Alterations of ubiquitylation and sumoylation in conventional renal cell carcinomas after the Chernobyl accident: a comparison with Spanish cases.

We determined whether ubiquitylation and sumoylation processes are involved in conventional renal cell carcinogenesis associated with chronic, long-term, persistent low doses of ionizing radiation (IR) in patients living for more than 20 years in cesium-137 ((137)Cs)-contaminated areas after the Chernobyl accident in Ukraine. To this end, we assessed the immunohistochemical expression of ubiquitin (Ub), SUMO1, SUMO E2 conjugating enzyme Ubc9, and the cell cycle regulators p53, mdm2, and p14(ARF) in 38 conventional renal cell carcinomas from Ukrainian patients with different degrees of radiation exposure after the Chernobyl accident. As control cases, 18 conventional renal carcinoma (cRCC) tissues from a Spanish cohort were analyzed. No significant differences between the Ukrainian and Spanish groups were found regarding Ub overexpression, although being higher in the Ukrainian cases. Furthermore, this expression was inversely associated with SUMO1 and Ubc9, with no correlation with tumor nuclear grade. There was also a direct relationship between Ubc9 and inflammatory response. These findings do not allow us to consider the immunohistochemical expression of ubiquitylation and sumoylation as valuable markers for discriminating the effects of long-term, low-dose IR exposure in cRCC carcinogenesis.

Urinary bladder carcinogenesis induced by chronic exposure to persistent low-dose ionizing radiation after Chernobyl accident.

Urinary bladder urothelium as well as cells in the microenvironment of lamina propria (endothelial elements, fibroblasts and lymphocytes) demonstrate a number of responses to chronic persistent long-term, low-dose ionizing radiation (IR). Thus, oxidative stress occurs, accompanied by up-regulation of at least two signaling pathways (p38 mitogen-activated protein kinase and nuclear factor-kappaB cascades) and activation of growth factor receptors, in the bladder urothelium of people living in Cesium 137-contaminated areas of Ukraine, resulting in chronic inflammation and the development of proliferative atypical cystitis, so-called Chernobyl cystitis, which is considered a possible pre-neoplastic condition in humans. Furthermore, significant alterations in regulation of cell cycle transitions are associated with increased cell proliferation, along with up-regulated ubiquitination and sumoylation processes as well as inefficient DNA repair (base and nucleotide excision repair pathways) in the affected urothelium. The microenvironmental changes induced by chronic long-term, low-dose IR also appear to promote angiogenesis and remodeling of the extracellular matrix that could facilitate invasion as well as progression of pre-existing initiated cells to malignancy. Based on the available findings, new strategies have been developed for predicting and treatment of Chernobyl cystitis-a first step in urinary bladder carcinogenesis in humans.

Upregulation of fibroblast growth factor receptor 3 and epidermal growth factor receptors, in association with Raf-1, in urothelial dysplasia and carcinoma in situ after the Chernobyl accident.

The present study was carried out in order to examine the molecular status of selected growth factor receptors (GFR) in urinary bladder lesions, recently described by our group as representing 'Chernobyl cystitis'. Fibroblast growth factor receptor 3 (FGFR3), epidermal growth factor receptor 1 (EGFR1), EGFR2neu (a member of the same family), p53 and Raf-1 serine/threonine kinase expression were evaluated immunohistochemically in urinary bladder biopsies from 22 men with benign prostate hyperplasia (group 1). For comparison, 16 men with benign prostate hyperplasia and five women with chronic cystitis living in non-radio-contaminated areas of the country were also investigated as controls (group 2). Additionally, 14 patients with dysplasia, carcinoma in situ (CIS) and primary urothelial carcinoma (UC) operated before the Chernobyl accident as well as 23 patients with UC living in the radio-contaminated areas were included as pre- and post-Chernobyl UC groups 1 and 2, respectively. Chronic proliferative atypical cystitis ('Chernobyl cystitis') was observed in group 1 patients. Foci of dysplasia and CIS were found in 22 (100%) and 19 (86%) of the 22 cases, respectively; moreover, two small UC were also detected. Elevated levels of FGFR3, EGFR2/neu, p53 and to a lesser extent EGFR1 and Raf-1 expression in the urothelial dysplasia and CIS were evident for patients of group 1. Statistically significant differences in immunohistochemical scores for FGFR3, EGFR1, p53 and Raf-1 were observed between groups 1 and 2 and between group 1 and the post-Chernobyl UC group 2, where a change in expression of EGFR2/neu was also noted. A significant decrease in FGFR3 expression in additional pre-Chernobyl UC group 1 with dysplasia, CIS and UC compared with group 1 Chernobyl cystitis cases was detected. Our findings suggest that FGFR and EGFR signaling pathways, associated with p53 and Raf-1 activation, may contribute to multistage urothelial carcinogenesis caused by irradiation, through autocrine or paracrine growth stimulation.

Molecular and immunohistochemical analysis of the prognostic value of cell-cycle regulators in urothelial neoplasms of the bladder.

OBJECTIVE: To evaluate the prognostic and predictive value of molecular and immunohistochemical markers related to cell-cycle control in terms of recurrence, progression, and survival in urothelial neoplasms of the bladder (UNB). PATIENTS AND METHODS: Clinical and pathological findings of 84 patients with UNB were assessed. Homozygous deletion (HD) and promoter methylation of p14ARF, p15INK4B, p16INK4A, loss of heterozygosity of the locus 9p21, p53 mutations, and immunohistochemical expression of p53, p16, p14, p21, p27, pRb, Ki67, MDM2, and cyclin D1 proteins were evaluated in relation to overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS). RESULTS: In the univariate analysis, RFS was shorter in cases with p14ARF (p=0.006), p15INK4B (p=0.003), p16INK4A (p=0.03) HD, low p14 immunoreactivity index (IRI) (p=0.01) and high Ki67 IRI (p=0.04); HD of the 9p21 locus genes and p14 IRI remained as independent prognostic factors for early UNB recurrence (p=0.006) whereas tumour stage (p=0.00001) and cyclin D1 IRI (p=0.049) were related to worse PFS in the multivariate analysis. In the univariate analysis, IRI for Ki67 (p=0.002), cyclin D1 (p=0.06), p53 (p=0.00008), p16 (p=0.02), p27 (p=0.0005) MDM2 (p=0.01) and p53 mutations (p=0.03) were related to poor OS, and only the Ki67 IRI retained their independent value in the multivariate analysis. CONCLUSION: 9p21 HD and p14 IRI constitute independent predictive factors for UNB recurrence and cyclin D1 IRI and tumour stage for progression. In addition, Ki67 IRI and tumour stage are independent prognostic factors for overall survival in UNB.

Extracellular matrix alterations in conventional renal cell carcinomas by tissue microarray profiling influenced by the persistent, long-term, low-dose ionizing radiation exposure in humans.

The present study was carried out in order to examine molecular alterations of extracellular matrix (ECM), associated with cell-cell communication in conventional (clear-cell) renal cell carcinomas (cRCCs) influenced by persistent long-term, low-dose ionizing radiation (IR) exposure to patients living more than 19 years after the Chernobyl accident in Cesium 137 (137Cs)-contaminated areas of Ukraine. The ECM major components such as fibronectin, laminin, E-cadherin/beta-catenin complexes and p53 tumor suppressor gene protein, and transforming growth factor beta 1 (TGF-beta1) were immunohistochemically (IHC) evaluated in cRCCs from 59 Ukrainian patients, which represented 18 patients living in non-contaminated areas and 41 patients from 137Cs-contaminated areas. In contrast, a control group of 19 Spanish patients with analogue tumors were also investigated. For IHC evaluation, a tissue microarray technique was used. Decrease or loss and abnormal distribution of fibronectin, laminin, E-cadherin/beta-catenin complexes accompanied by elevated levels of p53 and TGF-beta1 were detected in the Ukrainian cRCCs from 137Cs-contaminated areas with statistically significant differences. Thus, our study suggests that chronic long-term, low-dose IR exposure might result in global remodeling of ECM components of the cRCCs with disruption in peri-epithelial stroma and epithelial basement membranes.

Involvement of ubiquitination and sumoylation in bladder lesions induced by persistent long-term low dose ionizing radiation in humans.

PURPOSE: We determined whether ubiquitination and sumoylation processes are up-regulated in bladder urothelium by chronic, long-term, persistent low doses of ionizing radiation in male patients with benign prostate hyperplasia and females with chronic cystitis living more than 19 years in 137Cs contaminated areas after the Chernobyl accident in Ukraine. MATERIALS AND METHODS: Bladder urothelial biopsies from 45 patients were subjected to histopathological and immunohistochemical study of Ub, SUMO1, SUMO E2 conjugating enzyme Ubc9, and the cell cycle inhibitors p53 and p27(Kip1). RESULTS: Of 25 group 1 patients from radio contaminated areas chronic proliferative atypical cystitis (Chernobyl cystitis), featuring multiple foci of dysplasia, and carcinoma in situ were observed in 23 (92%) and 19 (76%), respectively, in addition to 1 small pTa grade 1 urothelial carcinoma. Chronic cystitis with areas of dysplasia and urothelial hyperplasia were detected in 2 (10%) and 3 (15%), respectively of the 20 patients in control group 2 from clean (without radio contamination) areas of Ukraine. Greatly increased levels of Ub, SUMO1, Ubc9 and p53 as well as decreased levels of p27(Kip1) were evident in patients in group 1 compared to those in group 2 (all p <0.001). CONCLUSIONS: These findings support the hypothesis that up-regulated ubiquitination and sumoylation processes might be an adaptive response to unscheduled proteolysis of aberrant p53 and p27(Kip1) cell cycle regulators occurring with long-term low dose rate ionizing radiation exposure with a possible contribution to urothelial carcinogenesis.

Aberrant expression of E-cadherin and beta-catenin in association with transforming growth factor-beta1 in urinary bladder lesions in humans after the Chernobyl accident.

This study examines the molecular pathways of cell-cell communication in chronic inflammatory processes associated with long-term low-dose urinary bladder exposure to ionizing radiation in people without major disease living more than 19 years in radio-contaminated areas of Ukraine after the Chernobyl accident. Patterns of components of the E-cadherin/beta-catenin complex, and transforming growth factor-beta1 (TGF-beta1) and inducible nitric oxide synthase (iNOS) expression were immunohistochemically evaluated in urinary bladder biopsies from 52 males with benign prostate hyperplasia and 8 females with chronic cystitis (group 1). For comparison, 25 males and 6 females living in non-contaminated areas of Ukraine were also investigated (group 2). Fourteen patients with primary urothelial carcinomas, which were operated on before the Chernobyl accident, were included as a carcinoma group. Chronic proliferative atypical cystitis ('Chernobyl cystitis') was observed in group 1 patients. Foci of dysplasia and carcinoma in situ were found in 51 (85%) and 34 (57%) of the 60 cases, respectively. Chronic cystitis with areas of dysplasia was detected in only 4 (13%) cases of 31 group 2 patients. Statistically significant differences in immunohistochemical scores for TGF-beta1 in the urothelium and lamina propria, iNOS in the urothelium and both beta-catenin and E-cadherin in the cytoplasm were observed between groups 1 and 2 with marked expression in group 1. Furthermore, TGF-beta1 overexpression and alteration in E-cadherin/beta-catenin complexes in bladder urothelium might play a crucial role in urinary bladder carcinogenesis in humans exposed to long-term low-dose ionizing radiation.

Alteration of Apoptotic Regulatory Molecules in Conventional Renal Cell Carcinoma Influenced by Chronic Long-term Low-dose Ionizing Radiation Exposure in Humans Revealed by Tissue Microarray.

Compared to the 19-year period subsequent to the Chernobyl accident, the morbidity of malignant renal tumors in the Ukraine has increased from 4.7 to 9.0 per 100,000 of the total population. Cesium 137 (137Cs), which accounts for 90% of the internal radioactivity in the Ukrainian population exposed to long-term low-dose radiation and 90% of the more labile pool of 137Cs, is excreted via the kidneys. Our present study aimed to evaluate the status of pro- and anti-apoptotic regulatory molecules in conventional renal cell carcinomas (cRCCs) in Ukrainian patients. To achieve this objective, Bcl-2, Bcl-x, BAX, death receptor (DR5) and transcriptional nuclear factor kappa B (NF-κB, with p50 and p65 subunits) were immunohistochemically investigated using a tissue microarray technique in cRCCs from a group of 56 Ukrainian patients, comprising 18 patients living in non-contaminated areas and 41 patients from 137Cs-contaminated areas. As a comparison, 19 Spanish patients with analogous tumors were also investigated. It was shown that BAX and DR5-positive cRCCs tended to increase among the Ukrainian patients living in the radio-contaminated areas, along with the suppression of anti-apoptotic molecules (Bcl-2 and Bcl-x) and with p65 and p50 overexpression in the same tumors. This study suggested that chronic long-term, low-dose radiation exposure might result in the alteration of the apoptotic regulatory mechanisms, which, in turn, could lead to enhanced tumor progression and resistance to apoptosis.

The INK4a /ARF locus: role in cell cycle control for renal cell epithelial tumor growth after the Chernobyl accident.

Previous studies have shown that during the period subsequent to the Chernobyl accident, increases in morbidity, aggressivity and proliferative activity of renal-cell carcinomas (RCCs) in Ukrainian patients were recognized. The present paper describes the molecular alterations of those tumor suppressor genes located on chromosome 9p21 ( INK4a/ARF locus and p15(INK4B)) in 26 primary renal-cell epithelial tumors from patients with different degrees of radiation exposure after the Chernobyl accident in Ukraine. Radiometric measurement of Cesium 137 ((137)Cs) was conducted with 1-day urine from all patients before surgery. Our results demonstrate that RCCs from patients living in the radio-contaminated areas showed aberrant hypermethylation of p14(ARF) and p16(INK4A) genes, associated with increased p38MAPK, p14(ARF), mdm2, cyclinD1 and Ki67 protein expression levels. Present findings show the possibility that chronic long-term low-dose radiation activates the INK4a/ARF locus, targeted by activation of the p38MAPK cascade. These actions could lead to disruptions and loss of cell cycle checkpoints and, thereby, to cellular transformation.

Possible distinct molecular carcinogenic pathways for bladder cancer in Ukraine, before and after the Chernobyl disaster.

After the Chernobyl accident in 1986, the incidence of urinary bladder cancers in the Ukraine increased gradually from 26.2 to 43.3 per 100,000 people between 1986 and 2001. In the areas of low level but persistent cesium-137 (137Cs) radio-contamination, a unique atypical radiation-related urinary bladder cystitis named 'Chernobyl cystitis', a possible pre-neoplastic condition in humans, has been detected. We have previously documented high incidences of bladder lesions, including severe dysplasias and/or carcinoma in situ, in association with this cystitis and correlating with oxidative DNA damage. To further investigate the molecular mechanisms underlying bladder carcinogenesis with this specific etiology, mutation analysis of p53 gene (exon 5-8) was performed for 11 and 18 paraffin-embedded bladder cancers in Ukrainians, respectively collected before and after the Chernobyl disaster. DNAs were extracted and subjected to nested PCR-single-strand conformational polymorphism analysis followed by direct DNA sequencing, as well as p53 immunohistochemistry (IHC). The incidences of p53 gene mutation were 54.5 and 16.7% for before and after the Chernobyl disaster, respectively, the difference being statistically significant. Also a tendency for higher p53 IHC score was apparent in the earlier group of lesions. No significant difference was noted for the proportions of historical types. These results point to possible distinct molecular carcinogenic pathways of bladder cancer formation, before and after the Chernobyl disaster, on the basis of variation in p53 gene alteration.

Urinary bladder lesions induced by persistent chronic low-dose ionizing radiation.

The incidence of urinary bladder cancer in the Ukraine increased from 26.2 to 43.3 per 100,000 population between 1986 and 2001 after the Chernobyl accident. The present study was conducted to evaluate the development of radiation-dependent lesions in the urinary bladders of people living in cesium 137 ((137)Cs) radio-contaminated areas of the Ukraine. Bladder urothelial biopsies from 159 male and 5 female patients were subjected to histological examination and immunohistochemical study of p38 mitogen-activated protein kinase (MAPK), as well as the p50 and p65 subunits of nuclear factor kappa B (NF-kappa B). A pattern of chronic proliferative atypical cystitis accompanied with large areas of sclerosis of connective tissue in the lamina propria was commonly observed in all cases. Interestingly, these lesions were associated with a dramatic increase in the incidences of dysplasia/carcinoma in situ, and, moreover, small urothelial carcinomas were incidentally detected. We defined the overall condition as "Chernobyl cystitis." Greatly elevated levels of p38, p65 and p50 expression in the urothelium were evident and the patients showed increased (137)Cs in urine. The data support conclusions from our previous studies of a critical role for increased oxidative stress in generation of urinary bladder urothelial lesions in individuals chronically exposed to low-dose (137)Cs radiation. Alterations in the p38 MAPK cascade and accumulation of NF-kappa B subunits could be crucial early molecular events in the pathogenesis of Chernobyl cystitis.

P16INK4A and p15INK4B gene alteration associated with oxidative stress in renal cell carcinomas after the chernobyl accident (pilot study).

Our study was undertaken to better understand the role of G1/S transition abnormalities in the malignant progression of renal cell carcinomas (RCCs), exposed to long-term low doses of ionizing radiation (IR), from patients living in radiocontaminated areas of the Ukraine after the Chernobyl accident. We studied p16 and p15 gene alteration in association with oxidative stress markers, including inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). We analyzed 88 samples collected from 22 patients with RCCs and with different exposure to IR. Homozygous deletion of the p16 and p15 genes, as well as hypermethylation of the 5CpG island in the promoter region of the same genes, were analyzed by differential PCR and Methylation-Specific PCR respectively, in association with histopathology and immunohistochemical analysis of p16 and p15 proteins. COX2 and iNOS expression in the same tumors were likewise analyzed. Aberrant hypermethylation was observed in 7 (32%) and 5 (23%) cases accompanied, by immunohistochemical loss of expression for p16 and p15 genes respectively, in both high stage and grade tumors from patients living in radiocontaminated areas, this being especially outstanding for the p16 gene. An association with COX2 and less iNOS overexpression in the same tumors was observed. Our data suggest that inactivation of p16 gene, but not p15, induced by increased oxidative stress generated by persistent chronic exposure to IR, could be one of the major pathways responsible for RCCs malignant progression.

DNA damage repair in bladder urothelium after the Chernobyl accident in Ukraine.

PURPOSE: We determined whether base and nucleotide excision repair is activated in bladder urothelium by chronic persistent low doses of ionizing radiation in male patients with benign prostate hyperplasia and females with chronic cystitis living more than 15 years in Cs contaminated areas after the Chernobyl accident in Ukraine. MATERIALS AND METHODS: Bladder urothelial biopsies from 204 patients were subjected to histological examination and biopsies from 35 were subjected to immunohistochemical study of 8-hydroxy-2'deoxyguanosine, 8-oxoguanine-DNA-glycosylase, apurinic/apyrimidinic endonuclease and xeroderma pigmentosum A endonuclease. RESULTS: Chronic proliferative atypical cystitis with multiple foci of dysplasia and carcinoma in situ were observed in 139 (89%) and in 91 (58%) of 156 group 1 patients from radio contaminated areas, respectively, as well as 10 small transitional cell carcinomas. Chronic cystitis with areas of dysplasia was detected in 9 of 48 patients (19%) in control group 2 from clean (without radio contamination) areas of Ukraine. Greatly elevated levels of 8-hydroxy-2'deoxyguanosine, 8-oxoguanine-DNA-glycosylase, apurinic/apyrimidinic endonuclease and xeroderma pigmentosum A were evident in the urothelium in group 1, accompanied by increased Cs in the urine. CONCLUSIONS: These findings support the hypothesis that significant activation of DNA damage repair (base and nucleotide excision repair) is induced by the oxidative stress generated by long-term low doses of ionizing radiation. The levels of DNA oxidative adducts pointing to mutagenic and carcinogenic potential were in line with the histopathologically diagnosed urothelial lesions.