Abstract
Carcinogenic molecules from cigarettes are known to cause DNA damage to bladder epithelial cells, but such damage can be corrected by some DNA repair mechanisms such as base and nucleotide excision repair, double-strand repair and mismatch repair. Various gene products play a role in these DNA repair systems. The aim of this study was to investigate six of these genes (XRCC1, XRCC3, XPD, XPG, APE1, hOGG1) each of which has a separate role in these repair mechanisms. The study was performed on 83 bladder cancer patients and 45 healthy controls. The genes were amplified by polymerase chain reaction (PCR) and restriction fragment polymorphism determinations were used to elucidate the specific changes in the gene region. There was no difference in smoking status between patient and control groups. It was found that there was a statistical significance in XRCC3 T carriers between patient and control groups and so there was a 4.87-fold protective role by the XRCC3 T allele against bladder cancer. The AA genotype and A allele carriers of the APE gene were more frequent in the transitional epithelial carcinoma group than in the adenocarcinoma group. The genotype distribution for the APE gene was determined to be significantly different between local and invasive cases; G allele carriers for this gene were significantly higher in invasive cancer types.
Exposure to tobacco smoke and occupational carcinogens is a major risk factor for urothelial cell carcinoma of the bladder. The carcinogenic molecules of cigarettes directly or indirectly create free oxygen radicals and cause DNA damage in bladder epithelial cells. DNA is a huge molecule that can be damaged by endogenous and exogenous mutagens, however the damage can be repaired through several ways such as base excision repair, nucleotide excision repair, double-strand repair and mismatch repair (1). Several different kinds of gene products play roles in these DNA repair systems. Apurinic endonuclease 1 (APE1), X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) and 8-oxoguanine DNA-glycosylase 1 (OGG1) genes express proteins for base excision repair (BER) (2). BER operates on slight damage such as oxidation or reduction. Polynucleotide kinase, apurinic/apyrimidinic endonuclease (APE), DNA polymerase-β and XRCC1 molecules are involved in the restoration phase of BER (3). Xeroderma pigmentosum D (XPD), excision repair cross-complementing rodent repair deficiency and complementation group 5 (ERCC5, XPG) genes express proteins having roles in the nucleotide excision repair (NER) pathway that can repair bulky lesions such as pyrimidine dimers, other photo-products, larger chemical adducts and cross-links. Double-strand breaks can also be produced by replication errors and by exogenous agents such as ionizing radiation; repair of those double-strand breaks is intrinsically more difficult than other types of DNA damage because no undamaged template is available (3). X-ray repair complementing defective repair in Chinese hamster cells 3 (XRCC3) gene expresses a protein that has a role in repair of double-strand DNA breaks by homologous recombination (4, 5).
The aim of this investigation is to study polymorphisms of XRCC1, XRCC3, XPD, XPG, APE1 and hOGG1 to determine whether there might be an increase in bladder cancer incidence in individuals with polymorphisms of these genes or not.
Patients and Methods
Study Groups. The patient group consisted of 83 bladder cancer patients referred to Haydarpaşa Numune Hospital, Urology Department aged between 26-86 years and the control group consisted of 45 healthy individuals 48-83 years old that had no family history of bladder cancer. The bladder cancer patients were operated by transurethral resection or radical cystectomy. Questionnaires, medical records and pathology reports were received to confirm the diagnoses and cancer status. All study participants were informed about the study and their approval was obtained.
Details of PCR and RFLP procedures and expected products.
Polymorphism snalysis. From all participants, blood specimens were taken by using EDTA-containing tubes, and their DNA materials were prepared by using a salt-extraction method. All materials were protected in 4°C refrigerators for later analyses. PCR-RFLP method was used to determine possible gene polymorphisms, the procedures of polymerase chain reaction (PCR) and restriction fragment polymorphism procedures (RFLP) are given in Table I (6-11). For each of the studied parameters, the appropriate primers were used to amplify the corresponding gene by PCR and the reaction products were digested by using the appropriate enzyme at 37°C. Finally, DNA fragments were redissolved in agarose gels stained by ethidium bromide to be visualised. The expected results after restriction for each gene are given in Table I.
Statistically, univariate analysis was performed to compare the distribution of age and gender and the frequencies of alleles and genotypes. Multivariate logistic regression analysis with adjustment of age, gender and smoking status were then performed.
Results
The demographic information of the two groups and tumor status of the patient group are given in Table II. Each group was divided into age groups and respectively compared with regards to age, gender and smoking aspects. The patient group was further classified by their tumour types and grades. There was no significant difference between patient and control groups for age and smoking status (p>0.05). There was no significant difference between the ages of two groups for any of the genotype and/or allele determinations. No statistically significant difference was determined between the patient and control groups either in APE A allele carriers or APE G allele carriers (p=0.771; p=0.264) (Table III).
Demographic information of both groups and tumor status of the patient group.
Although there was no significant difference between patient and control groups in XRCC3 C allele carriers (p=0.695) it was found that there was a statistical significance in XRCC3 T carriers between patient and control groups (p=0.002). It was also found that there is a 4.87-fold protective role of the XRCC3 T allele from bladder cancer. There were no significant differences between any genotypes or alleles in the control and patient groups for XRCC1, XPG and XPD genes (Table III).
There was a statistically significant difference between the control and patient groups in genotype for hOGG1 (p=0.003) and hOGG1 Ser allele carriers (p=0.022).
Grouping accordings to the pathological types of the tumours, the AA genotype (p=0.01) and A allele carriers (p=0.04) for APE gene were more frequent in the transitional epithelial carcinoma group than in the adenocarcinoma group (χ2: 4.21; OR: 6.88; CI 95%: 0.86-55.19). There was no significant difference between other groups. The genotype distribution for the APE gene was determined to be significantly different between local and invasive cases (p=0.002); G allele carriers for this gene were significantly more frequent in invasive cancer (p:0.001). There were no other significant differences between any of the groups for any of the genes (Table IV).
Genotype distributions of studied genes in control and patient groups.
Discussion
The bladder, due to being the urine collecting area, is prone to contact with carcinogens. It has been shown that smoking increases bladder cancer risk four-fold (12). It is thought that smoking increases the risk due to chemicals such as hydrocarbons, arylamines, nitrosamines and the formation of reactive oxygen species as by-products of the above compounds (13). It is very important to protect DNA from damage by these chemicals; DNA repair mechanisms are paramount in correcting the changes on DNA and provide unmutated DNA while replication goes on (14). It is known that there are more than a hundred genes participating in four main DNA repair mechanisms (15). Closas et al. determined the relationship of XPC and ERCC1-1 to -5 gene polymorphisms in bladder cancer (16). It is thought that the polymorphisms in DNA repair genes change their repair capacity, leading to cancer (3). The most frequent DNA damage caused by free oxygen radicals is the formation of 8-hydroxydeoxyguanosine (8-OHdG) and is normally repaired by 8-oxoguanine DNA glycosylase1 (hOGG1) by removing the bond between the damaged base and sugar, thereby creating an abasic region (17, 18). Kim et al. demonstrated that Ser326Ser and Ser326Cys genotypes had an increased probability of recurrency in bladder cancer over Cys326Cys cases. In this study, there was a statistically significant difference in genotype distribution between control and patient groups in hOGG1 Ser carriers, but there was no significance between hOGG1 Cys carrying cases (p=0.187).
Genotype distributions of patient group according to the pathological types and stages of the tumors.
Shen et al. previously determined that XRCC3 codon Thr241Met variant genotype had protective effects for bladder cancer, but no relationship of XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms with bladder cancer (19). XRCC1 gene is located on chromosome 13 and its protein is an important component in the repair of base excision caused by ionising radiation and alkylating agents (20). This protein recognizes single-strand breaks and binds onto those broken areas (21). Other studies on XRCC1 gene polymorphisms in bladder cancer and smoking have yielded no meaningful results (21, 22). In parallel to these previous studies, no relationship between XRCC1 gene polymorphisms and bladder cancer could be found in this study. XRCC3 gene protein has a role in the repair of DNA double helix breaks by cross-binding homologous recombination, as well as a role in maintainig chromosome stability (23). Sanyal et al. could not find any statistically significant relationship between XRCC1 Thr241Met polymorphism and bladder cancer (24). In the present study, a significant relationship between control and patient groups was found for XRCC3 codon 241 polymorphism, but there was no relationship for the XRCC3 C allele in those groups. Importantly, a 4.87-fold protection in XRCC3 T allele carriers was found in this study indicating that XRCC3 T allele carriers have 4.87 times more protection against bladder cancer.
Sanyal et al. previously investigated the relationship between several genes and bladder cancer and found that some XPC and XPD gene polymorphisms were increased in bladder cancer patients (24). XPD protein works in nucleotide excision repair to recognition and repair of tymidine dimers. XPD gene distortions increase repair and transcription defects and cause an abnormal response to apoptosis (25). There are various previous studies on the relationship between XPD genes and bladder cancer (24, 26, 27). In the present study, no difference in any of the XPD genotype and allele carriers between patient and control groups was found.
The XPG gene product has a role in nucleotide excision repair. It has been previously reported that the polymorphisms of this gene are related to lung cancer (9), but there was no obvious relationship between this polymorphism and bladder cancer (24). In parallel, no relationship was found in either genotype and allele distributions of this gene and bladder cancer.
The APE1 gene product is the rate-limiting enzyme in base excision repair and works in coordination with the product of XRCC1 gene. Damaged bases on DNA skeleton are removed by a specific glycosylase and an abasic region is formed (28).
This study is a rare investigation of several genes in bladder cancer in the Turkish population. The novel findings of this study require further investigation in larger study groups.
- Received October 10, 2008.
- Revision received December 23, 2008.
- Accepted January 19, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
