N7-methyldeoxyguanosine levels in DNA isolated from cervical cytology samples are associated with smoking

Smoking has been associated, in epidemiological studies, with an increased risk of cervical neoplasia. This may be in part due to the presence of tobacco-specific nitrosamines in cervical mucous of smokers, which may result in carcinogenic DNA damage. We have thus examined whether cervical DNA contains alkylation damage arising from exposure to methylating agents (N7-methyldeoxyguanosine, N7-MedG). DNA was extracted from cervical cytology samples and N7-MedG levels were measured using an immunoslotblot assay. Ninety percentage of the DNA samples were alkylated and N7-MedG levels (mean, 95% CI) in ever-smokers (1.27, 0.90-1.81 micromol/mol dG) were significantly higher than those in nonsmokers (0.42, 0.20-0.91 micromol/mol dG: p = 0.005). N7-MedG adduct levels were significantly correlated with number of cigarettes smoked per day and pack years of cigarette smoking in current smokers. There was no association with N7-MedG levels and cervical intraepithelial neoplasia status, age, parity or contraception use. Our study suggests that cervical DNA contains alkylation damage that can arise from exposure to cigarette smoke.     

Immunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues.

Three different activated supports were used to immobilize alpha-O6-methyldeoxyguanosine (O6-MedG) monoclonal antibody. Affinity gels based on CNBr Sepharose, Affigel Hz and periodate-activated Sepharose (PIAS) were able to bind 1.4, 1.5 and 6.2 nmol [3H]O6-MedG/mg immobilized IgG respectively, and recovery of bound material exceeded 95% in all cases. Significant non-specific binding of normal nucleosides occurred only when using the CNBr gel. PIAS alpha-O6-MedG affinity gel was able to purify O6-MedG-3'-monophosphate from digested synthetic oligonucleotides and in vitro-methylated calf thymus DNA to allow subsequent detection by 32P-postlabelling and two-dimensional TLC. The combined method was applied to three human samples and O6-MedG levels of 0.39, 0.38 and 0.45 mumol/mol 2'-deoxyguanosine were found. The minimum detection limit of the combined method is expected to be approximately 1 O6-MedG in 10(8) normal 2'-deoxyguanosines (i.e. approximately 30 lesions per human cell) when 100 micrograms of DNA is used.     

Formation and loss of O6-methyldeoxyguanosine in human leucocyte DNA following sequential DTIC and fotemustine chemotherapy.

There is increasing evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain anti-tumour alkylating agents and that the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide inter-individual variation in the depletion and subsequent regeneration of ATase in human peripheral blood lymphocytes following sequential DTIC (400 mg m-2) and fotemustine (100 mg m-2) treatment, with the nadir ATase activity occurring approximately 4 h after DTIC administration. We have now measured the formation and loss of O6-methyldeoxyguanosine (O6-MedG) in the DNA of peripheral leucocytes of eight patients receiving this treatment regimen. O6-MedG could be detected within 1 h and maximal levels occurred approximately 3-5 h after DTIC administration. Following the first treatment cycle, considerable inter-individual variation was observed in the peak O6-MedG levels, with values ranging from 0.71 to 14.3 mumol of O6-MedG per mol of dG (6.41 +/- 5.53, mean +/- s.d.). Inter- and intra-individual variation in the extent of O6-MedG formation was also seen in patients receiving additional treatment cycles. This may be a consequence of inter-patient differences in the capacity for metabolism of DTIC to release a methylating intermediate and could be one of the determinants of clinical response. Both the pretreatment ATase levels and the extent of ATase depletion were inversely correlated with the amount of O6-MedG formed in leucocyte DNA when expressed either as peak levels (r = -0.59 and -0.75 respectively) or as the area under the concentration-time curve (r = -0.72 and -0.73 respectively). One complete and one partial clinical response were seen, and these occurred in the two patients with the highest O6-MedG levels in the peripheral leucocyte DNA, although the true significance of this observation has yet to be established.     

Effects of a high fat diet on liver DNA methylation in rats exposed to N-nitrosodimethylamine

Previous experiments have shown that a high fat diet changes incidence and tumour sites by N-nitroso-dialkylamines. The purpose of this study was to examine the effect of high and low fat diet on DNA methylation 6 weeks after the end of a chronic N-nitrosodimethylamine (NDMA) exposure (total dose 150 mg/kg). The concentration of O6-methyldeoxyguanosine (O6-MedG) in liver DNA was measured by immunoassays. The level of O6-MedG persisted 6 weeks after the last dose of NDMA and was 6-fold higher (P less than 0.05) in animals on high fat as compared to low fat diet. In another experiment, in which rats on a low and high fat diet received a single NDMA dose (2 mg/kg), the time-dependent removal of O6-MedG from liver and the hepatic O6-methylguanine DNA-alkyltransferase activity was not modified by the type of diet. These results indicate that a high fat diet enhances DNA methylation in the liver, after chronic treatment by NDMA, and that this effect is likely to be responsible for an increased incidence of liver haemangiosarcomas.     

Frequency of Ki-ras mutations and DNA alkylation in colorectal tissue from individuals living in Manchester

Most human colorectal cancers arise through the accumulation of a series of genetic alterations such as point mutations within the Ki-ras and p53 genes, but the chemical carcinogens that may be implicated in these events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O⁶-methyldeoxyguanosine (O⁶-MedG), a promutagenic lesion arising from exposure to as yet unidentified methylating agents. To address whether such exposure may result in oncogene activation in human colorectal tumors, we examined another series of paired normal and tumor DNA samples from the lower intestinal tract for the presence of O⁶-MedG in DNA (as a marker of exposure) and for mutations within the Ki-ras gene. After isolation by high pressure liquid chromatography, O⁶-MedG was quantified by a radioimmunoassay with a limit of detection of 0.01 μmol O⁶-MedG/mol dG. The frequencies of methylation were 33%, 52%, and 48% for normal DNA and 58%, 32%, and 63% for tumor DNA isolated from the cecum, sigmoid colon, and rectum, respectively. Overall, 35% of the individuals had no detectable O⁶-MedG in the DNA from both their tumor and normal tissue. Ki-ras mutations were initially identified by a restriction site mutation assay and then sequenced to ascertain the mutations thus detected. The frequencies of mutations in tumor DNA isolated from the cecum, sigmoid colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolated from macroscopically normal tissue was found to contain Ki-ras mutations in 14% of sigmoid colon samples and 12% of rectal samples. Most base mutations were in codon 12 (72%), and 64% were GC→AT transitions: 28% and 8% were GC→TA and GC→CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 except for a single GC→TA transversion at the first base of codon 13 in a rectal tumor sample. There was no association between the presence of O⁵-MedG in DNA from either normal or tumor tissue or both normal and tumor tissue and the incidence of Ki-ras mutations or GC→AT transitions in mutated Ki-ras genes. It remains to be determined, however, whether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O⁶-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational activation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susceptibility to a mutational event after exposure, (iii) the predominant mutagen in the colon and rectum may not be a methylating agent (e.g., nitric oxide), and (iv) exposure to methylating agents need not result in oncogene activation in human tissues but may perhaps promote the emergence of the mutator phenotype. © 1996 Wiley-Liss, Inc.     

Effects of ethanol and various alcoholic beverages on the formation of O6-methyldeoxyguanosine from concurrently administered N-nitrosomethylbenzylamine in rats: a dose-response study.

Consumption of alcoholic beverages has been identified as a major cause of oesophageal cancer in industrialized countries, with an exceptionally high risk associated with apple-based liquors (calvados). In the present study, we have determined the dose--activity relationship of the effects of coincident ethanol on the formation of O6-methyldeoxyguanosine (O6-MEdG) by the oesophageal carcinogen N-nitrosomethylbenzylamine (NMBzA). Male Fischer 344 rats received a single intragastric dose of NMBzA (2.5 mg/kg body wt; 7.4 ml/kg body wt) in tap water containing 0-20% ethanol (v/v). Survival time was 3 h. In controls, concentrations of O6-MEdG were similar in oesophagus, lung and liver (11-14.9 mumol/mol dG). In oesophagus, coincident ethanol increased levels of O6-MEdG from 15.2 mumol/mol (0.1% ethanol) to 46.0 mumol/mol (20%). This increase was dose dependent for 1-20% ethanol; however, low doses produced a larger effect per gram of ethanol than higher doses. In lung, concentrations of O6-MEdG increased from 11 mumol/mol (0.1%) to a plateau value of 24 mumol/mol (greater than or equal to 5%). In nasal mucosa, an increase in O6-MEdG from 3.9 mumol/mol (controls) to 30.7 mumol/mol was observed with 4% ethanol. Effects of ethanol on hepatic DNA methylation were statistically non-significant. Modulation of NMBzA bioactivation by various alcoholic beverages (adjusted to 4% ethanol) was also investigated. Increases in oesophageal O6-MEdG were similar (+50% to +116%) with pear brandy, rice wine (sake), farm-made calvados, gin, Scotch whisky, white wine, Pilsner beer and aqueous ethanol. Significantly higher increases were elicited by commercially distilled calvados (+125%) and red burgundy (+162%). In contrast to its effects at an ethanol content of 4%, farm-made calvados diluted to 20% ethanol produced significantly higher (+200%) increases in oesophageal DNA methylation than aqueous ethanol (+148%). Our results show that ethanol is an effective modulator of nitrosamine bioactivation in vivo at intake levels equivalent to moderate social drinking, and that some alcoholic beverages contain congeners that amplify the effects of ethanol, suggesting that modulation of nitrosamine metabolism by acute ethanol may play a role in the etiology of human cancer.     

O-6-alkylguanine-DNA alkyltransferase activity in tissues of mice treated with antischistosomal agents

The activity of O-6-alkylguanine-DNA alkyl transferase (ATase), the enzyme responsible for repairing promutagenic methylation damage in DNA, was measured at various time intervals in tissue extracts of mice administered (in vivo) a single therapeutic dose of the antischistosomal agents hycanthone, oxaminiquine and metrifonate. In control animals, liver contained the highest levels of ATase activity (15.8+/-1.8 fmole ATase/mu g DNA) followed by spleen (11.0+/-1.7 fmole/mu g DNA), intestine (2.3+/-0.3 fmole/mu g DNA) and bladder (0.22+/-0.04 fmole/mu g DNA). With hycanthone, ATase activity was reduced by 6 and 24 h post treatment to 74% and 27% below the control value, respectively. Bladder exhibited a 25% inactivation in the ATase level at 6 h time point. Spleen and bladder did not show any alteration in the ATase activity. In animals administered oxaminiquine, liver and bladder had a near identical pattern to that observed for hycanthone. Spleen and intestine, however, revealed activation in ATase by 50% and 42%, respectively after 6 h of treatment. This activation was also observed in the bladder of metrifonate-treated mice. In a previous study (Badawi et al, Cancer Lett 75: 167, 1993), DNA-alkylation damage (O-6-methyldeoxyguanosine; O-6-MedG) was evaluated in these tissues and there was an inverse correlation between the levels of methylation damage and ATase activity in liver (r=-0.85, p<0.01), intestine (r=-0.62, p<0.01) and bladder (r=-0.59, p<0.05).     

Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients.

PURPOSE: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments. EXPERIMENTAL DESIGN: We analyzed the methylation status of the 5' regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients. RESULTS: The 5' regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P < or = 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P < or = 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P < or = 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P < or = 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals. CONCLUSIONS: Our results indicate that methylation of the 5' region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.     

O6-methyldeoxyguanosine in DNA of the adjacent epithelium in human esophageal cancer in Linxian County

Radioimmunoassay of monoclonal antibodies against O6-methyldeoxyguanosine (O6-MedG) was used to detect the presence of these DNA adducts in the human esophageal epithelium. The analysis comprised 48 adjacent epithelial specimens of the esophageal and cardiac cancer resected in Linxian County and 30 specimens of the fetal esophageal epithelium and 4 of the normal esophageal epithelium from autopsy as collected from the hospital in Beijing. The results show that O6-MedG was detected in all the specimens from the esophageal and cardiac cancer patients. In 7 samples in the adjacent epithelium of esophageal cancer, the level of O6-MedG ranged from 0.5 to 1.0 pmol/mgDNA. 19 showed higher levels up to 37.4 pmol/mgDNA with a mean of 4.72 +/- 6.08 pmol/mgDNA. 5 samples of gastric mucosa showed the level of O6-MedG ranging 0.3-1.0 pmol/mgDNA and the remaining 6 showed a higher level of 1.2-13.4 pmol/mgDNA. The mean was 3.31 +/- 3.97. In all the 11 patients, O6-MedG was detected in the para-cancerous gastric mucosa of the cardiac cancer. 4 normal autopsied esophageal epithelial samples were too low for detection. Samples from the fetal esophageal epithelium showed lower level of O6-MedG, the mean was 0.4 +/- 0.57 pmol/mgDNA. The results mentioned above give us the new evidence that the effect of N-nitrosamines is most likely a causative factor in the carcinogenesis of human esophageal cancer.     

Modification of esophageal epithelium DNA in the human fetus by N-nitrosomethylbenzylamine (NMBzA)

Human fetal esophageal epithelium, after being exposed to NMBzA, was found to contain O6-methyldeoxyguanine (O6-MedG), a NMBzA-modified DNA adduct, in tissue DNA by radioimmunoassay and monoclonal antibody, which is highly specific to O6-MedG. The highest level of O6-MedG was 58.83 pMol/mg DNA after adding 5.0 mM NMBzA in vitro. The level of O6-MedG and the concentration of NMBzA followed the dose-effect relationship. O6-MedG could be eliminated from DNA by normal human fetal esophageal epithelium. About 50% of O6-MedG was cleared away in the first 1-2 hours during the post-treatment incubation, which was followed by a slower phase of elimination with 18% left in 24 hours. The results indicate that the human fetal esophageal epithelium can metabolically activate NMBzA in vitro and form O6-MedG, which, as well known, can cause mutagenesis and carcinogenesis and, hence, may most likely be related to the development of human esophageal cancer.     

Promutagenic methylation damage in liver DNA of mice infected with Schistosoma mansoni

Male and female BK-TO mice were infected with different numbersof Schistosoma mansoni cercariae under standard environmentalconditions. Promutagenic methylation damage (O⁶-methyldeoxyguanosine;O⁶-MedG) was detected in liver DNA, but not in kidney, spleenor bladder DNA of infected animals. It was shown that levelsof hepatic O⁶-MedG increased with increasing intensities ofschistosomal infection. Possible mechanisms of action are discussed.These include the activating effects of schistosomes and theirproducts on murine macrophages and subsequent endogenous formationof N-nitroso compounds by the activated macrophages.     

Human papillomavirus and non-muscle invasive urothelial bladder cancer: potential relationship from a pilot study

The relationship between urothelial bladder cancer and high-risk human papillomaviruses (HR-HPV) is still a poorly understood entity, even if some studies have supposed a probably correlation. The aim of the present study was to assess the potential relationship between the presence of HR-HPV and non-muscle invasive urothelial bladder cancers (NMIBC). One hundred and thirty-seven subjects (78 patients affected by NMIBC and 59 controls) were recruited in this prospective study. HR-HPV DNA was evaluated both in urine and tumour tissues. Data from patients were compared with data from controls. The relationship between patients and controls, in terms of HR-HPV presence was performed. The relationship between all pathological data and HR-HPV presence in patient group was carried out. HR-HPV DNA in tissue was found in 27 of 78 (34.6%) tumour samples and in 6 of 59 (10.1%) specimens from TUR-P, with a statistically significant difference (p=0.0009; dF=1; χ2=10.98). HR-HPV DNA in urine was found in 36 of 78 (46.1%) samples obtained from patients, whereas in only 8 of 59 (13.5%) samples from controls (p<0.0001: dF=1; χ2=16.37). A statistical significant difference in terms of HR-HPV frequency between high-grade and low-grade urothelial bladder cancer, was found (p=0.032; RR=0.52-95% CI 0.27-0.93; OR=0.34-95% CI 0.13-0.90). In conclusion, this study highlights the correlation between urothelial bladder cancer and high-risk type HPV infection, suggesting the potential etiopathogenetic role of HR-HPV in urothelial bladder cancer development.     

Prognostic relevance of methylation markers in patients with non-muscle invasive bladder carcinoma

There is increasing evidence for the role of epigenetic gene silencing in superficial bladder cancer. The aim of the current study was to investigate the prognostic value of epigenetic alterations in patients with non-muscle invasive bladder carcinoma. We checked the methylation status of 20 cancer associated genes (p14ARF, p16 CDKN2A, STAT-1, SOCS-1, DR-3, DR-6, PIG-7, BCL-2, H-TERT, BAX, EDNRB, DAPK, RASSF-1A, FADD, TMS-1, E-Cadherin, ICAM-1, TIMP-3, MLH-1, COX-2) for DNA methylation. We analysed microdissected tumour samples from 105 consecutive patients with primary non-muscle invasive bladder carcinoma. Quantitative methylation analysis of CpG sites in the promoter region of the genes was performed with methylation sensitive quantitative real time PCR ('Methylight'). Univariate analysis for association with tumour recurrence was carried out with the Kaplan-Meier analysis and the log-rank test. Follow-up data were available in 95/105 patients (91.4%). A tumour recurrence was observed in 26 patients (27.3%). We could identify six genes (SOCS-1, STAT-1, BCL-2, DAPK, TIMP-3, E-Cadherin), where methylation was associated with tumour recurrence. In Kaplan-Meier analysis, TIMP-3 showed a significant association with recurrence free survival. Methylation of TIMP-3 predicted prolonged disease free interval. In this study, we report a comprehensive analysis on prognostic relevance of gene methylation in non-muscle invasive bladder cancer. We identified one gene (TIMP-3) where methylation was associated with a more favourable outcome. Our data strongly support the usefulness of gene methylation as a prognostic marker in patients with non-muscle invasive bladder cancer.     

用尿沉淀细胞DNA甲基化状态的分析检测膀胱癌

目的：确定尿沉淀细胞DNA中的13个肿瘤相关基因启动子的甲基化谱式分析在膀胱癌诊断中的价值。方法：用定性甲基化特异性（methylation specific polymerase chain reaction，MSP）的方法，对92例临床确诊的膀胱癌患者、23例非肿瘤性尿路疾病患者、6例脑外科患者、7例健康志愿者检测了尿沉淀细胞DNA中肿瘤相关基因启动子的甲基化状态。结果：在临床确诊的92例膀胱癌患者中被检测的13个基因的高甲基化状态出现频率显著高于23例非肿瘤性尿路疾病患者，差异有统计学意义（P〈0．05）。而6例脑外科患者和7例正常健康人的尿沉淀细胞DNA中，上述基因均为去甲基化状态。若以任一个基因高甲基化为膀胱癌的指征，88．0％（81／92例）的膀胱癌可被检出。结论：MSP法分析尿沉淀细胞DNA中肿瘤相关基因启动子的甲基化状态可有效地检出膀胱癌。     

Detection of O6-methyldeoxyguanosine in human placental DNA

A monoclonal antibody specific for O6-methyldeoxyguanosine (O6-MedGuo) was developed. When used in a competitive enzyme-linked immunosorbent assay, 50% inhibition of binding was achieved with 0.51 pmol O6-MedGuo. When the competitive enzyme-linked immunosorbent assay was coupled with high-performance liquid chromatography, 2 mg of DNA could be analyzed giving a lower limit of detection of 0.5 mumol O6-MedGuo/mol deoxyguanosine. This assay was used to test for O6-MedGuo in DNA from placentas of smoking and nonsmoking women. Two of 10 DNA samples from smoking women and three of 10 from nonsmoking women had detectable concentrations of O6-MedGuo. Concentrations ranged from 0.6 to 1.6 mumol O6-MedGuo/mol deoxyguanosine. Activity of O6-alkylguanine DNA alkyltransferase was also measured. There was no apparent relationship between O6-alkylguanine DNA alkyltransferase activity and O6-MedGuo concentrations in the 20 subjects, nor did mean O6-alkylguanine DNA alkyltransferase activity differ between the two groups. Although no apparent relationship between smoking history and O6-MedGuo concentration was found in this preliminary study, this is the first report of a structurally identified DNA adduct in human placenta.     

Methylational urinalysis: a prospective study of bladder cancer patients and age stratified benign controls

Tumour suppressor gene (TSG) methylation has been proposed as a diagnostic marker for urothelial cancer (UC). Here, we compare the frequency of urinary TSG methylation in young and elderly patients, with and without UC. Urine samples were obtained prospectively from 35 UC patients, 35 benign controls over the age of 70 years and 34 healthy volunteers under the age of 40 years. Methylation analysis was performed for eight gene promoters using quantitative methylation-specific PCR. Methylation was detected in urine DNA from all three patient groups. The highest frequencies were seen in UC patients. Significantly less methylation was present in control samples than UC cases for RASSF1a and APC (P < 0.034). The 'methylation index' and level of methylation was highest in the UC group and lowest in the young control group. A marker panel of RASSF1a, E-cad and APC generated a sensitivity of 69%, a specificity of 60% and a diagnostic accuracy of 86%. TSG methylation is detectable in urine DNA from patients with and without bladder cancer. The frequency and extent of methylation appears to increase with age and malignancy. The lack of tumour specificity suggests that further investigation is required before this test is introduced into clinical practice.     

Increased levels of fibroblast growth factor-like activity in urine from patients with bladder or kidney cancer

Growth factor activity was partially purified from human renal tumors and a human bladder cancer cell line by heparin-Sepharose chromatography. This activity stimulated bovine capillary endothelial cell proliferation and DNA synthesis in BALB/c 3T3 cells. Partially purified growth factor preparations from these tumors contained a protein with an approximate molecular weight of 17,000 which was recognized by a polyclonal antiserum raised against a peptide fragment of basic fibroblast growth factor (FGF). This growth factor activity appears to be related to basic fibroblast growth factor. Measurement of FGF-like activity in 50 urine samples from 32 adult males showed that 55% (6 of 11) of the urine samples from patients with bladder cancer and 100% (7 of 7) of the urine samples from patients with kidney cancer contained activity equivalent to more than 20 ng of basic FGF/h of urine production. In contrast, only 6% (2 of 32) of the urine samples from controls, patients with a benign disease, or patients with a history of bladder or kidney cancer contained this level of growth factor activity. These results suggest that patients with bladder or kidney cancer release an FGF-like factor into urine which may be used as a marker for these tumors.     

膀胱肿瘤组织及尿液标本中TWIST1基因的甲基化水平研究

背景与目的：众多遗传学及表观遗传性的改变引发癌基因的激活剂抑癌基因的失活是膀胱肿瘤发生、发展的重要原因,本研究旨在揭示膀胱肿瘤患者肿瘤组织及尿液标本TWIST1基因的甲基化模式。方法：78例经病理确诊的膀胱肿瘤标本、癌旁正常组织及配对75例尿液标本组成实验组,75例年龄及性别比例匹配的非肿瘤个体组成对照组。从肿瘤、癌旁及尿液标本提取DNA,甲基化特异性聚合酶链反应(methylationspecificpolymerase chain reaction,MSP)检测肿瘤组织、癌旁组织及尿液标本中TWIST1基因的甲基化水平,检测结果与尿细胞学检测结果进行对比,比较两种检测方法的灵敏度及特异度。结果：甲基化检测结果显示,88.5%的肿瘤标本及84.0%的尿液标本出现TWIST1基因的甲基化,11.5%的癌旁正常组织及5.3%的对照尿液标本出现甲基化。尿细胞学检测结果显示,49.3%的肿瘤患者尿液标本检测出瘤细胞,17.3%的对照者被诊断为肿瘤或疑似肿瘤。尿液标本甲基化检测灵敏度及特异度显著高于尿细胞学检测方法。针对低级别肿瘤,TWIST1基因甲基化检测灵敏度为66.7%,高于尿细胞学检测(33.3%)。结论：TWIST1基因甲基化水平检测可作为一种简单、非侵入、敏感及特异的方法应用于早期膀胱肿瘤诊断筛查。     

Detection of DNA adducts by high-performance liquid chromatography with electrochemical detection

A method for the detection of rare adducts in DNA has been developed by combining the resolution of high-performance liquid chromatography with the specificity and sensitivity of electrochemical detection. Many adducts are electrochemically active, while the normal bases, except for guanine, are not. Enzymatic hydrolysis of DNA is used to obtain the deoxynucleosides for analysis, or where appropriate, acid hydrolysis or thermal depurination of DNA is used to free the adduct base for analysis. Various types of DNA damage have been induced by in vitro exposure of DNA to acrolein, dimethyl sulfate, sodium nitrite, ascorbate/Cu2+ and gamma-irradiation. Several adducts are detected at a level of one adduct in 10(5)-10(6) normal bases in micrograms of DNA. The method is also useful for measuring O6-methylguanine (O6MeGua) in DNA from rats treated with N-nitrosodimethylamine and 8-hydroxydeoxyguanosine (oh8dG), and O6-MeGua in DNA from bacteria treated with hydrogen peroxide and dimethyl sulfate. oh8dG, which appears to be the most suitable marker for measuring the steady-state level of oxidative DNA damage, can be measured at fmol levels in DNA from normal rat tissues. The method is applicable to the analysis of DNA base damage caused by major endogenous processes relevant to aging, such as deamination, methylation and oxidation. The analysis of DNA adducts with this simple assay also may be potentially useful for studies on carcinogenesis and as a tool in studies on the epidemiology of cancer.     

Determination of O6-butylguanine in DNA by immunoaffinity extraction/gas chromatography-mass spectrometry

A sensitive, specific, and rapid method for quantitating the minor adduct O6-butylguanine (O6BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against O6BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific O6-alkylguanine. After addition of O6BuG and its deuterium labeled analogue (O6BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O6BuG recovery from this column were 1.53 nmol O6BuG/column and 62 +/- 5%, respectively. The method was applied to evaluate O6BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylamine. In the first case the level of modifications present in calf thymus DNA was 104 mumol O6BuG/mol guanine, and in the second case O6BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 mumol O6BuG/mol guanine) than after N-nitrosodibutylamine (0.34 mumol O6BuG/mol guanine) treatment. These results indicate that O6BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms.