The Promoter Hypermethylation of DAPK Gene and pl6 Gene in Sera from Chinese Non-small Cell Lung Cancer Patients

Objective： To evaluate the clinical significance of the aberrant methylation of DAPK gene and p16 gene in sera from 65 NSCLC patients from Nanjing General Hospital of Nanjing Command, China. Methods： A methylation-specific PCR （MSP） was performed for the detection of promoter hypermethylation of DAPK gene and p16 gene in blood DNA from 65 cases of NSCLC, and to analyze the relation of the aberrant methylation of DAPK gene and p16 gene and the clinicopathological data. Results： 30.8% （20/65） of the sera from 65 cases of NSCLC showed hypermethylation for DAPK promoter and 43.1% （28/65） the same for p16 promoter, whereas no methylated DAPK gene promoter and p16 gene promoter were found in sera from the patients with lung benign diseases and normal controls. Methylated DAPK gene promoter and p16 gene promoter in sera were not closely correlated with the pathological classification, stage, metastasis and differentiation in NSCLC. Conclusion： Detection of the aberrant methylation of DAPK gene and p16 gene in blood DNA from NSCLC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.

p53 gene in treatment of hepatic carcinoma:Status quo

Hepatocellular carcinoma (HCC) is one of the 10 most common cancers worldwide. There is no ideal treatment for HCC yet and many researchers are trying to improve the effects of treatment by changing therapeutic strategies. As the majority of human cancers seem to exhibit either abnormal p53 gene or disrupted p53 gene activation pathways, intervention to restore wild-type p53 (wt-p53) activities is an attractive anti-cancer therapy including HCC. Abnormalities of p53 are also considered a predisposition factor for hepatocarcinogenesis. p53 is frequently mutated in HCC. Most HCCs have defects in the p53-mediated apoptotic pathway although they carry wt-p53. High expression of p53 in vivo may exert therapeutic effects on HCC in two aspects: (1) High expression of exogenous p53 protein induces apoptosis of tumor cells by inhibiting proliferation of cells through several biologic pathways and (2) Exogenous p53 renders HCC more sensitive to some chemotherapeutic agents. Several approaches have been designed for the treatment of HCC via the p53 pathway by restoring the tumor suppression function from inactivation, rescuing the mutated p53 gene from instability, or delivering therapeutic exogenous p53. Products with p53 status as the target have been studied extensively in vitro and in vivo . This review elaborates some therapeutic mechanisms and advances in using recombinant human adenovirus p53 and oncolytic virus products for the treatment of HCC.

Inhibition of pancreatic carcinoma cell growth in vitro by DPC4 gene transfection

AIM: To detect the expression of DPC4 in malignant and non-malignant specimens of human pancreas,and observe the inhibition of retroviral pLXSN containing DPC4 on pancreatic carcinoma cells in vitro.METHODS: The expression of DPC4 was determined in 40 pancreatic adenocarcinoma and 36 non-malignant pancreatic specimens by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohisto-chemistry.Furthermore,we constructed retroviral vectors containing DPC4,which then infected the pancreatic carcinoma cell line BxPC-3.Cell growth in vitro after being infected was observed,and the vascular endothelial growth factor (VEGF) mRNA level in the daughter cells was determined by semi-quantitative PCR assay.RESULTS: The RT-PCR assay showed a positive rate of DPC4 mRNA in 100% (36/36) of normal specimens,compared to 40% (16/40) in adenocarcinoma specimens.The regional and intense positive cases of DPC4 expression in adenocarcinoma detected by immunohistochemistry were 10 and four,whereas it was all positive expression in normal tissues.There was a significant difference of DPC4 expression between them.The stable expression of DPC4 in the pancreatic carcinoma cells BxPC-3 could be resumed by retroviral vector pLXSN transfection,and could inhibit cell growth in vitro.Rather,DPC4 could decrease VEGF mRNA transcription levels.CONCLUSION: The deletion of DPC4 expression in pancreatic carcinoma suggests that loss of DPC4 may be involved in the development of pancreatic carcinoma.The retroviral vector pLXSN containing DPC4 can inhibit the proliferation of pancreatic carcinoma cells,and down-regulate the level of VEGF.

Expression pattern of leptin and leptin receptor (OB-R) in human gastric cancer

AIM: To examine the expression of leptin and its receptor, OB-R, in normal gastric mucosa and neoplasia. METHODS: By immunohistochemical staining using specifi c antibodies, we evaluated the expression of leptin and OB-R in 207 gastric carcinomas (100 early and 107 advanced carcinomas) and analyzed their relationship with clinicopathological features. RESULTS: Both normal gastric epithelium and carci- noma cells expressed a significant level of leptin. In cases with OB-R staining, carcinoma cells showed OB-R- positive expression, but the intensity was weaker than that in normal mucosa. The expression of OB-R showed a signifi cant correlation with the level of leptin expres- sion. The expression levels of both leptin and OB-R tend- ed to increase as the depth of tumor invasion or TMN stage increased (P < 0.01). Lymph node metastasis was detected in 49.5% (47/95) of leptin-strong cases and in 50.5% (48/95) of OB-R-positive cases, and the rate was 33% (37/112) in leptin-weak cases and 17% (19/112) in OB-R-negative cases. Both venous and lymphatic inva- sion also tended to be observed frequently in positive tumors as compared with negative tumors. Interestingly, in the 96 leptin- or OB-R-positive tumors, hematogenous metastasis was detected preoperatively in 3 (3.1%) pa- tients. In contrast, none of the carcinomas that lacked expression of leptin and OB-R showed hematogenous metastasis. CONCLUSION: Overexpression of leptin and expres- sion of OB-R may play a positive role in the process of progression in gastric cancer. Functional upregulation of leptin/OB-R may have a positive role in the development and initial phase of progression in gastric cancer.

Gene therapy for gastric cancer:Is it promising?

Gastric cancer is one of the most common tumors worldwide. The therapeutic outcome of conventional therapies is inefficient. Thus, new therapeutic strategies are urgently needed. Gene therapy is a promising molecular alternative in the treatment of gastric cancer, including the replacement of defective tumor suppressor genes, the inactivation of oncogenes, the introduction of suicide genes, genetic immunotherapy, anti-angiogenetic gene therapy, and virotherapy. Improved molecular biological techniques and a better understanding of gastric carcinogenesis have allowed us to validate a variety of genes as molecular targets for gene therapy. This review provides an update of the new developments in cancer gene therapy, new principles, techniques, strategies and vector systems, and shows how they may be applied in the treatment of gastric cancer.

Gene therapy that inhibits NF-κB results in apoptosis of human hepatocarcinoma by recombinant adenovirus

AIM： To investigate whether the recombinant adenovirus induces the TNF-α-mediated apoptosis in vivo. METHODS： Human hepatocarcinoma cell line （HepG2） cells were transfected into BALB/c nude mice, and the tumor growth curve was drawn. We analyzed apoptosis in HepG2 cells by TUNEL, HE staining and electron microscopy. RESULTS： AdIκBαM was expressed stably and efficiently in HepG2 and could not be degraded by induction of TNF-α. Tumor growth in mice could be reduced remarkably if treated by AdIκBαM plus TNF-α. There was apoptosis of 〉 70% of cells treated with AdIκBαM plus TNF-α and about 50% of cells treated with AdIκBαM. In contrast, there was few cell apoptosis in HepG2 cells treated with phosphate buffered saline and AdIκBαM. HepG2 cells in mice also exhibited a high level of apoptosis after in vivo injection with AdIκBαM. The tumor growth curve indicated the tumor transfected with AdIκBαM could be restrained. CONCLUSION： AdIκBαM gene therapy greatly enhances apoptosis due to inhibition of an NF-κB-mediated antiapoptosis signaling pathway.

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were 】15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P【0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P【0.05, P【0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was 】15000 and 214.5+/-31.3 micromol.L(-1), P【0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

An armed oncolytic adenovirus system,ZD55-gene,demonstrating potent antitumoral efficacy

ONYX-015 is an attractive therapeutic adenovirus for cancer because it can selectively replicate in tumor cells and kill them. To date, clinical trials of this adenovirus have demonstrated marked safety but not potent enough when it was used alone. In this paper, we put forward a novel concept of Gene-ViroTherapy strategy and in this way, we constructed an armed therapeutic oncolytic adenovirus system, ZD55-gene, which is not only deleted of ElB 55-kD gene similar to ONYX-015, but also armed with foreign antitumor gene. ZD55-gene exhibited similar cytopathic effects and replication kinetics to that of ONYX-015 in vitro. Importantly, the carried gene is expressed and the expression level can increase with the replication of virus. Consequently, a significant antitumoral efficacy was observed when ZD55-CD/5-FU was used as an example in nude mice with subcutaneous human SW620 colon cancer. Our data demonstrated that ZD55-gene, which utilizing the Gene-ViroTherapy strategy, is more efficacious than each individual component in vivo.

Successful management of postoperative recurrence of hepatocellular carcinoma with p53 gene therapy combining transcatheter arterial chemoembolization

Transcatheter arterial chemoembolization (TACE) has become the standard treatment for unresectable hepatocellular carcinoma (HCC). But this method has some shortages. p53 gene, which was found to be mutant in many human tumors, has been proved with broadspectrum anti-tumor effects. We reported a 23-year-old patient with recurrent HCC after irregular hepatectomy. The p53 gene was applied to this patient. We injected percutaneously and infused transcatheterally p53 gene (Gendicine, Shenzhen Sibiono Bentech, China) into his recurrent nodules in liver respectively and 4 d later, the patient received TACE therapy. In the 2 mo follow-up, the patient was in good clinical condition with normal liver function and no recurrence was identified. The case report proposed that recurrent HCC could be successfully treated with p53 gene therapy combining TACE.

A study on p53 gene alterations in esophageal squamous cell carcinoma and their correlation to common dietary risk factors among population of the Kashmir valley

AIM： To systematically examine the extent of correlation of risk factors, such as age, consumed dietary habit and familial predisposition with somatic Tp53 molecular lesion causal to elevate carcinogenesis severity of esophageal squamous cell carcinoma （ESCC） among the Kashmiri population of Northern India. METHODS： All cases （n = 51） and controls （n = 150） were permanent residents of the Kashmir valley. Genetic alterations were determined in exons 5-8 of Tp53 tumor suppressor gene among 45 ESCC cases histologically confirmed by PCR-SSCP analysis. Data for individual cancer cases （n = 45） and inpatient controls （n = 150） with non-cancer disease included information on family history of cancer, thirty prevailing common dietary risk factors along with patient＇s age group. Correlation of genetic lesion in p53 exons to animistic data from these parameters was generated by Chi-square test to all 45 histologically confirmed ESCC cases along with healthy controls.RESULTS： Thirty-five of 45 （77.8%） histologically characterized tumor samples had analogous somatic mutation as opposed to 1 of 45 normal sample obtained from adjacent region from the same patient showed gerrnline mutation. The SSCP analysis demonstrated that most common p53 gene alterations were found in exon 6 （77.7%）, that did not correlate with the age of the individual and clinicopathological parameters but showed significant concordance （P 〈 0.05） with familial history of cancer （CD = 58）, suggesting germline predisposition at an unknown locus, and dietary habit of consuming locally grown Brassica vegetable ＂Hakh＂ （CD = 19.5）, red chillies （CD = 20.2）, hot salty soda tea （CD = 2.37） and local baked bread （CD = 1.1）. CONCLUSION： Our study suggests that somatic chromosomal mutations, especially in exon 6 of Tp53 gene, among esophageal cancer patients of an ethnically homogenous population of Kashmir valley are closely related to continued exposure to various common dietary risk factors, especially hot salty tea, meat, baked bread and ＂Hakh＂, that are rich in nitrosoamines and familial cancer history.

Liposome transfected to plasmid-encoding endostatin gene combined with radiotherapy inhibits liver cancer growth in nude mice

AIM： To evaluate whether intratumoral injection of liposome-endostatin complexes could enhance the antitumor efficacy of radiation theapy in human liver cardnoma （BEL7402） model.METHODS： Recombinant plasmid pcDNA3.End was transfected into human liver carcinoma cell line （BEL7402） with lipofectamine to produce conditioned medium. Then BEL7402 cells and human umbilical vein endothelial cells （HUVECs） were treated with the conditioned medium. Cell cycle and apoptosis were analyzed by flow cytometer and endothelial cell proliferation rates were determined by MTT assay. The antitumor efficacy of endostatin gene combined with ionizing radiation in mouse xenograft liver tumor was observed.RESULTS： Endostatin significantly suppressed the S phase fraction and increased the apoptotic index in HUVECs. In contrast, endostatin treatment had no effect on BEL7402 cell apoptosis （2.1±0.3% vs 8.9±1.3%, t= 8.83, P= 0.009〈0.01） or cell cycle distribution （17.2±2.3% vs 9.8±1.2%, t = 4.94,P = 0.016〈0.05）. The MTT assay showed that endostatin significantly inhibited the proliferation of HUVECs by 46.4%. The combination of local endostatin gene therapy with radiation therapy significantly inhibited the growth of human liver carcinoma BEL7402 xenografts, the inhibition rate of tumor size was 69.8% on d 28 compared to the untreated group. The tumor volume in the pcDNA3.End combined with radiation therapy group （249±83 mm^3） was significantly different from that in the untreated group （823±148 mm^3, t= 5.86, P= 0.009〈0.01） or in the pcDNA3 group （717±94 mm^3, t= 6.46, P= 0.003〈0.01）. Endostatin or the radiation alone also inhibited the growth of liver tumor in vivo, but their inhibition effects were weaker than those of endostatin combined with radiation, the inhibition rates on d 28 were 44.7% and 40.1%, respectively.CONCLUSION： Endostatin not only significantly suppresses tumor growth but also enhances the antitumor efficacy of radiation therapy in human carcinoma xenograft.

Selective tropism of liver stem cells to hepatocellular carcinoma in vivo

AIM： To investigate the selective tropism of liver stem cells to hepatocellular carcinoma （HCC） in an animal model and its feasibility as a vector to deliver therapeutic genes for targeted therapy of HCC.METHODS： WB-F344, a kind of rat liver stem cell, was infected with recombinant virus to establish a cell line with stable, high-level expressing enhanced green fluorescent protein （EGFP）. An animal model of HCC in Wistar rats was established by implanting HCC cells （CBRH7919） combined with an immunosuppressive drug. EGFP labeled liver stern cells were injected into caudal veins of the animals and distribution was observed at different time points after injection. SDF-1 and c-kit expression in non-tumor liver and tumor tissue were analysed by immunohistochemistry for the relationshiop between the expression and migration of liver stem cells. Furthermore, hepatic stern cells were injected via the portal vein, hepatic artery, caudal vein, or directly into the pericancerous liver tissue, respectively, and effects on migration, localization, and proliferation of the hepatic stern cells within the tumor tissue were observed and analyzed.RESULTS： Recombinant adenovirus could deliver the EGFP gene to hepatic stem cells. A new stem cell line, named WB-EGFP, was established that stably expressed EGFP. WB-EGFP cells still showed selective tropism towards HCC and EGFP expression was stable in vivo. According to immunohistochemistry results, SDF-1 may not be related to the mechanisms of tropism of hepatic stem cells. Different application sites affected the distribution of liver stem cells. Injection via the portal vein was superior with regard to selective migration, localization, and proliferation of the hepatic stem cells within the tumor tissue.CONCLUSION： Liver stem cells have the biological behavior of selective migration to HCC in vivo and they could localize and proliferate within HCC tissue stably expressing the target gene. Liver stem cells are a potential tool for a targeted gene therapy of HCC.

Single-tube-genotyping of gastric cancer related SNPs by directly using whole blood and paper-dried blood as starting materials

AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials. METHODS: PCR amplification is directly carried out from the whole blood or paper-dried blood sample without any DNA extraction step. Before PCR, a blood sample, four primers, and all of biological reagents necessary for PCR were added at a time; After PCR, the amplified products were directly separated by slab gel electrophoresis or microchip CE without any purification. SNP typing was performed by tetra-primer PCR with two inner primers specific to each allele and two outer primers defining the length of allele-specific amplicons. Genotypes were directly discriminated by the size of amplicons specific to each allele, thereby avoiding any post-PCR process. RESULTS: Using a special PCR buffer, inhibitory substances in blood (including the anticoagulant in blood) and filter paper were effectively suppressed; a 'true' single-tube-genotyping is thus realized. We successfully determined genotypes IL-1B-511 and IL-1B-31 polymorphisms at the gene IL-1B by using whole-blood and paper-dried blood samples as starting materials respectively. The method is so sensitive that 0.5-1.0μL of blood sample is enough to give a satisfactory typing results. The genotyping results were confirmed by RFLP-PCR using purified genome DNA, indicating that amplification specificity was not affected by inhibitory components (including coagulants) in blood or filter paper. CONCLUSION: Compared with SNP typing methods based on purified DNA, the proposed method is labor saving, simple, inexpensive, and less cross-contaminated. It is promising to use this method to type other SNPs.

Effect of fragile histidine triad gene transduction on proliferation and apoptosis of human hepatocellular carcinoma cells

AIM:To evaluate the inhibitory effects of human fragile histidine triad(FHIT) gene on cell proliferation and apoptosis in human hepatocellular carcinoma line Hep3B in vitro. METHODS:A recombinant pcDNA3.1(+) /FHIT including the functional region of FHIT gene was constructed and transferred into human hepatocellular carcinoma cells in vitro. mRNA and protein expression of the FHIT gene in the transfected cells was detected by RT-PCR and Western blot,respectively. The effect of FHIT on proliferation was detected by MTT assay. Changes in cell cycle and apoptosis were assayed by flow cytometry. Five mice received subcutaneous transplantation of Hep3B-FHIT;5 mice received subcutaneous transplantation of normal Hep3B and Hep3B-C as controls. The body weight of nude mice and tumor growth were measured. RESULTS:RT-PCR and Western blot analysis showed that the expression level of FHIT-mRNA and FHIT protein was higher in Hep3B cells after infection withpcDNA3.1(+) /FHIT. The growth of Hep3B cells treated with pcDNA3.1(+) /FHIT was significantly inhibited. The pcDNA3.1(+) /FHIT-transfected Hep3B cells showed a significantly higher cell rate at G0-G1 phase and increased apoptosis in comparison with controls(P < 0.05) . The growth of transplanted tumor was inhibited markedly by FHIT. Tumors arising from the Hep3B-FHIT cells occurred much later than those arising from the Hep3B and Hep3B-C cells. The growth of Hep3B-FHIT cells was slow and the tumor volume was low. CONCLUSION:Transduction of FHIT gene inhibits the growth of human hepatocellular carcinoma cells and induces cell apoptosis in vivo and in vitro.

Adenoviral gene therapy in gastric cancer: A review

Gastric cancer is one of the most common malignancies worldwide. With current therapeutic approaches the prognosis of gastric cancer is very poor, as gastric cancer accounts for the second most common cause of death in cancer related deaths. Gastric cancer like almost all other cancers has a molecular genetic basis which relies on disruption in normal cellular regulatory mechanisms regarding cell growth, apoptosis and cell division. Thus novel therapeutic approaches such as gene therapy promise to become the alternative choice of treatment in gastric cancer. In gene therapy, suicide genes, tumor suppressor genes and anti-angiogenesis genes among many others are introduced to cancer cells via vectors. Some of the vectors widely used in gene therapy are Adenoviral vectors. This review provides an update of the new developments in adenoviral cancer gene therapy including strategies for inducing apoptosis, inhibiting metastasis and targeting the cancer cells.

Lack of association between UGT1A7,UGT1A9,ARP,SPINK1 and CFTR gene polymorphisms and pancreatic cancer in Italian patients

AIM： To investigate simultaneously UGT1A7, UGT1A9, ARP, SPINK and CFTR genes to verify whether genetic polymorphisms predispose to the development of pancreatic cancer （PC）.METHODS： Genomic DNA of 61 pancreatic cancer patients and 105 healthy controls （HC） were analyzed. UGT1A7 genotyping was determined by PCR-RFLP analysis. Specific PCR and sequencing were used to analyze genetic variants of UGT1A9, ARP, SPINK1 and CFTR genes.RESULTS： Four different alleles （^＊1：WT; ＂2： N129K and R131K; ＂3： N129K, R131K, and W208R; and ＂4： W208R） in UGT1A7 and three different alleles （^＊1：WT;^＊4： Y242X; and ^＊5： D256N） in UGT1A9 were detected. All UGT1A polymorphisms were observed at similar frequency in PC patients and HC. Seven different alleles in ARP were found in PC patients and HC at similar frequency. The SPINK1 mutations N34S and P55S occurred in five PC patients with a prevalence （4.1%） not significantly different from that observed （2.0%） in HC. The only CFTR AFS08 mutation was recognized in three PC patients with a prevalence （4.9%） similar to HC.CONCLUSION： UGT1A7, UGT1A9, ARP, SPINK1 and CFTR gene polymorphisms are not associated with PC in Italian patients.

DNMT3B 579 G>T promoter polymorphism and risk of esophagus carcinoma in Chinese

AIM:To investigate the relationship between 579 G>T polymorphisms in the DNMT3B gene, which is involved in de novo methylation and associated with the risk of esophagus cancer (EC) in Chinese. METHODS:DNMT3B 579 G>T genotypes were determined by PCR-RFLP in 194 EC patients and 210 healthy controls matched for age and sex, who did not receive radiotherapy or chemotherapy for newly diagnosed and histopathologically confirmed EC. RESULTS:In control subjects, the frequency of T/T and G/T genotypes, and T and G alleles was 81.4%, 18.1%, 90.05% and 9.55%, respectively. The distribution of genotypes and allelotypes in the EC patients was not significantly different from that in the controls. When stratified by sex and age, there was still no significant association between the risks of EC and GT and GG genotypes. This study also showed a distinct difference in the distribution of DNMT3B and single nucleotide polymorphism (SNP) between Chinese and Koreans.CONCLUSION:DNMT3B 579 G>T polymorphism may not be a stratification marker to predict the susceptibility to EC, at least in Chinese. DNMT3B promoter SNP is diverse in ethnic populations.

Effect of mutant p27^(kip1) gene on human cholangiocarcinoma cell line, QBC_(939)

AIM：To investigate the effects of exogenously mutated p27^kip1 （p27） on proliferation and apoptosis of human cholangiocarcinoma cell line, QBC939 in vivo.METHODS： Adenviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line. Expression of p27 was detected by RT-PCR. Western blot. Cell growth, morphological change, cell cycle, apoptosis and cloning formation were determined by MTT assay and flow cytometry.RESULTS： The expression of p27 protein and mRNA was increased signifi cantly in QBC939 cell line transfected with Ad-p27mt. The transfer of Ad-p27mt could signifi cantly inhibit the growth of QBC939 cells, decrease the cloning formation rate and induce apoptosis. p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Ad-p27mt.CONCLUSION： p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis. Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.