Hyperphosphorylation of pRb: a mechanism for RB tumour suppressor pathway inactivation in bladder cancer

Abstract

Loss of heterozygosity, mutations or deletions of the RB1 gene usually result in loss of pRb expression, which has been regarded as an indicator of loss of pRb function in human tumours. It has previously been shown that in addition to loss of pRb expression, aberrantly high (pRb2+) pRb expression also indicates loss of pRb function in bladder tumours compared with moderate (normal, pRb1+) pRb expression. The aim of this study was to elucidate the mechanism by which pRb is functionally inactivated in bladder tumours expressing aberrantly high levels of pRb. Constitutive phosphorylation was therefore investigated as a mechanism of pRb inactivation in bladder tumours. Of 28 bladder tumours examined, western blotting demonstrated pRb hyperphosphorylation in 5/7 (71%) pRb2+ bladder tumours compared with only 4/11 (36%) pRb1+ tumours (p = 0.002). All cases with undetectable pRb showed moderate to high p16 expression and none showed cyclin D1 expression by immunohistochemistry. All pRb1+ tumours with underphosphorylated pRb showed p16 but not cyclin D1 expression. All pRb2+ tumours with hyperphosphorylated pRb showed loss of p16 expression and/or cyclin D1 overexpression. Thus, elevated pRb expression was associated with pRb hyperphosphorylation, which, in turn, was associated with loss of p16 expression and/or increased cyclin D1 expression. In order to analyse this association in vitro, T24 cells, which express high levels of pRb, were transfected with p16 cDNA. Transfection with p16 cDNA resulted in a marked decrease in pRb phosphorylation, decreased cell proliferation, and a change in expression of pRb from high to moderate phenotype as assessed by immunohistochemistry. This paper gives the biological basis for constitutive alteration of pRb function in human tumours in the presence of an intact, expressed pRb protein; the mechanism of pRb inactivation is through hyperphosphorylation, which results from loss of p16 expression and/or cyclin D1 overexpression. Immunohistochemical expression of pRb appears to be a reliable indicator of pRb function.