Regulation of the activation of the Fanconi anemia pathway by the p21 cyclin-dependent kinase inhibitor.

Fanconi anemia (FA) is a rare disease characterized by congenital defects, progressive bone marrow failure and heightened cancer susceptibility. The FA proteins, BRCA1 and FANCD1/BRCA2 function cooperatively in the FA-BRCA pathway to repair damaged DNA. Activation of the FA-BRCA pathway occurs via the monoubiquitination of the FANCD2 and FANCI proteins, targeting these proteins to discrete nuclear foci where they function in DNA repair. The cellular regulation of FANCD2/I monoubiquitination, however, remains poorly understood. In this study, we have examined the roles of the p53 tumor suppressor protein, as well as its downstream target, the p21(Cip1/Waf1) cyclin-dependent kinase inhibitor, in the regulation of the activation of the FA-BRCA pathway. We demonstrate that, in contrast to p53, p21 has a major role in the regulation of the activation of the FA-BRCA pathway: p21 promotes S-phase and DNA damage-inducible FANCD2/I monoubiquitination and nuclear foci formation. Several lines of evidence establish that this effect is not a consequence of a defective G1-S checkpoint or altered cell-cycle progression in the absence of p21. Instead, we demonstrate that p21 is required for the transcriptional repression of the USP1 deubiquitinating enzyme upon exposure to DNA-damaging agents. In the absence of p21, persistent USP1 expression precludes the DNA damage-inducible accumulation of monoubiquitinated FANCD2 and FANCI. Consequently, p21(-/-) cells exhibit increased levels of mitomycin C-inducible complex chromosomal aberrations and elevated γH2AX nuclear foci formation. Our results demonstrate that p21 has a critical role in the regulation of the activation of the FA-BRCA pathway and suggest a broader role for p21 in the orchestration of DNA repair processes following exposure to DNA crosslinking agents.

Assessing the daily consistency of bladder filling using an ultrasonic Bladderscan device in men receiving radical conformal radiotherapy for prostate cancer.

OBJECTIVE: Consistency in target organ and organ at risk position from planning to treatment is an important basic principle of radiotherapy. This study evaluates the effectiveness of bladder-filling instructions in achieving a consistent and reproducible bladder volume at the time of planning CT and daily during the course of radical radiotherapy for prostate cancer. It also assessed the rate of bladder filling before and at the end of radiotherapy. METHODS: 30 men attending for radiation therapy planning for prostate cancer received written and verbal bladder-filling instructions. They had their bladder volume assessed using a bladder ultrasound scanner post-void, immediately prior to planning CT scan and then daily immediately prior to treatment while in the therapy position. The inflow was calculated using the void and full bladder volumes and the time for the bladder to fill. RESULTS: The mean bladder volume at the time of planning was 282 ml (range 89-608 ml, standard deviation (SD) = 144.5 ml). This fell during treatment, with a mean value for all treatments of 189 ml (range 11-781 ml, SD = 134 ml). During radiotherapy, 76% (828/1090), 53% (579/1090) and 36% (393/1090) of bladder volumes had >50 ml, >100 ml and >150 ml difference, respectively when compared with their volume at the time of planning. Inflow reduced from 4.6 ml min(-1), SD = 2.9 min(-1) at planning to 2.5 min(-1), SD = 1.8 min(-1) after radiotherapy. CONCLUSION: The Bladderscan device (BVI 6400 Bladderscan, Verathon Medical UK, Sandford, UK) provides an effective means of assessing bladder volume prior to radiotherapy for prostate cancer. The evaluated bladder-filling protocol does not produce consistent, reproducible bladder volumes for radiotherapy.