Pending Duplicate Bibliography Entries

The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair.

The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNAdamage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

Defective DNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1.

Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a neurodegenerative disease that results from mutation of tyrosyl phosphodiesterase 1 (TDP1). In lower eukaryotes, Tdp1 removes topoisomerase 1 (top1) peptide from DNA termini during the repair of double-strand breaks created by collision of replication forks with top1 cleavage complexes in proliferating cells. Although TDP1 most probably fulfils a similar function in human cells, this role is unlikely to account for the clinical phenotype of SCAN1, which is associated with progressive degeneration of post-mitotic neurons. In addition, this role is redundant in lower eukaryotes, and Tdp1 mutations alone confer little phenotype. Moreover, defects in processing or preventing double-strand breaks during DNA replication are most probably associated with increased genetic instability and cancer, phenotypes not observed in SCAN1 (ref. 8). Here we show that in human cells TDP1is required for repair of chromosomal single-strand breaks arising independentlyof DNA replication from abortive top1 activity or oxidative stress. We report that TDP1 is sequestered into multi-protein single-strand break repair (SSBR) complexes by direct interaction with DNA ligase IIIalpha and that these complexes are catalytically inactive in SCAN1 cells. These data identify a defect in SSBR in a neurodegenerative disease, and implicate this process in the maintenance ofgenetic integrity in post-mitotic neurons.

Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapsereplication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients aredefective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

Tumor vascular changes mediated by inhibition of oncogenic signaling.

Many inhibitors of the epidermal growth factor receptor (EGFR)-RAS-phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway are in clinical use or under development for cancer therapy. Here, we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3K, or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion, and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results, as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target, as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor but did respond with vascular alterations to RAS or PI3K inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3K inhibition with decreased tumor hypoxia, increased vascular flow, and morphologic alterations oftheir vessels, including increased vascular maturity and acquisition of pericytemarkers. These changes are similar to the vascular normalization that has been described after the antiangiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

Tumor vascular changes mediated by inhibition of oncogenic signaling.

Many inhibitors of the epidermal growth factor receptor (EGFR)-RAS-phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway are in clinical use or under development for cancer therapy. Here, we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3K, or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion, and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results, as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target, as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor but did respond with vascular alterations to RAS or PI3K inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3K inhibition with decreased tumor hypoxia, increased vascular flow, and morphologic alterations oftheir vessels, including increased vascular maturity and acquisition of pericytemarkers. These changes are similar to the vascular normalization that has been described after the antiangiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy.

Tumor vascular changes mediated by inhibition of oncogenic signaling.

Many inhibitors of the epidermal growth factor receptor (EGFR)-RAS-phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway are in clinical use or under development for cancer therapy. Here, we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3K, or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion, and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results, as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target, as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor but did respond with vascular alterations to RAS or PI3K inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3K inhibition with decreased tumor hypoxia, increased vascular flow, and morphologic alterations oftheir vessels, including increased vascular maturity and acquisition of pericytemarkers. These changes are similar to the vascular normalization that has been described after the antiangiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

In vivo magnetic resonance imaging of acute brain inflammation using microparticles of iron oxide.

Multiple sclerosis is a disease of the central nervous system that is associatedwith leukocyte recruitment and subsequent inflammation, demyelination and axonalloss. Endothelial vascular cell adhesion molecule-1 (VCAM-1) and its ligand, alpha4beta1 integrin, are key mediators of leukocyte recruitment, and selective inhibitors that bind to the alpha4 subunit of alpha4beta1 substantially reduce clinical relapse in multiple sclerosis. Urgently needed is a molecular imaging technique to accelerate diagnosis, to quantify disease activity and to guide specific therapy. Here we report in vivo detection of VCAM-1 in acute brain inflammation, by magnetic resonance imaging in a mouse model, at a time when pathology is otherwise undetectable. Antibody-conjugated microparticles carryinga large amount of iron oxide provide potent, quantifiable contrast effects that delineate the architecture of activated cerebral blood vessels. Their rapid clearance from blood results in minimal background contrast. This technology is adaptable to monitor the expression of endovascular molecules in vivo in variouspathologies.

MRI reveals that early changes in cerebral blood volume precede blood-brain barrier breakdown and overt pathology in MS-like lesions in rat brain.

Magnetic resonance imaging (MRI) is an established clinical tool for diagnosing multiple sclerosis (MS), the archetypal central nervous system neuroinflammatorydisease. In this study, we have used a model of delayed-type hypersensitivity inthe rat brain, which bears many of the hallmarks of an MS lesion, to investigatethe development of MRI-detectable changes before the appearance of conventional indices of lesion development. In addition, we have correlated the MRI-detectable changes with the developing histopathology. Significant increases in regional cerebral blood volume (rCBV) preceded overt changes in blood-brain barrier (BBB)permeability, T2 relaxation and the diffusion properties of tissue water. Thus, changes in rCBV might be a more sensitive indicator of lesion onset than the conventional indices used clinically in MS patients, such as contrast enhancement. In addition, we show that BBB breakdown, and consequent edema formation, are more closely correlated with astrogliosis than any other histopathologic changes, while regions of T1 and T2 hypointensity appear to reflect hypercellularity.

Confounding effects of anesthesia on functional activation in rodent brain: a study of halothane and alpha-chloralose anesthesia.

Functional magnetic resonance imaging (fMRI) in animal models provides a platform for more extensive investigation of drug effects and underlying physiological mechanisms than is possible in humans. However, it is usually necessary for the animal to be anesthetized. In this study, we have used a rat model of direct cortical stimulation to investigate the effects of anesthesia in rodent fMRI. Specifically, we have sought to answer two questions (i) what is the relationship between baseline neuronal activity and the BOLD response to stimulation under halothane anesthesia? And (ii) how does the BOLD response change after transferring from halothane to the commonly used anesthetic alpha-chloralose? Inthe first set of experiments, we found no significant differences in the amplitude of the BOLD response at the different halothane doses studied, despiteelectroencephalography (EEG) recordings indicating a dose-dependent reduction inbaseline neuronal activity with increasing halothane levels. In the second set of experiments, a reduction in the spatial extent of the BOLD response was apparentimmediately after transfer from halothane to alpha-chloralose anesthesia, although no change in the peak signal change was evident. However, several hoursafter transfer to alpha-chloralose, a significant increase in both the spatial extent and peak height of the BOLD response was observed, as well as an increased sensitivity to secondary cortical and subcortical activation. These findings suggest that, although alpha-chloralose anesthesia is associated with a greater BOLD response for a fixed stimulus relative to halothane, there is substantial variation in the extent and magnitude of the response over time that could introduce considerable variability in studies using this anesthetic.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

Mechanism of action of an imidopiperidine inhibitor of human polynucleotide kinase/phosphatase.

The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4- b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphataseactivity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNAfrom binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp(402) of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNAagainst PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase beta or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

Mechanism of action of an imidopiperidine inhibitor of human polynucleotide kinase/phosphatase.

The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4- b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphataseactivity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNAfrom binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp(402) of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNAagainst PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase beta or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.

Mechanism of action of an imidopiperidine inhibitor of human polynucleotide kinase/phosphatase.

The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4- b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphataseactivity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNAfrom binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp(402) of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNAagainst PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase beta or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers.

Germ-line mutations in breast cancer 1, early onset (BRCA1) result in predisposition to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify p53-binding protein 1 (53BP1) as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell-cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Notably, loss of 53BP1 partially restores the homologous-recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.

53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers.

Germ-line mutations in breast cancer 1, early onset (BRCA1) result in predisposition to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify p53-binding protein 1 (53BP1) as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell-cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Notably, loss of 53BP1 partially restores the homologous-recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.

A sequence variant at 4p16.3 confers susceptibility to urinary bladder cancer.

Previously, we reported germline DNA variants associated with risk of urinary bladder cancer (UBC) in Dutch and Icelandic subjects. Here we expanded the Icelandic sample set and tested the top 20 markers from the combined analysis inseveral European case-control sample sets, with a total of 4,739 cases and 45,549 controls. The T allele of rs798766 on 4p16.3 was found to associate with UBC (odds ratio = 1.24, P = 9.9 x 10(-12)). rs798766 is located in an intron of TACC3, 70 kb from FGFR3, which often harbors activating somatic mutations in low-grade, noninvasive UBC. Notably, rs798766[T] shows stronger association withlow-grade and low-stage UBC than with more aggressive forms of the disease and is associated with higher risk of recurrence in low-grade stage Ta tumors. The frequency of rs798766[T] is higher in Ta tumors that carry an activating mutation in FGFR3 than in Ta tumors with wild-type FGFR3. Our results show a link betweengermline variants, somatic mutations of FGFR3 and risk of UBC.

A sequence variant at 4p16.3 confers susceptibility to urinary bladder cancer.

Previously, we reported germline DNA variants associated with risk of urinary bladder cancer (UBC) in Dutch and Icelandic subjects. Here we expanded the Icelandic sample set and tested the top 20 markers from the combined analysis inseveral European case-control sample sets, with a total of 4,739 cases and 45,549 controls. The T allele of rs798766 on 4p16.3 was found to associate with UBC (odds ratio = 1.24, P = 9.9 x 10(-12)). rs798766 is located in an intron of TACC3, 70 kb from FGFR3, which often harbors activating somatic mutations in low-grade, noninvasive UBC. Notably, rs798766[T] shows stronger association withlow-grade and low-stage UBC than with more aggressive forms of the disease and is associated with higher risk of recurrence in low-grade stage Ta tumors. The frequency of rs798766[T] is higher in Ta tumors that carry an activating mutation in FGFR3 than in Ta tumors with wild-type FGFR3. Our results show a link betweengermline variants, somatic mutations of FGFR3 and risk of UBC.

A sequence variant at 4p16.3 confers susceptibility to urinary bladder cancer.

Previously, we reported germline DNA variants associated with risk of urinary bladder cancer (UBC) in Dutch and Icelandic subjects. Here we expanded the Icelandic sample set and tested the top 20 markers from the combined analysis inseveral European case-control sample sets, with a total of 4,739 cases and 45,549 controls. The T allele of rs798766 on 4p16.3 was found to associate with UBC (odds ratio = 1.24, P = 9.9 x 10(-12)). rs798766 is located in an intron of TACC3, 70 kb from FGFR3, which often harbors activating somatic mutations in low-grade, noninvasive UBC. Notably, rs798766[T] shows stronger association withlow-grade and low-stage UBC than with more aggressive forms of the disease and is associated with higher risk of recurrence in low-grade stage Ta tumors. The frequency of rs798766[T] is higher in Ta tumors that carry an activating mutation in FGFR3 than in Ta tumors with wild-type FGFR3. Our results show a link betweengermline variants, somatic mutations of FGFR3 and risk of UBC.

A sequence variant at 4p16.3 confers susceptibility to urinary bladder cancer.

Previously, we reported germline DNA variants associated with risk of urinary bladder cancer (UBC) in Dutch and Icelandic subjects. Here we expanded the Icelandic sample set and tested the top 20 markers from the combined analysis inseveral European case-control sample sets, with a total of 4,739 cases and 45,549 controls. The T allele of rs798766 on 4p16.3 was found to associate with UBC (odds ratio = 1.24, P = 9.9 x 10(-12)). rs798766 is located in an intron of TACC3, 70 kb from FGFR3, which often harbors activating somatic mutations in low-grade, noninvasive UBC. Notably, rs798766[T] shows stronger association withlow-grade and low-stage UBC than with more aggressive forms of the disease and is associated with higher risk of recurrence in low-grade stage Ta tumors. The frequency of rs798766[T] is higher in Ta tumors that carry an activating mutation in FGFR3 than in Ta tumors with wild-type FGFR3. Our results show a link betweengermline variants, somatic mutations of FGFR3 and risk of UBC.

The vascular basement membrane as "soil" in brain metastasis.

Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Paget's "seed and soil" concept. However, there is little direct evidence for this "neurotropic" growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the "soil" for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM) as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesionand invasion of malignant cells and was sufficient for tumor growth prior to anyevidence of angiogenesis. Blockade or loss of the beta1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies.

Phase II study of conformal hypofractionated radiotherapy with concurrent gemcitabine in muscle-invasive bladder cancer.

PURPOSE The aim of this prospective, phase II trial was to determine the response of muscle-invasive bladder cancer (MIBC) to concurrent chemoradiotherapy of weekly gemcitabine with 4 weeks of radiotherapy (RT; GemX). PATIENTS AND METHODS. Fifty patients with transitional cell carcinoma, stage T2-3, N0, M0 after transurethral resection and magnetic resonance imaging, were recruited. Gemcitabine was given intravenously at 100 mg/m(2) on days 1, 8, 15, and 22 of a28-day RT schedule that delivered 52.5 Gy in 20 fractions. Chemotherapy was stopped for Radiation Therapy Oncology Group (RTOG) grade 3 bladder or bowel toxicity. The primary end points were tumor response, toxicity, and survival. Results All patients completed RT; 46 tolerated all four cycles of gemcitabine. Two patients stopped after two cycles, and two stopped after three cycles, because of bowel toxicity. Forty-seven patients had a post-treatment cystoscopy;44 (88%) achieved a complete endoscopic response. At a median follow-up of 36 months (range, 15 to 62 months), 36 patients were alive, and 32 of these had a functional and intact bladder. Fourteen patients died; seven died as a result ofmetastatic MIBC, five died as a result of intercurrent disease, and two died as a result of treatment-associated deaths. Four patients underwent cystectomy; threebecause of recurrent disease and one because of toxicity. One patient required abowel resection for late toxicity. By using Kaplan-Meier analyses, 3-year cancer-specific survival was 82%, and overall survival was 75%. CONCLUSION Concurrent gemcitabine-based chemoradiotherapy (ie, GemX) produces a high response rate in MIBC and has durable local control and acceptable toxicity, which allows patients to preserve their own bladder. This treatment modality warrants additional investigation in a phase III setting.

A sequence variant at 4p16.3 confers susceptibility to urinary bladder cancer.

Previously, we reported germline DNA variants associated with risk of urinary bladder cancer (UBC) in Dutch and Icelandic subjects. Here we expanded the Icelandic sample set and tested the top 20 markers from the combined analysis inseveral European case-control sample sets, with a total of 4,739 cases and 45,549 controls. The T allele of rs798766 on 4p16.3 was found to associate with UBC (odds ratio = 1.24, P = 9.9 x 10(-12)). rs798766 is located in an intron of TACC3, 70 kb from FGFR3, which often harbors activating somatic mutations in low-grade, noninvasive UBC. Notably, rs798766[T] shows stronger association withlow-grade and low-stage UBC than with more aggressive forms of the disease and is associated with higher risk of recurrence in low-grade stage Ta tumors. The frequency of rs798766[T] is higher in Ta tumors that carry an activating mutation in FGFR3 than in Ta tumors with wild-type FGFR3. Our results show a link betweengermline variants, somatic mutations of FGFR3 and risk of UBC.

Common predisposition alleles for moderately common cancers: bladder cancer.

Bladder cancer is the 5th commonest cancer and two major risk factors are smoking and occupational chemical exposure. There is also evidence of a genetic component to its aetiology. Candidate gene studies have mostly focused on genes involved in adduct metabolism and DNA repair, including a recent consortium-based meta-analysis. Recently, two genome-wide association studies in bladder cancer have been published and a third is awaited with interest. These first two studies have identified three SNPs of genome-wide significance, two located within the 8q24 'gene desert'. These SNPs are positioned near or within loci of genes potentially implicated in cancer predisposition, namely MYC, TP63 and PSCA, although the functional significance of this is as yet unclear.

Radiation response of cancer stem-like cells from established human cell lines after sorting for surface markers.

PURPOSE: A subpopulation of cancer stem-like cells (CSLC) is hypothesized to exist in different cancer cell lines and to mediate radioresistance in solid tumors. METHODS AND MATERIALS: Cells were stained for CSLC markers and sorted (fluorescence-activated cell sorter/magnetic beads) to compare foci and radiosensitivity of phosphorylated histone H2AX at Ser 139 (gamma-H2AX) in sorted vs. unsorted populations in eight cell lines from different organs. CSLC properties were examined using anchorage-independent growth and levels of activated Notch1. Validation consisted of testing tumorigenicity and postirradiation enrichment of CSLC in xenograft tumors. RESULTS: The quantity ofCSLC was generally in good agreement with primary tumors. CSLC from MDA-MB-231 (breast) and Panc-1 and PSN-1 (both pancreatic) cells had fewer residual gamma-H2AX foci than unsorted cells, pointing to radioresistance of CSLC. However, only MDA-MB-231 CSLC were more radioresistant than unsorted cells. Furthermore, MDA-MB-231 CSLC showed enhanced anchorage-independent growth and overexpression of activated Notch1 protein. The expression of cancer stem cell surface markers in the MDA-MB-231 xenograft model was increased after exposure to fractionated radiation. In contrast to PSN-1 cells, a growth advantage for MDA-MB-231 CSLC xenograft tumors was found compared to tumors arising from unsorted cells. CONCLUSIONS: CSLC subpopulations showed no general radioresistant phenotype, despite the quantities of CSLC subpopulations shown to correspond relatively well in other reports. Likewise, CSLC characteristics were found in some but not all of the tested cell lines. The reported problems in testing for CSLC in cell lines may be overcome by additional techniques, beyond sorting for markers.

Radiation response of cancer stem-like cells from established human cell lines after sorting for surface markers.

PURPOSE: A subpopulation of cancer stem-like cells (CSLC) is hypothesized to exist in different cancer cell lines and to mediate radioresistance in solid tumors. METHODS AND MATERIALS: Cells were stained for CSLC markers and sorted (fluorescence-activated cell sorter/magnetic beads) to compare foci and radiosensitivity of phosphorylated histone H2AX at Ser 139 (gamma-H2AX) in sorted vs. unsorted populations in eight cell lines from different organs. CSLC properties were examined using anchorage-independent growth and levels of activated Notch1. Validation consisted of testing tumorigenicity and postirradiation enrichment of CSLC in xenograft tumors. RESULTS: The quantity ofCSLC was generally in good agreement with primary tumors. CSLC from MDA-MB-231 (breast) and Panc-1 and PSN-1 (both pancreatic) cells had fewer residual gamma-H2AX foci than unsorted cells, pointing to radioresistance of CSLC. However, only MDA-MB-231 CSLC were more radioresistant than unsorted cells. Furthermore, MDA-MB-231 CSLC showed enhanced anchorage-independent growth and overexpression of activated Notch1 protein. The expression of cancer stem cell surface markers in the MDA-MB-231 xenograft model was increased after exposure to fractionated radiation. In contrast to PSN-1 cells, a growth advantage for MDA-MB-231 CSLC xenograft tumors was found compared to tumors arising from unsorted cells. CONCLUSIONS: CSLC subpopulations showed no general radioresistant phenotype, despite the quantities of CSLC subpopulations shown to correspond relatively well in other reports. Likewise, CSLC characteristics were found in some but not all of the tested cell lines. The reported problems in testing for CSLC in cell lines may be overcome by additional techniques, beyond sorting for markers.

HIV protease inhibitors block Akt signaling and radiosensitize tumor cells both in vitro and in vivo.

In tumor cells with mutations in epidermal growth factor receptor (SQ20B), H-Ras(T24), or K-Ras (MIAPACA2 and A549), the inhibition of Akt phosphorylation increases radiation sensitivity in clonogenic assays, suggesting that Akt is a potential molecular target when combined with therapeutic radiation. Insulin resistance and diabetes are recognized side effects of HIV protease inhibitors (HPIs), suggesting that these agents may inhibit Akt signaling. Because activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is common in human cancers, we hypothesized that HPIs can inhibit Akt activity resulting in increased tumor cell sensitivity to ionizing radiation-induced celldeath. Five first-generation HPIs were subsequently tested and three of the five(amprenavir, nelfinavir, and saquinavir but not ritonavir or indinavir) inhibited Akt phosphorylation at Ser473 at serum concentrations routinely achieved in HIV patients. In both tumor cell colony formation assays and tumor regrowth delay experiments, combinations of drug and radiation exerted synergistic effects compared with either modality alone. In addition, in vivo, doses of amprenavir or nelfinavir comparable with the therapeutic levels achieved in HIV patients were sufficient to down-regulate phosphorylation of Akt in SQ20B and T24 xenografts. Finally, overexpression of active PI3K in cells without activation of Akt resulted in radiation resistance that could be inhibited with HPIs. Because there is abundant safety data on HPIs accumulated in thousands of HIV patients over the last 5 years, these agents are excellent candidates to be tested as radiation sensitizers in clinical trials.

Pancreatic cancer cell radiation survival and prenyltransferase inhibition: the role of K-Ras.

Activating K-ras mutations are found in approximately 90% of pancreatic carcinomas and may contribute to the poor prognosis of these tumors. Because radiotherapy is frequently used in pancreatic cancer treatment, we assessed the contribution of oncogenic K-ras signaling to pancreatic cancer radiosensitivity.Seven human pancreatic carcinoma lines with activated K-ras and two cell lines with wild-type ras were used to examine clonogenic cell survival after Ras inhibition. Ras inhibition was accomplished by small interfering RNA (siRNA) knockdown of K-ras expression and by blocking Ras processing using a panel of prenyltransferase inhibitors of differing specificity for the two prenyltransferases that modify K-Ras. K-ras knockdown by siRNA or inhibition of prenyltransferase activity resulted in radiation sensitization in vitro and in vivo in tumors with oncogenic K-ras mutations. Inhibition of farnesyltransferasealone was sufficient to radiosensitize most K-ras mutant tumors, although K-Ras prenylation was not blocked. These results show that inhibition of activated K-Ras can promote radiation killing of pancreatic carcinoma in a superadditive manner. The finding that farnesyltransferase inhibition alone radiosensitizes tumors with K-ras mutations implies that a farnesyltransferase inhibitor-sensitive protein other than K-Ras may contribute to survival in the context of mutant K-ras. Farnesyltransferase inhibitors could therefore be of use as sensitizers for pancreatic carcinoma radiotherapy.

Oncogenic K-Ras signals through epidermal growth factor receptor and wild-type H-Ras to promote radiation survival in pancreatic and colorectal carcinoma cells.

Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cellsthat express oncogenic K-Ras. In these experiments, inhibiting the expression ofoncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) ledto similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor alpha was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

Tumor vascular changes mediated by inhibition of oncogenic signaling.

Many inhibitors of the epidermal growth factor receptor (EGFR)-RAS-phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway are in clinical use or under development for cancer therapy. Here, we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3K, or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion, and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results, as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target, as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor but did respond with vascular alterations to RAS or PI3K inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3K inhibition with decreased tumor hypoxia, increased vascular flow, and morphologic alterations oftheir vessels, including increased vascular maturity and acquisition of pericytemarkers. These changes are similar to the vascular normalization that has been described after the antiangiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy.

Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy.

A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major DNA polymerase involved in base excision repair. The underlyingmechanism and biological consequences of overexpression of this protein are unknown. We examined whether Pol beta, expressed at levels found in tumor cells,is involved in the repair of DNA damage induced by oxaliplatin treatment and whether the expression status of this protein alters the sensitivity of cells tooxaliplatin. DNA damage induced by oxaliplatin treatment of HCT116 and HT29 colon cancer cells was observed to be associated with the stabilization of Pol beta protein on chromatin. In comparison with HCT116 colon cancer cells, isogenic oxaliplatin-resistant (HCT-OR) cells were found to have higher constitutive levels of Pol beta protein, faster in vitro repair of a DNA substrate containinga single nucleotide gap and faster repair of 1,2-GG oxaliplatin adduct levels incells. In HCT-OR cells, small interfering RNA knockdown of Pol beta delayed the repair of oxaliplatin-induced DNA damage. In a different model system, Pol beta-deficient fibroblasts were less able to repair 1,2-GG oxaliplatin adducts and were hypersensitive to oxaliplatin treatment compared with isogenic Pol beta-expressing cells. Consistent with previous studies, Pol beta-deficient mouse fibroblasts were not hypersensitive to cisplatin treatment. These data provide the first link between oxaliplatin sensitivity and DNA repair involving Pol beta. They demonstrate that Pol beta modulates the sensitivity of cells to oxaliplatintreatment.

Molecular targets for altering radiosensitivity: lessons from Ras as a pre-clinical and clinical model.

Ras activation has been correlated with malignant and metastatic cancer phenotypes and poor prognosis for cancer patients. In the preclinical setting, Ras activation by mutation or EGFR amplification results in increased clonogeniccell survival and decreased tumor growth delay following irradiation. Activationof the Ras pathway has also been associated with increased risk of local failureand decreased overall survival in patients receiving radiotherapy. Prenyltransferase inhibitors target the post-translational processing of Ras andhave been shown to increase the radiosensitivity of human cancer cell lines. In the clinical setting, these inhibitors have been used with concurrent radiotherapy in a small number of phase I clinical trials with acceptable toxicity. Therefore, inhibiting Ras activation represents a promising molecular approach for radiosensitization in cancer therapy.

Molecular targets for altering radiosensitivity: lessons from Ras as a pre-clinical and clinical model.

Ras activation has been correlated with malignant and metastatic cancer phenotypes and poor prognosis for cancer patients. In the preclinical setting, Ras activation by mutation or EGFR amplification results in increased clonogeniccell survival and decreased tumor growth delay following irradiation. Activationof the Ras pathway has also been associated with increased risk of local failureand decreased overall survival in patients receiving radiotherapy. Prenyltransferase inhibitors target the post-translational processing of Ras andhave been shown to increase the radiosensitivity of human cancer cell lines. In the clinical setting, these inhibitors have been used with concurrent radiotherapy in a small number of phase I clinical trials with acceptable toxicity. Therefore, inhibiting Ras activation represents a promising molecular approach for radiosensitization in cancer therapy.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

CHIP-mediated degradation and DNA damage-dependent stabilization regulate base excision repair proteins.

Base excision repair (BER) is the major pathway for processing of simple lesions in DNA, including single-strand breaks, base damage, and base loss. The scaffold protein XRCC1, DNA polymerase beta, and DNA ligase IIIalpha play pivotal roles in BER. Although all these enzymes are essential for development, their cellular levels must be tightly regulated because increased amounts of BER enzymes lead to elevated mutagenesis and genetic instability and are frequently found in cancer cells. Here we report that BER enzyme levels are linked to and controlled by the level of DNA lesions. We demonstrate that stability of BER enzymes increases after formation of a repair complex on damaged DNA and that proteins not involved in a repair complex are ubiquitylated by the E3 ubiquitin ligase CHIP and subsequently rapidly degraded. These data identify a molecular mechanism controlling cellular levels of BER enzymes and correspondingly the efficiency and capacity of BER.

The role of microRNA-binding site polymorphisms in DNA repair genes as risk factors for bladder cancer and breast cancer and their impact on radiotherapy outcomes.

MicroRNAs (miRNAs) are involved in post-transcriptional regulation of gene expression through binding to messenger RNAs (mRNA) thereby promoting mRNA degradation or altered translation. A single-nucleotide polymorphism (SNP) located within a miRNA-binding site could thus alter mRNA translation and influence cancer risk and treatment response. The common SNPs located within the 3'-untranslated regions of 20 DNA repair genes were analysed for putative miRNA-binding sites using bioinformatics algorithms, calculating the difference in Gibbs free binding energy (DeltaDeltaG) for each wild-type versus variant allele. Seven SNPs were selected to be genotyped in germ line DNAs both from a bladder cancer case-control series (752 cases and 704 controls) and 202 muscle-invasive bladder cancer radiotherapy cases. The PARP-1 SNP rs8679 was also genotyped in a breast cancer case-control series (257 cases and 512 controls). Without adjustment for multiple testing, multivariate analysis demonstrated an association with increased bladder cancer risk with PARP1 rs8679 (P(trend) = 0.05) while variant homozygotes of PARP1 rs8679 were also noted to have an increased breast cancer risk (P = 0.03). In the radiotherapy cases, carriers of the RAD51 rs7180135 minor allele had improved cancer-specific survival (hazard ratio 0.52, 95% confidence interval 0.31-0.87, P = 0.01). This is the first report of associations between DNA repair gene miRNA-binding site SNPs with bladder and breast cancer risk and radiotherapy outcomes. If validated, these findings may give further insight into the biology of bladder carcinogenesis, allow testing of the RAD51 SNP as a potential predictive biomarker and also reveal potential targets for new cancer treatments.