant in the development of novel therapeutic approaches
to IC.