human gliomas represent 50% to 60% of all intracranial tumors . according to the world health organization ( who ) guidelines , gliomas are histologically classified into four grades : pilocytic astrocytoma ( grade i ) , low - grade diffuse astrocytoma ( grade ii ) , anaplastic astrocytoma ( grade iii ) , and glioblastoma multiforme ( gbm , grade iv ) . both diagnostic technologies and therapeutic strategies the 5-year survival rates of low - grade ( grade i~ii ) and high - grade ( grade iii~iv ) glioma patients in china are 75.4% and 18.2% , respectively . especially , the median survival time for patients with gbm is still only 12 months . indeed , early diagnosis and prolonging survival in glioma patients remains a great challenge for clinicians in the field of neurooncology . there have been several prognostic factors for glioma patients , such as age , preoperative duration of symptoms , karnofsky performance status ( kps ) score , histologic grade , tumor necrosis , surgical resection extent , use of postoperative radiation therapy , and , probably , adjuvant chemotherapy . however , these clinical parameters can not completely account for the observed variation in survival because of the heterogeneity of glioma patients . thus , there is an urgent need to further investigate the molecular mechanisms of glioma and to identify the effective prognostic indicators for survival prediction.the dna - base excision repair ( ber ) pathway is responsible for the repair of exogenous and endogenous alkylating and oxidative dna damage , which may lead to carcinogenesis , cell death , and aging if left unrepaired this pathway involves the recognition and removal of damaged bases by a dna glycosylase , followed by incision of the resulting abasic ( ap ) site by ap endonuclease , dna synthesis by polymerase , and strand ligation by dna ligase . thus , the ber pathway is an important candidate for intervention into the cellular responses to dna change . n - methylpurine - dna glycosylase ( mpg ) as a dna repair enzyme is a main component in the ber pathway . in previous study aimed at understanding the significance of initiating lesions removed by the ber pathway , kaina et al .   identified the over - expression of the human mpg in chinese hamster ovary cells . in the n - alkylpurine repair process , mpg is responsible for the glycolytic removal of the modified base , which leads to the formation of apurinic sites . although n - alkylpurines have not been found to be directly mutagenic , apurinic sites left by this repair process can block replication and lead to mutation . mpg also participates in the repair of 8-hydroxyguanine and hypoxanthine . because of the potential role of dna base lesions in mutagenesis and carcinogenesis , a number of studies have been performed to investigate the association of mpg with various human cancers . ( 1998 )   detected the increased mpg gene and protein expression in the breast cancer cells versus normal breast epithelial cells by northern analysis , southern blots , immunofluorescence , immunohistochemistry , and western blot analysis . in 2001 , sohn et al . reported that the expression of mpg was increased in high - risk hpv - infected cervical neoplasias and the intracellular distribution of mpg protein was altered , suggesting a role of mpg in carcinogenesis . in an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change , many researchers have been interested in the effects of mpg in tumor sensitivity to the clinical chemotherapeutic agents . as their results , mpg - overexpressing ovarian cancer , osteosarcoma , and breast cancer   cells are significantly more sensitive to the clinical chemotherapeutic agents , suggesting that the overexpression of mpg may be a possible gene therapy approach to sensitize tumor cells to the cell - killing effects of chemotherapeutic alkylating agents . the biological mechanism behind the increase of sensitivity to the chemotherapeutic agents in mpg overexpressing cell lines may be that the balance between glycosylase activity , leading to apurinic sites and formation of strand breaks , and subsequent excision repair processes may play an important role in determining cellular cytotoxicity and resistance to alkylating agents . of our interests , we focus on the involvement of mpg in human gliomas . recent study has demonstrated that mpg mrna expression was higher in astrocytic tumor tissues than in brain tissues adjacent to tumor , and mpg protein localization in immunohistochemical study was detected only in the nucleus of all tumor tissues , suggesting an mpg 's role in human astrocytic tumors and raise the possibility that the altered mpg expression and intracellular localization could be associated with astrocytic tumorigenesis . tang et al .   further demonstrated that mpg overexpression , together with the inhibition of ber , could sensitize glioma cells to the alkylating agent . however , little is known about the expression level of mpg in human gliomas with different clinical grades and its prognostic significance . to address this problem , we used quantitative real - time pcr , immunohistochemistry assay , and western blot analysis to investigate the expression pattern of mpg gene and protein in glioma specimens and normal control brain tissues . next , we analyzed the relationship between mpg expression and the glioma stage as well as the survival of patients . this study was approved by the research ethics committee of the institute for functional neurosurgery p.l.a , tangdu hospital , fourth military medical university , xi'an , p.r . china . written informed consent a total of 128 formalin - fixed , paraffin - embedded specimens of gliomas resected between 2000 and 2010 were retrieved from the archives of the pathology department of tangdu hospital , fourth military medical university , p.r . all the slides were reevaluated according to who classifications   by two pathologists , with differences resolved by careful discussion . a total of 76 males and 52 females ( 1.46  :  1 ) were enrolled in this study , and the median age was 42 years ( range , 1271 ) . thirty - two of the 128 gliomas were classified as low - grade [ 18 pilocytic astrocytomas ( who i ) and 14 diffuse astrocytomas ( who ii ) ] , and 96 were classified as high - grade gliomas [ 38 anaplasia astrocytomas ( who iii ) , and 58 primary glioblastomas ( who iv ) ] . the clinicopathological features , and the treatment strategies of all the patients were indicated in table 1 . paraffin and snap - frozen sections of nonneoplastic brain tissues from 10 patients with intractable epilepsy were also included as controls . five - year followup was performed , and all patients had complete followup until death . overall survival time was calculated from the date of the initial surgical operation to death . patients , who died of diseases not directly related to their gliomas or due to unexpected events , were excluded from this study . in addition , 20 glioma specimens [ 5 pilocytic astrocytomas ( who i ) , 3 diffuse astrocytomas ( who ii ) , 3 anaplasia astrocytomas ( who iii ) , and 9 primary glioblastomas ( who iv ) ] were snap - frozen in liquid nitrogen and stored at 80c following surgery for mrna and protein isolation . total rna purified from all 20 glioma tissues and 10 control brain tissues real - time monitoring of polymerase chain reactions ( pcrs ) was performed using the abi 7900ht ( idaho technology , idaho falls , i d , usa ) . cdna was synthesized using random primers and oligo 18dt and superscript ii reverse transcriptase ( invitrogen ) . gene expression was determined using the sybr green i dye ( biogene ) , which binds preferentially to double - stranded dna , and 10  ng of template . fluorescence signals , which are proportional to the concentration of the pcr product , are measured at the end of each cycle and immediately displayed on a computer screen , permitting real - time monitoring of the pcr . the reaction is characterized by the point during cycling when amplification of pcr products is first detected , rather than the amount of pcr product accumulated after a fixed number of cycles . the higher the starting quantity of the template , the earlier a significant increase in fluorescence is observed . the threshold cycle is defined as the fractional cycle number at which fluorescence passes a fixed threshold above the baseline . the primers 5-gtc cta gtc cgg cga ctt cc-3 and 5-ctt gtc tgg gca ggc cct ttg c-3 were used to amplify 603-bp transcripts of mpg , and the primers 5-ggt ggc ttt tag gat ggc aag3 and 5-act gga acg gtg aag gtg aca g3 were used to amplify 161-bp transcripts of -actin . the pcr profile consisted of an initial melting step of 1  min at 94c , followed by 38 cycles of 15  s at 94c , 15  s at 56c , and 45  s at 72c , and a final elongation step of 10  min at 72c . fluorescence data were converted into cycle threshold measurements using the sds system software and exported to microsoft excel . thermal dissociation plots were examined for biphasic melting curves , indicative of whether primer dimers or other nonspecific products could be contributing to the amplification signal . twenty glioma tissues and 10 control brain tissues were homogenized in lysis buffer [ pbs , 1% nonidet p-40 ( np-40 ) , 0.5% sodium deoxycholate , 0.1% sodium dodecyl sulfate ( sds ) , 100  ug / ml aprotinin , 100  g / ml phenylmethylsulfonyl fluoride ( pmsf ) , sodium orthovanadate ] at 4c throughout all procedures , and sonicated for 70  s , then add 300  g pmsf per gram of tissue and incubate on ice for 30  min , followed by centrifugation at 15,000  rpm for 20  min at 4c . the protein content was determined according to bradford 's method , with bovine serum albumin used as a standard . protein samples ( 30  g ) were boiled with 2  sample buffer containing 5%  -mercaptoethanol for 5  min , separated by size on 15% polyacrylamide gel under sds denaturing conditions , and transferred to a nitrocellulose membrane at 90  v for 2  h. the nitrocellulose membranes were stained with ponceau s to assess the efficiency of transfer . nonspecific binding was blocked by incubation in block buffer ( 5% nonfat dry milk , 0.05% tween-20 , 1  tris - cl - buffered saline ) overnight at 4c , the membranes were hybridized with a mouse monoclonal antibody to mpg ( ab55461 ; abcam ) , then incubated with a horseradish peroxidase - labeled goat anti - mouse igg ( 1  :  500 ) . the bound secondary antibody was detected by enhanced chemiluminescence ( amersham life science , little chalfont , uk ) . positive immunoreactive bands were quantified densitometrically ( leica q500iw image analysis system ) and expressed as ratio of mpg to -actin in optical density units . immunohistochemical assay was performed using the conventional immunoperoxidase technique according to the protocol of the department of neurosurgery , institute for functional neurosurgery p.l.a , tangdu hospital , fourth military medical university , xi'an , p.r . briefly , following peroxidase blocking with 0.3% h2o2/methanol for 30  min , specimens were blocked with phosphate - buffered saline ( pbs ) containing 5% normal horse serum ( vector laboratories inc . , all incubations with a mouse monoclonal antibody to mpg ( ab55461 ; abcam ) at 1  :  500 dilution were carried out overnight at 4c . then the specimens were briefly washed in pbs and incubated at room temperature with the anti - mouse antibody and avidin - biotin peroxidase ( vector laboratories inc . , the specimens were then washed in pbs and color developed by diaminobenzidine solution ( dako corporation , carpinteria , ca , usa ) . after washing with water , specimens were counterstained with meyer 's hematoxylin ( sigma chemical co. , st louis , mo , usa ) . nonneoplastic brain tissues were used as control tissues , and nonimmune igg was also used as negative control antibody for immunohistochemical staining . immunostaining was scored by two independent experienced pathologists , who were blinded to the clinicopathologic parameters and clinical outcomes of the patients . 5% scored 0 , 625% scored 1 , 2650% scored 2 , 5175% scored 3 , > 75% scored 4 ; ( 2 ) intensity of stain : colorless scored 0 , pallide - flavens scored 1 , yellow scored 2 , brown scored 3 . the staining score was stratified as  ( 0 score , absent ) , + ( 14 score , weak ) , + + ( 58 score , moderate ) , and + + + ( 912 score , strong ) according to the proportion and intensity of positively stained cancer cells . all computations were carried out using the software of spss version13.0 for windows ( spss inc , il , usa ) . randomized block design anova was used to analyze the statistical difference among different tissue types . in the analysis of glioma morbidity for all patients , we used the kaplan - meier estimator and univariate cox regression analysis to assess the marginal effect of each factor . a spearman 's analysis was carried out to analyze the correlation between mpg mrna and protein expression levels . the gene expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was detected by real - time pcr . as shown in figure 2 , the expression levels of mpg gene in human glioma tissues were conspicuously higher than those in control brain tissues ( p < 0.001 ) . interestingly , mpg gene expression increased with the advancement of who grades from i to iv ( p < 0.001 ) . the protein expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was also detected by western blot analysis . as the results , the expression levels of mpg protein tended to increase from the glioma to the normal tissue ( figures 3(a ) and 3(c ) ) . mpg expression was highest in grade iv and lowest in grade i ( figures 3(b ) and 3(c ) ) , indicating a close correlation of mpg protein expression with who grade . there was a significant positive correlation between the expression of mpg gene and protein expression levels from the same glioma tissues ( rs = 0.82 , p < 0.001 ) . the expression and the subcellular localization of mpg protein in 10 nonneoplastic brain and 128 glioma tissues were detected by immunohistochemistry assay . as shown in figure 4 , the positive staining for mpg was mainly observed in the nuclei of tumor cells in glioma tissues , which was consistent with the previous study of wang et al . the representative photographs were shown in figures 4(a ) and 4(b ) . among the glioma specimens , 102 ( 79.7% ) glioma specimens were positively stained for mpg , whereas its immunoreactivities in nonneoplastic brain sections ranged from undetectable to low ( figure 4(c ) ) . additionally , the immunostaining of mpg in nonneoplastic brain tissues is below detection levels of both real - time pcr and western blot . according to the statistical analysis , the expression level of mpg protein in glioma tissues was significantly higher ( p < 0.001 ) than that in nonneoplastic brain tissues , which was consistent with the results of western blot analysis . in addition , mpg expression was not significantly affected by the gender and age ( both p > 0.05 ) of the patients . in contrast , the mpg expression was the closely correlated with who grade , as well as kps . the expression levels of mpg protein in glioma tissues with higher who grade ( table 1 ; p = 0.002 ) and lower kps ( table 1 ; p = 0.01 ) were significantly higher than those with lower who grade and higher kps . moreover , we reviewed clinical information of these mpg - positive or -negative glioma patients . during the follow - up period , 100 of the 128 glioma patients ( 78.1% ) had died ( 24 from the mpg - negative group and 76 from the mpg - positive group ) . as determined by the log - rank test , the survival rate of patients with positive mpg staining was lower than those without mpg staining ( p < 0.001 ; figure 5(a ) ) . the median survival time of patients with negative expression of mpg could not be estimated by statistical analysis because all patients survived better than the overall median level , and those patients with strong positive ( + + + ) , moderate positive ( + + ) , and weak positive ( + ) of mpg were 9.1  1.5 months , 12.3  1.1 months , and 22.6  2.2 months ( log - rank test : p < 0.001 ) . furthermore , figure 5(b ) shows the postoperative survival curve of patients with glioma and mpg expression after adjusting for age , gender , who grade , and kps . by multivariate analysis ( table 2 ) , the loss of mpg expression was a significant ( p < 0.001 ) and independent prognostic indicator for patients with glioma besides age , who grade and kps . the cox - proportional hazards model showed that mpg over - expression was associated with poor overall survival . because of the dismal survival in the patients with glioma , especially in gbm patients , there is an urgent need to find novel specific and effective prognostic markers in order to apply individualized treatments to specifically target the pathophysiologic and molecular properties of glioma patients . in the current study , we investigated the expression of mpg in 128 cases of human glioma and compared the expression with tumor grade and survival rates of patients . our data demonstrated that mpg gene and protein were both increased in glioma compared to nonneoplastic brain tissue . we found an increased trend of both mpg protein level and gene level from who grade i to who grade iv glioma . these results suggest that the transcriptional and translational activation of human mpg might participate in the tumorigenesis and progression of glioma . dna in all somatic and germ cells in the body are continuously exposed to exogenous and endogenous alkylating and oxidative agents that result in damage to dna . if dna damage was not repaired , it may lead to genetic mutation , chromosome aberration , aging , and carcinogenesis . the lack of precise dna repair activities may lead to defective embryogenesis , tissue - specific dysfunction , hypersensitivity to dna - damaging agents , premature senescence , genetic instability , and elevated cancer rates . the repair of dna bases damaged by alkylation is initiated in mammalian cells by mpg , as an alkyladenine dna glycosylase . the majority of repair that is initiated by mpg occurs via short - patch ber , a mechanism whereby only one nucleotide is replaced . the majority of base damage induced by methylation is removed by mpg , including the principal adducts 3-mea and 7-meg . it has been reported that the expression of mpg could be induced by viral damage and a variety of dna - damaging agents . for example , the increased expression of mpg gene was studied in breast cancer , cervical neoplasia , and thymic carcinoma in sv 40 t - antigen - expressing transgenic mice . as previous investigation , mammalian cells actively regulated dna repair enzymes and genes during cell proliferation . dna repair enzymes are expressed in the cell cycle in a defined temporal pattern relative to the induction of dna replication . in brain cells that are not undergoing dna replication , kim et al .   indicated that mpg expression in the brain was relative high in 1 week after birth , and the level remained low in day 400 mature adults , suggesting that brain tissue is terminally differentiated and nonproliferating tissues . it has been demonstrated that dna repair enzymes play an important role in the carcinogenesis of several cancers . of our interests , mpg was overexpressed in breast cancer up to 24-fold as compared to normal primary breast epithelium , suggesting the role of mpg in breast carcinogenesis . in astrocytomas , kim et al .   in 2003 reported that mpg mrna level was significantly higher than that in tumor - adjacent brain tissues . with the similar results , we in this study also found the upregulation of mpg protein and gene in glioma tissues . additionally , we provide evidence that mpg protein is found in nuclear localization of the glioma tissues , which was consistent with the results of kim et al . . the nuclear localization of mpg in tumor cells of glioma tissues may be suggestive of a role of mpg in the regulation of cell division and cell cycle . furthermore , the most important finding of this study was the correlation of mpg expression and survival rates of patients . kaplan - meier and multivariate analysis both showed a significantly worse overall survival for patients whose tumors had high mpg levels , indicating that high mpg protein level is a marker of poor prognosis for patients with gliomas . this is the first investigation to demonstrate the prognostic value of mpg in human cancers . our data showed the over - expression of mpg gene and protein in human gliomas and also suggested for the first time that mpg be an unfavorable independent prognostic indicator for glioma patients .
aim . to examine the expression of n - methylpurine - dna glycosylase 
 ( mpg ) gene and protein in glioma samples with different who grades 
 and its association with patients ' survival 
 . methods . 
 immunohistochemistry assay , quantitative real - time pcr and western 
 blot analysis were carried out to investigate the expression of 
 mpg gene and protein in 128 glioma and 10 non - neoplastic brain 
 tissues . 
 results . 
 mpg gene expression level in glioma tissues was 
 significantly higher than that in non - neoplastic brain tissues 
 ( p < 0.001 ) . 
 immunohistochemistry also showed that mpg protein was 
 over - expressed in glioma tissues , which was consistent with the 
 resutls of western blot analysis . 
 additionally , the expression 
 levels of mpg gene and protein both increase from grade i to grade 
 iv glioma according to the results of real - time pcr , 
 immunohistochemistry and western blot analysis . 
 moreover , the 
 survival rate of mpg - positive patients was significantly lower 
 than that of mpg - negative patients ( p < 0.001 ) . 
 we further confirmed 
 that the over - expression of mpg was a significant and independent 
 prognostic indicator in glioma by multivariate analysis ( p < 0.001 ) . 
 conclusions . 
 our data showed the over - expression of mpg gene and 
 protein in human gliomas , and also suggested for the first time 
 that mpg be an unfavorable independent prognostic indicator for 
 glioma patients .