J Korean Med Sci.  2008 Oct;23(5):825-832. 10.3346/jkms.2008.23.5.825.

Impaired Delta NP63 Expression is Associated with Poor Tumor Development in Transitional Cell Carcinoma of the Bladder

• He Y
• Wu X
• Tang W
• Tian D
• Luo C
• Yin Z
• Du H

• KMID: 1783068
• DOI: http://doi.org/10.3346/jkms.2008.23.5.825

Abstract

The oncogenic isoform of the p63 protein, delta NP63, plays an important role in the pathogenesis of many epithelial carcinomas, and emerging evidences suggest that delta NP63 is a promising drug target. However, the functions of delta NP63 in transitional cell carcinoma of bladder (TCCB) are poorly defined. In this study, a delta NP63 shRNA expression vector was transfected into TCCB cell line 5637 and cell cycling, cell proliferation and protein expression were assessed by flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-dimethyl tetrazolium bromide (MTT) assay, and immunohistochemistry, respectively. The delta NP63 shRNA expression vector was also injected into 5637 cell xenograft tumors in nude mice, and tumor size was measured, tumor tissue morphology was assessed by immunohistopathology and transmission electron microscopy. In the in vitro study, delta NP63 shRNA transfection caused successful delta NP63 gene silencing and resulted in significant arrest of cell cycling and cellular proliferation (p<0.05) as well as cyclin D1 expression. In the nude mouse xenograft model, delta NP63 shRNA greatly inhibited tumor growth, induced tumor cell apoptosis (p<0.05) and resulted in cyclin D1 downregulation. Our data suggest that delta NP63 may play an oncogenic role in TCCB progression through promoting cell survival and proliferation. Intratumoral administration of delta NP63-specific shRNA suppressed tumor delta NP63 expression and cellular proliferation while promoted tumor cellular apoptosis, and therefore inhibited tumor growth and improved survival of xenograft-bearing mice, which was not accompanied by significant signs of systemic toxicity.

Keyword

Delta N p63; RNA Interference; Short Hairpin RNA; Urinary Bladder Neoplasms

MeSH Terms

Animals
Carcinoma, Transitional Cell/*genetics/metabolism
Cell Line, Tumor
Cell Proliferation
Cyclin D1/biosynthesis
Disease Progression
Female
Humans
Mice
Mice, Nude
Microscopy, Electron, Transmission
Models, Biological
Neoplasm Transplantation
Trans-Activators/*biosynthesis/*physiology
Tumor Suppressor Proteins/*biosynthesis/*physiology
Urinary Bladder Neoplasms/*genetics/metabolism

Figure

• 

  Fig. 1 The mRNA expression of ΔNp63 in 5637 cells 48 hr post-transfection, assayed by semiquantitative RT-PCR. (A) Products were analyzed on a 1.5% agarose gel. The 335 bp band is ΔNp63 mRNA. The 302 bp band is β-actin serving as the internal control. (B) Quantified expression levels of ΔNp63 as normalized to the β-actin level. *Statistically significant (p<0.05, mean±SD, n=10) relative to control shRNA.

• 

  Fig. 2 Expression of ΔNp63 protein in TCCB 5637 cell line was assayed by immunocytochemistry at 48 hr post-transfection with the indicated vector or medium (original magnification, ×200).

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  Fig. 3 Cell cycle progression of 5637 cells following different treatments as indicated. (A) Flow cytometry graphs. (B) Quantification of the percentages of cells in G0/G1 phase and S phase. *Statistically significant (p<0.05, mean±SD, n=10) relative to control shRNA.

• 

  Fig. 4 Proliferation curve of 5637 cells treated with pΔNp63-shRNA, control-shRNA or PBS over 120 hr. Measured by MTT assay. Data were mean±SD. n=10 per data point.

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  Fig. 5 Cyclin D1 protein expression in TCCB 5637 cell line at 48 hr post-transfection of ΔNp63 specific or control shRNA or PBS. Assayed by immunocytochemistry (original magnification, ×200).

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  Fig. 6 Cyclin D1 protein expression in TCCB 5637 xenograft tumors from nude mice treated with PBS, control shRNA vector or ΔNp63 specific shRNA vector. Tumor tissues were taken at the end of the experiment and assayed by immunocytochemistry (original magnification, ×200).

• 

  Fig. 7 Tumors were taken at 8 weeks from treatment from mice treated with pΔNp63-shRNA (A1), pΔNp63-cRNA (A2) or PBS (A3) and their sizes were measured and shown in B. *Statistically significant (p<0.05, mean±SD, n=6) relative to pΔNp63-cRNA group.

• 

  Fig. 8 Transmission electron microscopic examination of TCCB xenograft tumor tissues. Tumor tissues were from mice treated with pΔNp63-shRNA (A, B), pΔNp63-cRNA A (C) or PBS (D). (A) Tumor tissues treated with pΔNp63-shRNA; the arrows indicate the apoptotic cells. (B) Tumor tissues treated with pNp63-shRNA; the arrow indicate the inflammatory cells. (C) Tumor tissues treated with pΔNp63-cRNA. (D) Tumor tissues treated with PBS.

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