[Case of a Plasmacytoid Urothelial Carcinoma Identified Due to the Hardening of the Abdominal Wall].

The patient was a 75 year-old male. Noticing areas of hardening in the lower abdomen, and consequently feelings of systemic fatigue and difficulty in walking, the patient visited a clinic and was diagnosed with kidney failure prior to the visit to our clinic. Computed tomography and magnetic resonance imaging showed thickness of the rectus abdominis muscle and the bladder wall, and bilateral hydronephrosis was also identified. As no explicit tumor was identified in the bladder, the patient underwent biopsies of the abdominal wall and bladder membrane mucous, and was diagnosed with a plasmacytoid urothelial carcinoma primarily developed in the bladder. The patient displayed a poor general state of health and died five months after the diagnosis. It is known that plasmacytoid urothelial carcinomas progress rapidly and the prognosis is poorer than for the micropapillary variant. It is important to obtain a tissue specimen in the early stage of this disease because there are cases in which no explicit tumor can be identified. Furthermore, the value of carbohydrate antigen (CA) 19-9 of the patient was much higher than would be expected as normal at the first visit. It kept rising during the follow-up and was useful as a marker to indicate the progress of the disease.

[Renal arteriovenous fistula (AVF) following nephrectomy : a case of arterial embolization treatment].

The patient was a 78-year-old female. At the age of 76, the patient had undergone computed tomographic scanning for left lower abdominal pain. A retroperitoneal mass was detected on the right side of the abdomen. The patient had been subject to right nephrectomy due to renal calculus at the age of 33. With Doppler echocardiography and magnetic resonance imaging (MRI), based on the nephrectomy the patient was diagnosed with a renal arteriovenous fistula developed in the right renal bed. Following the wishes of the patient a follow-up examination was conducted. Because of poor heart condition and renal dysfunction, percutaneous arterial embolization was performed. After the embolization, the heart condition and renal dysfunction indicators improved and the blood flow in the arteriovenous fistula disappeared. Cases of renal arteriovenous fistulae after nephrectomy are rare, with only 90 reported worldwide, and percutaneous arterial embolization has been used as the first choice of treatment in recent years.

Restoring erectile function by antioxidant therapy in diabetic rats.

PURPOSE: Diabetes mediates an increase in reactive oxygen species that can lead to impaired endothelial function, decreased smooth muscle in the diabetic corpus cavernosum and increased apoptosis. We hypothesized that antioxidant therapy may restore erectile function by inhibiting apoptosis in diabetic rat crura. MATERIALS AND METHODS: A total of 40 male Sprague-Dawley rats were randomized to 5 groups of 8 each, including healthy controls, rats with diabetes, and rats with diabetes with the antioxidant tempol (4-hydroxytetramethyl-piperidine-1-oxyl) (Sigma-Aldrich), with insulin, and with tempol and insulin. Intracavernous pressure was measured for functional analysis. Smooth muscle and collagen fiber levels in the rat penile corpus cavernosum were assessed by hematoxylin and eosin, and Masson's trichrome staining. Endothelial cells were assessed by CD31 staining. Reactive oxygen species related genes were analyzed by cDNA microarray. We confirmed mRNA and protein expression profiles for these genes in diabetic and treated rats using real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. TUNEL assay was done to analyze apoptosis status. RESULTS: Intracavernous pressure in diabetic rats was significantly decreased vs controls. After treatment with tempol or insulin alone intracavernous pressure was significantly increased compared to that in untreated diabetic rats. In the diabetic group mean smooth muscle area significantly decreased but was restored after combined tempol and insulin. Endothelial cell area in diabetic rats significantly decreased and was not restored by any treatments. However, apoptosis was restored to normal by combined insulin and tempol. Of 84 reactive oxidative stress and antioxidant genes 32 were identified specific to diabetic rats compared to healthy controls. UCP3 expression was significantly increased in diabetic rats and normal levels were restored by all treatments. CONCLUSIONS: To our knowledge this is the first report that tempol and insulin can restore erectile function in diabetic rats by inhibiting apoptosis.

Validation study of a clinical diagnosis support tool for lumbar spinal stenosis.

BACKGROUND: A clinical diagnosis support tool for lumbar spinal stenosis was developed by the Japanese Society for Spine Surgery and Related Research. However, the use of this tool has not yet been validated. METHODS: Patients with symptoms in the lower extremities and who visited the Department of Orthopedics initially were recruited to the study. Orthopedic physicians who were not spine specialists completed the support tools. Spine specialists examined the patients, made a diagnosis, and completed the lumbar spine examination sheet made for the study. The support tool and lumbar spine examination sheet were sent to a central panel comprising four panelists who then decided on a final diagnosis. RESULTS: In total, 118 patients were evaluated, including 62 males and 56 females. Lumbar spinal stenosis was diagnosed in 58 and nonlumbar spinal stenosis in 60 patients. The mean score in the lumbar spinal stenosis group was 12.2 points (median 13 points). In the nonlumbar spinal stenosis group, the mean score was 7.5 points (median 7 points). Sensitivity was 0.948, and specificity was 0.40. CONCLUSIONS: Patients with lumbar spinal stenosis with a very low score were diagnosed with mild lumbar spinal stenosis, whereas nonlumbar spinal stenosis patients with a very high score were diagnosed as suffering from spine disease and needing special treatment by spine surgeons. Our results validate the use of the support tool for the diagnosis of lumbar spinal stenosis. Although the specificity observed in the present study was lower than that reported at development, we conclude that this support tool is useful for screening patients with lumbar spinal stenosis.

Epigenetic modifications of RASSF1A gene through chromatin remodeling in prostate cancer.

PURPOSE: The RAS-association domain family 1, isoform A (RASSF1A) gene is shown to be inactivated in prostate cancers. However, the molecular mechanism of silencing of the RASSFIA gene is not fully understood. The present study was designed to investigate the mechanisms of inactivation of the RASSF1A gene through the analysis of CpG methylation and histone acetylation and H3 methylation associated with the RASSF1A promoter region. EXPERIMENTAL DESIGN: Methylation status of the RASSF1A gene was analyzed in 131 samples of prostate cancer, 65 samples of benign prostate hypertrophy (BPH), and human prostate cell lines using methylation-specific PCR. Histone acetylation (acetyl-H3, acetyl-H4) and H3 methylation (dimethyl-H3-K4, dimethyl-H3-K9) status associated with the promoter region in prostate cells were analyzed by chromatin immunoprecipitation (ChIP) assay. RESULTS: Aberrant methylation was detected in 97 (74.0%) prostate cancer samples and 12 (18.5%) BPH samples. The methylation frequency of RASSF1A showed a significant increase with high Gleason sum and high stage. The ChIP assays showed enhancement of histone acetylation and dimethyl-H3-K4 methylation on the unmethylated RASSF1A promoter. TSA alone was unable to alter key components of the histone code. However, after 5-aza-2'-deoxy-cytidine treatment, there was a complete reversal of the histone components in the hypermethylated promoter. Levels of acetyl-H3, acetyl-H4, and dimethyl-H3-K4 became more enriched, whereas H3K9me2 levels were severely depleted. CONCLUSIONS: This is the first report suggesting that reduced histone acetylation or H3K4me2 methylation and increased dimethyl-H3-K9 methylation play a critical role in the maintenance of promoter DNA methylation-associated RASSF1A gene silencing in prostate cancer.

Wnt antagonist family genes as biomarkers for diagnosis, staging, and prognosis of renal cell carcinoma using tumor and serum DNA.

PURPOSE: We hypothesized that combined methylation analysis of Wnt antagonist genes could serve as a panel of biomarkers for diagnosis, staging, and prognosis in renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: Samples (n = 62) of RCC and corresponding normal renal tissue (NRT) were analyzed using methylation-specific PCR for methylation of six Wnt antagonist genes (sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1, and Dkk-3). To increase the sensitivity/specificity of RCC detection, the methylation score (M score) for multigene methylation analysis was developed. Receiver operator characteristic curve analysis was used to determine the optimal sensitivity/specificity of the M score. In addition, the M score was compared with the clinicopathologic outcome. Thirty-three serum DNA samples were also used to investigate the methylation status of Wnt antagonist genes. RESULTS: The methylation levels of all Wnt antagonists were significantly higher in RCC than in NRT. In multivariate regression analysis, the methylation level of sFRP-1 was a significant independent predictor of RCC, whereas for sFRP-2 and sFRP-4 there was a trend toward significance as independent predictors. The M score of Wnt antagonist genes was significantly higher in RCC than in NRT. Overall, the M score had a sensitivity of 79.0% and a specificity of 75.8% (area under the curve, 0.808) as a diagnostic biomarker. In addition, the M score could significantly distinguish grade, pT category, M category, and overall survival of RCC patients. The M score was independent of age and gender in predicting overall survival by the Cox proportional hazards model. In RCC patients, 72.7% of the methylation-specific PCR results had identical methylation in samples of tumor and serum DNA. No serum DNA in normal controls showed aberrant methylation of the Wnt antagonist genes. In addition, the methylation status of Wnt antagonist genes in serum DNA was significantly correlated with tumor grade and stage. CONCLUSIONS: This is the first report showing that M score analysis of Wnt antagonist genes can serve as an excellent epigenetic biomarker panel for detection, staging, and prognosis of RCC using serum DNA.

Combination analysis of hypermethylated Wnt-antagonist family genes as a novel epigenetic biomarker panel for bladder cancer detection.

PURPOSE: Aberrant promoter hypermethylation of Wnt-antagonist genes contributes to the pathogenesis of several cancers. We hypothesized that combined methylation analysis of Wnt-antagonist genes could improve their use as a panel of biomarkers for diagnosing and staging of bladder cancers. EXPERIMENTAL DESIGN: Samples (54 total) of bladder tumor and corresponding normal bladder mucosa were analyzed for the methylation and expression levels of six Wnt-antagonist genes (sFRP-1, sFRP-2, sFRP-4, and sFRP-5, Wif-1, and Dkk-3). To increase the sensitivity/specificity of bladder tumor detection, the methylation score (M score), a new method for multigene methylation analysis, was developed. The M score of each sample was calculated as the sum of the corresponding log hazard ratio coefficients derived from multivariate logistic regression analysis of the methylation status for each Wnt-antagonist gene. Receiver operator characteristic (ROC) curve analysis was used to determine the optimal sensitivity/specificity of the M score. Urine DNA from 24 matched patients with bladder tumor and 20 cancer-free volunteers was also used to investigate the methylation status of Wnt-antagonist genes. RESULTS: The methylation levels of Wnt-antagonists were significantly higher and mRNA levels were significantly lower in bladder tumor than in bladder mucosa. Each methylation level was inversely correlated with the corresponding mRNA level. In multivariate regression analysis, the methylation levels of sFRP-2 and Dkk-3 were significant independent predictors of bladder tumor (P < 0.05 and P < 0.01, respectively), whereas with sFRP-1, sFRP-5, and Wif-1 there was a trend towards significance as independent predictors. The M score of Wnt-antagonist genes was significantly higher in bladder tumor than in bladder mucosa (P < 0.05). Overall, the M score had a sensitivity of 77.2% and a specificity of 66.7% as a diagnostic biomarker (areas under the curve, 0.763). The M score could distinguish superficial from invasive bladder tumors with a sensitivity of 72.2% and a specificity of 61.1% as a staging biomarker (areas under the curve, 0.671). In patients with bladder tumor, 80.6% of the methylation-specific PCR results had identical methylation in samples of tumor- and urine-derived DNA. Most urine DNA in normal controls showed no aberrant methylation of the Wnt-antagonist genes. CONCLUSIONS: Hypermethylation of Wnt-antagonist genes plays an important role in the pathogenesis of bladder tumor and can be detected using cellular DNA extracted from urine samples. This is the first report demonstrating that M score analysis of Wnt-antagonist genes could serve as an excellent epigenetic biomarker panel for bladder tumors.

Epigenetic inactivation of Wnt inhibitory factor-1 plays an important role in bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway.

PURPOSE: Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the pathogenesis of several human cancers. Wnt inhibitory factor-1 (Wif-1) was identified as one of the secreted antagonists that can bind Wnt protein. We hypothesize that Wif-1 plays an important role in bladder cancer pathogenesis. EXPERIMENTAL DESIGN: To test this hypothesis, epigenetic and genetic pathways involved in the Wif-1 gene modulation and expression of Wnt/beta-catenin-related genes were analyzed in 4 bladder tumor cell lines and 54 bladder tumor and matched normal bladder mucosa. RESULTS: Wif-1 mRNA expression was significantly enhanced after 5-aza-2'-deoxycytidine treatment in bladder tumor cell lines. Wif-1 promoter methylation level was significantly higher and Wif-1 mRNA expression was significantly lower in bladder tumor samples than in bladder mucosa samples. In the total bladder tumor and bladder mucosa samples, an inverse correlation was found between promoter methylation and Wif-1 mRNA transcript levels. However, loss-of-heterozygosity at chromosome 12q14.3 close to the Wif-1 gene loci was a rare event (3.7%). Nuclear accumulation of beta-catenin was significantly more frequent in bladder tumor than in bladder mucosa and inversely correlated with Wif-1 expression. In addition, known targets of the canonical Wnt/beta-catenin signaling pathway, such as c-myc and cyclin D1, were up-regulated in bladder tumor compared with bladder mucosa, and this up-regulation was associated with reduced Wif-1 expression at both mRNA and protein levels. Furthermore, transfection of Wif-1 small interfering RNA into bladder tumor cells expressing Wif-1 mRNA transcripts had increased levels of c-myc and cyclin D1 and accelerated cell growth. CONCLUSION: This is the first report showing that CpG hypermethylation of the Wif-1 promoter is a frequent event in bladder tumor and may contribute to pathogenesis of bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway. The present study elucidates novel pathways that are involved in the pathogenesis of bladder cancer.

Promoter CpG hypomethylation and transcription factor EGR1 hyperactivate heparanase expression in bladder cancer.

Heparanase plays a critical role in the degradation of extracellular matrix and cell membrane and is frequently upregulated in malignant tumors. Transcription factor, early growth response 1 (EGR1), is closely associated with inducible transcription of the heparanase gene. We hypothesized that promoter CpG hypomethylation with increased EGR1 expression could determine heparanase expression during the pathogenesis of bladder cancer. Bladder cancer cell lines (J82, T24 and transitional cell carcinoma) significantly restored heparanase expression after 5-Aza-dC treatment. Transfection of EGR1 siRNA with T24 bladder cancer cell line significantly downregulated heparanase expression compared to the control siRNA transfection. In 54 bladder cancer and paired normal bladder samples, heparanase expression was significantly higher in bladder cancer than in normal bladder (P<0.01). We performed methylation-specific PCR targeting the CpG sites within the core-binding consensus motifs of EGR1 (GGCG) and Sp1 (GGGCGG). Methylation prevalence was significantly higher in normal bladder than in bladder cancer (P<0.05) and inversely correlated with heparanase expression (P=0.055). In the total series of bladder cancer and normal bladder samples, the combination of promoter CpG methylation and EGR1 expression regulated heparanase expression in a stepwise manner, where heparanase expression was the lowest in methylation-positive and EGR1-negative samples and the highest in methylation-negative and EGR1-positive samples. To our knowledge, this is the first study demonstrating that increased heparanase expression during the pathogenesis of bladder cancer is due to promoter hypomethylation and transcription factor EGR1.

Catechol-O-methyltransferase gene polymorphisms in benign prostatic hyperplasia and sporadic prostate cancer.

Various carcinogenic metabolites, including catechol estrogens, play a role in malignant transformation. An enzyme that is capable of neutralizing the genotoxic effects of these compounds is catechol-O-methyltransferase (COMT). A variant form of this enzyme has been shown to reduce its activity by up to 4-fold; thus, we hypothesize that single nucleotide polymorphisms of the COMT gene can be a risk factor for benign prostatic hyperplasia (BPH) and prostate cancer. To test this hypothesis, the genetic distribution of three different COMT polymorphisms at codon 62 (C-->T), codon 72 (G-->T), and codon 158 (G-->A) were analyzed in 131 normal healthy subjects, 134 BPH, and 178 sporadic prostate cancer samples from a Japanese population. Results of these experiments show that the variant genotype at codon 62 (P = 0.060) and codon 158 (P = 0.047) are risk factors for prostate cancer but not BPH when compared with normal controls. Odds ratio (OR) and 95% confidence interval (95% CI) for cancer were 3.24 and 1.38 to 7.61, respectively, for codon 62 T/T genotype when compared with wild type. At codon 158, the A/A variant for cancer had an OR of 3.00 with a 95% CI of 1.38 to 6.54 compared with wild type. Codons 62 and 158 were in linkage disequilibrium (LD), and when compared with the C-G haplotype, other types (C-A, T-G, T-A) were observed to be associated with prostate cancer (P = 0.040) but not BPH. Codon 72 on the other hand, was not in LD with either codon 62 or 158. The homozygous variant on codon 72 was rare in this Japanese population, and the heterozygous G/T at this codon was not associated with either prostate cancer or BPH. When evaluating the risk of COMT polymorphisms with stage or grade of cancer, no associations were observed for any of the genotypes with the exception of a tendency (P = 0.096) for the variant A allele on codon 158 to be correlated with higher stages (> or = T3) of cancer. This is the first report that shows the polymorphisms of COMT to be associated with sporadic prostatic carcinogenesis. These results are important in understanding the role of COMT polymorphisms in the pathogenesis of prostate cancer.

Increased heparanase expression is caused by promoter hypomethylation and up-regulation of transcriptional factor early growth response-1 in human prostate cancer.

PURPOSE: Heparanase degrades heparan sulfate and has been implicated in tumor invasion and metastasis. The transcription factor, early growth response 1 (EGR1), is associated with the inducible transcription of the heparanase gene. We hypothesize that CpG hypomethylation in the heparanase promoter coupled with up-regulation of EGR1 levels may induce heparanase expression in human prostate cancer. EXPERIMENTAL DESIGN: Cultured prostate cancer cell lines (Du145, DuPro, LNCaP, and PC-3) with and without 5'-aza-2-deoxycytidine treatment, 177 prostate cancer samples, and 69 benign prostatic hyperplasia (BPH) samples were used. The frequency and level of heparanase promoter methylation were analyzed by methylation-specific primers which covered the core binding motif of EGR1 (GGCG) or SP1 (GGGCGG) or both. RESULTS: In cultured Du145, DuPro, LNCaP, and PC-3 cell lines, mRNA transcripts of heparanase were significantly increased after 5'-aza-2-deoxycytidine treatment, suggesting that promoter methylation was involved in the regulation of heparanase mRNA transcript. Significantly higher methylation was found in BPH samples than in prostate cancer samples (P < 0.0001), whereas mRNA transcripts of the heparanase gene were inversely lower in BPH samples than in prostate cancer samples (P < 0.01). EGR1 expression in prostate cancer tissues was significantly higher than in BPH tissues (P < 0.001) and correlated with heparanase expression (P < 0.0001). Moreover, multiple regression analysis revealed that up-regulation of EGR1 contributed significantly more to heparanase expression than did promoter CpG hypomethylation in prostate cancer samples (P < 0.0001). CONCLUSIONS: To our knowledge this is the first comprehensive study demonstrating that increased heparanase expression in prostate cancer tissues is due to promoter hypomethylation and up-regulation of transcription factor EGR1.

Cytochrome P450 1B1 is overexpressed and regulated by hypomethylation in prostate cancer.

PURPOSE: Cytochrome P450 1B1 (CYP1B1), a dioxin inducible member of the CYP supergene family, is overexpressed in various human malignancies including prostate cancer. We hypothesized that promoter/enhancer CpG methylation contributes to the regulation of CYP1B1 expression in human prostate tissue. EXPERIMENTAL DESIGN: Expression and induction of the CYP1B1 gene in clinical prostate tissues and prostate cancer cell lines were investigated. The methylation status of the CYP1B1 gene was analyzed in 175 prostate cancer and 96 benign prostatic hyperplasia samples using methylation-specific PCR (MSP) and bisulfite-modified DNA sequencing. MSP primers covered dioxin response elements (DRE) and Sp1 sites that are important for the expression of CYP1B1. RESULTS: Expressions of CYP1B1 mRNA and protein were increased in prostate cancer. The aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) heterodimer complex activates gene transcription by binding to the DREs of CYP1B1. In prostate cancer cells, CYP1B1 mRNA was induced by 2,3,7,8-tetrachlorodigenzo-p-dioxin (TCDD) and/or demethylation agent (5-aza-2-deoxycytidine). There was no change in the expressions of AhR and ARNT. Methylation of promoter/enhancer regions was significantly higher in benign prostatic hyperplasia compared with prostate cancer. MSP-positive patients had significantly lower risk for prostate cancer as compared with MSP-negative patients. There was no correlation between CYP1B1 methylation status and clinicopathologic features. CONCLUSIONS: CYP1B1 is overexpressed in prostate cancer and regulated by hypomethylation of its promoter/enhancer region. This is the first report about CYP1B1 regulation in human clinical prostate samples showing that hypomethylation of the CYP1B1 gene may play an important role in prostate cancer.

CpG hypermethylation of MDR1 gene contributes to the pathogenesis and progression of human prostate cancer.

Multidrug resistance 1 (MDR1) gene encodes for P-glycoprotein (P-gp), a Mr 170,000 transmembrane calcium-dependent efflux pump that is inactivated in prostate cancer. We hypothesize that inactivation of the MDR1 gene through CpG methylation contributes to the pathogenesis and progression of prostate cancer. To test this hypothesis, CpG methylation status of the MDR1 promoter and its correlation with clinicopathological findings were evaluated in 177 prostate cancer samples and 69 benign prostate hypertrophy (BPH) samples. Cellular proliferation index and apoptotic index were determined by proliferating cell nuclear antigen (PCNA) and single-strand DNA immunostaining, respectively. After 5-aza-2'-deoxycytidine treatment, increased expression of MDR1 mRNA transcript was found in prostate cancer cell lines (DU145, DuPro, and ND1). MDR1 methylation frequency was significantly higher in prostate cancer samples compared with BPH samples (54.8 versus 11.6%, respectively, P < 0.001). Logistic regression analysis revealed that PC patients are 11.5 times more likely to have MDR1 methylation than BPH patients (95% confidence interval 4.87-27.0) and that MDR1 methylation is independent of the age. Significant correlation of MDR1 methylation was observed with high pT category (P < 0.001), high Gleason sum (P = 0.008), high preoperative prostate-specific antigen (P = 0.01), and advancing pathological features. In addition, PCNA-labeling index were significantly higher in methylation-specific PCR (MSP)-positive than in MSP-negative prostate cancer samples (P = 0.048). In contrast, no significant difference in apoptotic index was found between MSP-positive and -negative prostate cancer samples. These findings suggest that CpG hypermethylation of MDR1 promoter is a frequent event in prostate cancer and is related to disease progression via increased cell proliferation in prostate cancer cells.

Vascular endothelial growth factor restores erectile function through modulation of the insulin-like growth factor system and sex hormone receptors in diabetic rat.

Previous studies have shown that intracavernous injection of vascular endothelial growth factor (VEGF) restored erectile function in diabetic rats. However, the mechanism of VEGF in diabetes-related erectile dysfunction (ED) has not been fully investigated. We hypothesize that intracavernous injection of VEGF may reverse diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To test this hypothesis the erectile function of treated and control rats was analyzed by measurement of intracavernous pressure (ICP) following electrostimulation of the cavernous nerves. Mean ICP was significantly lower in non-treated diabetic rats compared to controls. After VEGF injection, ICP was significantly higher than in non-treated diabetic rats. IGFBP-3 mRNA and protein expression was significantly higher in non-treated diabetic rat crura than controls, while VEGF-treated animals had control levels. ER-beta and PR mRNA and protein expression was significantly lower in non-treated diabetic rat crura. After VEGF injection, ER-beta and PR mRNA and protein expression was similar to control levels. Expression of AR and ER-alpha was the same in all groups. These findings suggest that orthotopic injection of VEGF may improve the functional recovery of diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To our knowledge, this is the first study demonstrating that VEGF treatment restores erectile function through restoration of the insulin-like growth factor system and sex hormone receptor genes at the mRNA and protein levels in diabetic rat crura. These results may be important in understanding the pathogenesis of diabetes-related ED and also in providing better strategies for management of this disease.

Polymorphisms of DNA repair genes are associated with renal cell carcinoma.

DNA repair gene alterations have been shown to cause a reduction in DNA repair capacity and may influence an individual's susceptibility to carcinogenesis. Single nucleotide polymorphisms (SNPs) of DNA repair genes have been shown to cause a reduction in repair activity. We hypothesized that SNPs of DNA repair genes may be a risk factor for renal cell carcinoma (RCC). To test this hypothesis, DNA samples from 112 cases of renal cell cancer and healthy controls (n=180) were analyzed by PCR-RFLP to determine the genotypic frequency of six different polymorphic loci on five DNA repair genes (XRCC1, XPC, ERCC1, XRCC3, and XRCC7). The chi(2) test was applied to compare the genotype frequency between patients and controls. We found that the frequency of 399Gln variant at XRCC1 Arg399Gln was significantly higher in RCC cases than in controls (OR=2.83, 95%CI=1.24-6.49, P=0.01). The frequency of T-A haplotype of XRCC1 194 Trp and XRCC1 399Gln was significantly higher in RCC than controls. No differences in genotypes were observed at the other sites. This is the first report on SNPs of DNA repair genes in renal cell carcinoma that suggests XRCC1 399Gln polymorphism may be a risk factor for RCC. Our present data suggest that the XRCC1 399Gln allele may be linked to susceptibility for RCC.

Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation.

Methylation-specific PCR (MSP) is frequently used to distinguish methylated alleles in the genome. Sequences that have been incompletely converted during bisulfite treatment are frequently co-amplified during MSP. For accurate MSP, it is important to detect methylated sequences in a background of unconverted DNA with a high level of sensitivity. We report here sensitive techniques, bisulfite conversion-specific MSP (BS-MSP) to accurately evaluate CpG methylation. BS-MSP provides accurate results across a wide spectrum of bisulfite conversion levels. BS-MSP is also confirmed to be a useful technique for the routine analysis of clinical tumor specimens that were paraffin-embedded and microdissected. BS-MSP thus provides the powerful features of ease of use and compatibility with paraffin sections. We recommend that methylation analysis should include a step to eliminate unconverted DNA to avoid overestimation of the DNA methylation level in the samples.

Inactivation of the hMSH3 mismatch repair gene in bladder cancer.

Deficiency in the DNA mismatch repair (MMR) is frequently involved in various cancers. The hMSH3 gene is one of the human MMR genes whose role in bladder cancer is not known. We hypothesized that down-regulation of the hMSH3 gene might be involved in bladder cancer. In this study we analyzed this gene with regard to frame-shift mutation, single nucleotide polymorphism (SNP), a 9bp repeat in exon 1, loss of heterozygosity (LOH), immunohistochemistry, and methylation status in 102 bladder cancer samples. Immunohistochemistry revealed that hMSH3 expression in bladder cancer was significant decreased compared to normal epithelium (p<0.0001). An inverse correlation with pathological grade was found. The frame-shift mutation in the (A) 8 tract was lacking in bladder cancer. There was no significantly difference between bladder cancer samples and healthy controls' with regard to SNP and the 9bp repeat. In bladder cancer, presence of the codon 222 polymorphism, LOH, and the 9bp repeats in exon 1 had a correlation with either pathological stage or pathological grade. Presence of the codon 1036 polymorphism had significant correlation with pathological stage and a trend to correlation with pathological grade. After 5-aza-dC treatment, MSH3 expression was significantly enhanced in TCC and UMUC bladder cancer cells when compared to untreated cells. This is the first report suggesting that genetic and epigenetic alterations in the human MSH3 gene might play a significant role in the progression of bladder tumors.