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.

RB1 and TP53 pathways in radiation-induced sarcomas

“…INK4a expression, but this association is not systematic (Sartor et al, 1999;Chatterjee et al, 2004). In our series, various levels of phosphorylation of pRb were observed despite the lack of expression of INK4a.…”

“…Electrophoresis was run with 100 mg of protein extract on a NuPAGE 3-8% Tris-acetate gel (Invitrogen, Cergy Pontoise, France), and Western blotting was performed using a mouse anti-pRb antibody (14001A, BD Pharmigen, Erembodegem, Belgium, 1/250, overnight at 41C) after transfer on polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Saint Guentin en Yveline, France). The antibody detected underphosphorylated (110 kDa) and hyperphosphorylated (116 kDa) pRb (Chatterjee et al, 2004 (Sharpless, 2005). In this series of sarcomas, we can conclude that, in addition to the cases with direct gene inactivation, the loss of Rb function occurred by hyperphosphorylation of the protein in three of 11 other tested tumours.…”

Chromosome 9p21 gene copy number and prognostic significance of p16 in ESFT

“…Other studies have reported p16 promoter hypermethylation in ESFT to be rare; 1/24 (4%; Tsuchiya et al, 2000) and 2/19 (10%; Lopez-Guerrero et al, 2001). Heterogeneity of expression might therefore be effected through expression of wild-type p53, which can downregulate transcription from the p14 ARF and p16 promoter (Robertson and Jones, 1998), pRb hyperphosphorylation (Chatterjee et al, 2004) or overexpression of the bmi-1 gene (Jacobs et al, 1999). Homozygous and single copy deletions of the MTAP gene were observed in ESFT cell lines and primary tumour; homozygous deletions (but not single copy deletions) have previously been described with similar frequency to this study (Huang et al, 2005).…”

Posttranslational Protein Modifications

“…Phosphorylation of pRB1 is thus the key regulatory step in this pathway controlling cell division. Amplification of the cyclin D1 gene or mutations in p16 are often found in tumors contributing to a hyperphosphorylated state of pRB1 and a consequential commitment to continued cell growth (16,17).…”