DNA damage in cytologically normal urothelial cells of patients with a history of urothelial cell carcinoma

Abstract

In order to determine if patients with a history of previous urothelial cell carcinoma (UCC) but with current normal urinary cytology have DNA damage in urothelial cells, the single-cell gel electrophoresis (comet) assay was conducted with cells obtained by urinary bladder washings from 44 patients (28 with a history of previous UCC). Increased DNA damage was observed in cytologically "normal" urothelial cells of patients with a history of UCC when compared with referents with no similar history and after correcting the data for smoking status and age (P < 0.018). Increased DNA damage also correlated with the highest tumor grade, irrespective of time or course of the disease after clinical intervention (Kendall tau correlation, 0.37, P = 0.016). Moreover, aneuploidy, as assessed by DNA content ratio (DCR; 75th/25th percentile of total DNA fluorescence of 50 comets/patient) was unaltered by smoking status, but increased with UCC grade: 1.39 +/- 0.12 (median +/- 95% confidence interval; referents); 1.43 +/- 0.11 (Grade I UCC; P = 0.264, against referents); 1.49 +/- 0.16 (Grade II UCC; P = 0.057); 1.57 +/- 0.16 (Grade III UCC; P = 0.003). Micronucleated urothelial cells (MNC) were also scored on Giemsa-stained routine cytological smears and were found not to correlate with DNA damage or DCR. MNC frequencies were higher for patients with a history of UCC and/or smoking than referents with neither history, but there was no statistical difference between groups. Taken together, these results suggest that the normal-appearing urothelium of patients resected for UCC still harbor genetically unstable cells.

Genetic Instability Persists in Non-Neoplastic Urothelial Cells from Patients with a History of Urothelial Cell Carcinoma

“…These findings strengthen the notion that MN scoring could be used in cytopathological analyses to assist in the proper diagnosis of these cases [10]. We have previously shown that genetic instability could be detected in urothelial cells, even before cellular atypia was detectable by cytology [13]. In this previous study, increased MNC frequencies were observed in normal urothelial cells obtained from bladder washings of patients with a history of UCC and/or smoking, but this increase was not statistically significant when compared to urothelial cells from a control population, which was likely due to the relatively small sample size [13].…”

“…The micronuclei (MN) frequency is a widely used biomarker for chromosome instability [8]–[13]. MN are chromosome fragments or whole chromosomes that are not incorporated into daughter nuclei during cell division [8], and the frequency of micronucleated cells (MNC) may predict cancer risk, suggesting that increased MN formation may be associated with early carcinogenic events [9].…”

“…We have previously shown that genetic instability could be detected in urothelial cells, even before cellular atypia was detectable by cytology [13]. In this previous study, increased MNC frequencies were observed in normal urothelial cells obtained from bladder washings of patients with a history of UCC and/or smoking, but this increase was not statistically significant when compared to urothelial cells from a control population, which was likely due to the relatively small sample size [13]. This finding, together with recent data from Arora et al [10], prompted us to investigate the utility of a MN assay to detect genetic instability in urothelial cells with normal cytology in a new and larger cohort of patients with recurrent UCC.…”

Acute Bacterial Cystitis Does not Cause Deoxyribonucleic Acid Damage Detectable by the Alkaline Comet Assay in Urothelial Cells of Dogs

“…The liquid obtained from the bladder washing (total of 15 ml) was divided into two aliquots: one (5 ml) was used for routine urinary cytology and other for the comet assay. 3,12 Cell viability was evaluated by trypan blue exclusion. After centrifugation of the second aliquot (for comet assay), the cell pellet (approximately 5 ϫ 10 5 cells) was embedded in low-melting point agarose (0.5%), layered onto a microscope slide precoated with agarose, allowed to solidify at 4 C, and lysed for 24 hour at 4 C in a solution containing 2.5 M NaCl, 100 mM Na 2 ethylenediaminetetraacetic acid, 10 mM Tris, pH 10, 2% sodium sarcosinate with 1% Triton X-100, and 10% dimethyl sulfoxide added just before use.…”

“…5 Increased DNA damage, as detected by the comet assay, has been shown in the urothelial cells of human patients at increased risk for urothelial cell carcinoma, such as smokers 5 and patients with a history of urothelial cell carcinoma but with normal-looking cytology. 3 We are unaware of prospective studies in the literature evaluating primary DNA damage as a potential risk factor for cancer. The biologic significance of primary DNA damage in viable cells is the risk for mutation accumulation and its relationship with genomic instability in vitro 6 and in vivo.…”