p53 mediates interstitial cystitis antiproliferative factor (APF)-induced growth inhibition of human urothelial cells

Abstract

Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis, a urinary bladder disorder of unknown etiology that is characterized by chronic pelvic pain. The present study was directed toward uncovering a pathway through which APF signals. Treatment of human urothelial cells with native APF resulted in growth inhibition accompanied by blockade of cell cycle transit and increased p53. Reduced expression of p53 by RNA interference diminished, while ectopic expression of p53 mimicked, the effects of APF. These are the first findings implicating the network of p53 target genes in urothelial defects associated with interstitial cystitis.

Fig. 1

APF effect on cell proliferation and cell cycle progression. (A) Human urothelial cells were incubated in media containing 10 ng/ml APF or Mock APF control. At the indicated time points, cell number was counted by MTT assay. (B) FACS analysis was performed to determine the cell cycle transit 48 h after treatment.

Fig. 2

Increased p53 protein expression in response to APF. (A) Whole cell lysates were prepared from urothelial cells treated with 10 ng/ml APF or Mock APF for 7 d. Western blot analysis was performed using anti-p53 and GAPDH Abs. (B) Cells were stained with anti-p53 Ab (red), and nuclei were counterstained with DAPI (blue).

Fig. 3

Reversal of the APF effect by p53 knockdown. Urothelial cells were transiently transfected with p53 siRNA duplex and incubated in media containing 10 ng/ml APF or Mock APF (control) for 7 days. Cell number was determined by MTT analysis. The protein levels of p53 and p21Cip1/WAF1 under individual experimental conditions were determined by Western blot.

Fig. 4

Inhibition of T24 bladder cancer cell growth by APF. (A) T24 cells were serum-starved and treated with 10 ng/ml APF or Mock APF (control), followed by cell number determination by MTT assay at the indicated time points. (B) Serum-starved T24 cells were stimulated with APF or Mock APF in the presence of serum. Three days later, FACS analysis was performed to assess the cell cycle.

Fig. 5

Effects of altered p53 level. (A) T24 cells were transiently transfected with p53 or vector only. Cell counting was performed 7 days after treatment by MTT assay. p53 levels were determined by Western blotting. (B) p53 in T24 cells was knocked down using siRNA. Following 10 ng/ml APF or Mock APF treatment for 7 d, the cells were counted, and p53 and p21Cip1/WAF1 protein levels were assessed.