Functional significance of the hepaCAM gene in bladder cancer

Abstract

Background: The hepaCAM gene encodes a new immunoglobulin-like cell adhesion molecule, and its expression is suppressed in a variety of human cancers. Additionally, hepaCAM possesses properties often observed in tumor suppressor genes. However, the expression and biological function of hepaCAM has not been investigated in bladder cancer. Therefore we sought to examine hepaCAM expression and the relationship between its structure and function in human transitional cell carcinoma of bladder (TCCB).

Materials and methods: HepaCAM expression was evaluated in 28 normal and 34 TCCB bladder specimens and 2 TCCB cell lines using semi-quantitative RT-PCR. The wild-type hepaCAM and the extracellular domain-truncated mutant gene were transfected into the TCCB cell line T24, and the biological properties of both the wild-type gene and the domain-truncated mutant were then assessed.

Results: HepaCAM expression was down-regulated in 82% (28/34) of TCCB specimens and undetectable in the 2 TCCB cell lines tested. The localization of hepaCAM appeared to be dependent on cell density in T24 cells. In widely spread cells, hepaCAM accumulated on the perinuclear membrane and the cell surface protrusions, whereas in confluent cells, hepaCAM was predominantly localized at the sites of cell-cell contacts on the cell membrane. Functionally, hepaCAM expressed not only increased cell spreading, delayed cell detachment, enhanced wound healing and increased cell invasion; it also inhibited cell growth (P < 0.01). When the extracellular domain was deleted, the localization of hepaCAM was significantly altered, and it lost both its adhesive function and its influence on cell growth.

Conclusions: HepaCAM is involved in cell adhesion and growth control, and its expression is frequently silenced in TCCB. The extracellular domain of hepaCAM is essential to its physiological and biological functions.

Figure 1

The expression of hepaCAM gene as determined by semi-quantitative RT-PCR. Products were analyzed on a 1.5% agarose gel. The 461-bp band is the wild-type hepaCAM gene. Line 1 is the marker. Lines 2 to 5 exhibit the expression of hepaCAM gene in normal bladder tissue specimens, TCCB specimens, T24 cells, and BIU-87 cells(A). Quantified expression levels of hepaCAM normalized to β-actin expression levels. Groups 1 to 4 exhibit hepaCAM gene expression levels in normal bladder tissue specimens, TCCB specimens, T24 cells, and BIU-87 cells(B).

Figure 2

Subcellular localization of hepaCAM and its extracellular domain truncation mutant in T24 cells. In well-spread cells, hepaCAM and its extracellular domain truncation mutant were localized on the perinuclear membrane and at the tip of cell surface protrusions. [B, C (white arrows indicate the localization of wild-type hepaCAM and its mutant), A is the control group]. In confluent cells, hepaCAM predominantly accumulated at the sites of cell-cell contacts on the cell membrane (E). However, in confluent cells, hepaCAM-mt1 did not accumulate at the sites of cell-cell contact, but instead localized on the cell membrane non-specifically (F, white arrows indicate localization of hepaCAM-mt1). D is the control group. 1000× magnification.

Figure 3

Wound healing assay. Wounds were made with a pipette tip on confluent T24/pEGFP-N2 (left panel), T24/hepaCAM (middle panel), and T24/hepaCAM-mt1 (right panel) cells and allowed to heal for 12 and 24 h. 100× magnification(A). The diameters of wounds were measured on the microscopic photos at 0 h, 12 h, and 24 h after the wound was induced. Changes in wound diameter were computed into percentages (means ± SD) to represent wound closure. P < 0.01 as assessed by ANOVA(B).

Figure 4

Inhibition of cell growth by hepaCAM. The growth rate T24/hepaCAM and T24/hepaCAM-mt1 with T24/pEGFP-N2 cells measured over a seven day period by MTT assay. Data represent means ± SD. P < 0.01 as assessed by ANOVA.