Abstract
Aberrant Wnt gene expression is involved in the development of breast cancer, but its role in other tumours is unknown. Wnts regulate cadherin function, previously shown to be more commonly deregulated in invasive bladder cancer. This study investigated whether factors upstream of cadherins were aberrantly expressed in superficial bladder cancer. The expression of one transforming (Wnt7b) and one non-transforming (Wnt5a) Wnt gene in four human bladder carcinoma cell lines, and in normal human bladder tissues (n = 8) and bladder cancers (n = 48) were analysed by ribonuclease protection analysis. All cell lines expressed an approximately equal level of Wnt7b mRNA. Wnt5a and Wnt7b mRNAs were both expressed in normal bladder tissues and bladder tumours. The median expression of Wnt7b was fourfold higher in superficial tumours (n = 29) than in normal tissues (n = 8, P = 0.002) and five fold higher than in invasive tumours (n = 17, P = 0.003). There was no significant difference between normal tissues and invasive tumours (P = 0.3). The expression of Wnt5a did not vary significantly between normal tissues and superficial tumours (P = 0.4), normal tissues and invasive tumours (P = 0.3) or superficial tumours and invasive tumours (P = 0.2). The differential expression of Wnt7b suggests a role in the early events of superficial bladder tumorigenesis involving cell adhesion and provides further evidence of different pathways of evolution of superficial and invasive cancer.
Highlights
The level of Wnt5a mRNA was very high in the RT4 cell line that was obtained from a well-differentiated bladder carcinoma, moderately high and low in the 253J and T24 cell lines, respectively, which were obtained from poorly differentiated bladder carcinomas
There was no significant difference between normal tissues and invasive tumour (P = 0.3) (Figure 3B, Table 1)
There was no significant correlation between the levels of Wnt5a and Wnt7b mRNA expression in normal tissues and superficial or invasive tumours alone, or in combined tumours
Summary
Cell culture and RNA preparation The collection of normal human (n = 8) and tumour (n = 48) bladder tissues has been described previously (O'Brien et al, 1995). To assess Wnt expression in epithelial cells representing a pure population, human bladder carcinoma cell lines (T24, RT4, RTl 12 and 253J) were obtained from Dr MA Knowles, Marie Curie Research Institute, Oxsted, Surrey, UK. All the cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Imperial Cancer Research Fund, Clare Hall Laboratories) and 10% fetal calf serum (FCS) (Globepharm). Total RNA was prepared from tissues and cells using the acid guanidium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987), followed by a 5.7 M caesium chloride separation at 50 000 r.p.m. for 3 h using a SW50 or SW55 swing rotor (Beckman). The RNA pellet was resuspended in 200 ml of sterile water, treated with RNAase-free DNAase for 15 min at 37°C, extracted with an equal volume of phenol, ethanol precipitated with 0.1 x volume of sodium acetate, pH 5.2, and resuspended in water to the final concentration of 1 mg ml'
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