Correlation between hypermethylation and expression of Syk gene in colorectal cancer cell lines

AIM:To explore the relationship of methylation status and expression of spleen tyrosine kinase (Syk) gene in colorectal cancer cell lines. METHODS:Bisulfite sodium modification sequencing, methylation specific PCR and Western blotting were used to detect the methylation status and expression of Syk gene in 23 colorectal cancer (CRC) cell lines. Luciferase assay was applied to measure the activity of promoter with or without methylation in CpG islands. Meanwhile, methylation status and expression level were compared in Syk(-) HCT-15 cell line before or after 5-Aza-CdR administration. RESULTS:(1) Among 23 cell lines, loss expression of Syk gene in 9 cell lines due to hypermethylation in promoter, and 14 expression with unmethylation status were observed. The total methylation rate was 39.2%. (2) Microsatellite instability was found in 7 of 9 cell lines with promoter hypermethylation, 4 of 14 were wihtout hypermethylation. The difference between methylation and unmethylation group was significant (P<0.05). (3) 5-Aza-CdR restored methylated-Syk gene promoter activity. Compared to methylated-promoter, luciferase activities increased to 4.5 and 4.7 folds with Syk full size promoter and unmethylated-promoter, respectively. (4) 5-Aza-CdR restored methylated-Syk gene expression and the effect had time-course dependence. CONCLUSION:Hypermethylation of CpG islands in Syk gene promoter silences Syk expression in CRC cell lines, and 5-Aza-CdR restores Syk expression by demethylation.     

5’-GpG islands of p15 gene hypermethlation in non-Hodgkin’s lymphomas

AIM:To illustrate the expression of hypermethylation p15 gene in 53 non-Hodgkin’s lymphoma (NHL). METHODS:The methylation of p15 gene in 53 cases of non-Hodgkin’s lymphoma was detected by using methylation-specific PCR (MSP) technique. RESULTS:18.9% (10/53) in NHL were methylation in p15 gene. p15 gene was frequently in high malignant NHL patients (27.3%) compared with in low malignant patients (0%). CONCLUSION:The result suggested that the hypermethylation of p15 may play an important role in non-Hodgkin’s lymphoma.     

Significance and alterations of p14ARF gene of CDKN2A locus in gastric cancer

AIM:To investigate the significance and changes of p14^ARF gene in gastric cancer.METHODS:The tumors and gastric tissues neighboring carcinoma from 48 patients with gastric cancer were studied. The homozygous deletions, mutations, methylation of the CpG islands, and mRNA expression of p14^ARF gene were assessed by PCR, PCR-SSCP, PCR based methylation assay, and RT-PCR.RESULTS:①The homozygous deletion rate of p14^ARF was 31.3% (15/48), and no homozygous deletions were examined in all the gastric tissues neighboring tumor. ②There were no point mutations of p14^ARF in 33 gastric cancers without homozygous deletion and in the matched gastric tissues adjacent to tumor. ③Methylation rate of the CpG islands of p14^ARF was significantly higher in gastric cancers(47.9%, 23/48) than that in gastric tissues neighboring cancer (4.2%, 2/48)(P＜0.01).④ No expression of p14^ARF mRNA was detected in 45.8%(22/48) of gastric cancers. Moreover, the negative rate (100%, 3/3) of p14^ARF mRNA of gastric cancers with the combined methylation of exons 1β and 2 was significantly higher than that (15%, 3/20) of the sole methylation of exon2(P＜0.05). CONCLUSION:p14^ARF gene is frequently inactivated by homozygous deletion and methylation of the 5' CpG islands in gastric cancer, which may play an important role in the development of gastric cancer.     

Effect of p19ARF on growth and apoptosis of leukemia cells

AIM:To study the effect of p19ARF on the biological behavior of human leukemia cells. METHODS:p19ARF was cloned in eukaryotic expression vector pcDNA3.1 and transferred into INK4a/ARF locus-deficient leukemia cells HEL and K562. The changes in biological characteristics of the two p19ARF-transfected cells were observed.RESULTS:The growth of the p19ARF-transfected HEL cells was significantly inhibited compared with the vector-transfected cells; Cell cycle analysis showed that the expression of foreign p19ARF gene resulted in G1 phase cell cycle arrest and apoptosis cell death in some of HEL cells. However, p19ARF had no marked effects on the growth of K562 cells with p53 gene mutation and did not induce apoptosis in K562 cells.CONCLUSION:p19ARF suppressed the growth of leukemia cells by p53-dependent pathway.     

Aberrant DNA methylation in promoter region of p53 gene in acute leukemia

AIM: To investigate the significance of aberrant p53 gene promoter methylation in acute leukemia by detecting the occurrence of p53 gene promoter methylation. METHODS: Genomic DNA was digested using restriction endonuclease MspⅠ, HpaⅡ, EcoRⅡ, BstNⅠ, respectively. PCR amplification was conducted and the products after digestion and genomic DNA were used as template. The PCR product was subjected to electrophoresis and the results were analyzed by gel imaging and analysis system. Parts of the separated DNA were sequenced after purification from gel. RESULTS: The prevalence of methylation in acute leukemia group was 38.7％, of which ALL was 45.5％ (5 of 11) and ANLL 35.0％ (7 of 20). No methylation was detected in normal control group. There was significant difference between the prevalence of methylation in acute leukemia group and the normal control group (Fisher′s exact test, P<0.05). However, the prevalence between ALL and ANLL was not significantly different (Fisher′s exact test, P>0.05). Compared the relationship between aberrant methylation of p53 gene and clinical data, statistical significance between aberrant methylation of p53 gene and enlargement of lymph nodes, liver or spleen(P<0.05) was observed. CONCLUSION: ①Aberrant DNA methylation in P1 promoter region of p53 gene exits in part of acute leukemic patients, but not in health people. ②The prevalence of aberrant DNA methylation between ALL and ANLL is not significantly different. ③The patients with aberrant methylation of p53 gene seem to show more frequently the manifestations of enlarged lymph nodes, liver or spleen than usual.     

Relationship between mutated k-ras and biological behavior of colorectal cancer

AIM: To investigate mutations of oncogene k-ras in colorectal cancer tissues and the relationship between mutations of k-ras and biological behavior of colorectal carcinoma. METHODS: The specimens of 123 patients with colorectal cancer were collected. Real-time fluorescence quantitative PCR were performed to detect k-ras mutations at codon 12 and codon 13 of exon 1, and the results were analyzed with the corresponding clinical pathological data. RESULTS: Among 123 colorectal cancer cases, point mutations were detected in 53 cases (40.8%), point mutations at codon 12 were found in 42 (34.1%) cases, and 11(8.9%) cases at codon 13. No closely relationship between mutations of k-ras and tumor size, location, invasive depth and differentiation extent was observed. The rate of k-ras mutation in the cases with more invaded lymph nodes was higher than that in the cases without invaded lymph nodes (P<0.05), and the rate of k-ras gene mutation in the cases with hepatic metastases was higher than that in no hepatic metastases (P<0.05). The rate of k-ras gene mutation was higher in TNM staging Ⅲ/Ⅳ than that in Ⅰ/Ⅱ(P<0.05). CONCLUSION: Mutation of oncogene k-ras plays an important role in the carcinogenesis and development of colorectal cancer, and it is closely associated with invaded lymph notes and hepatic metastases, suggesting that mutation of k-ras indicates a poor prognosis.     

Clinical significance of detection of p16 gene methylation in early diagnosis of lung cancer

AIM: To investigate the aberrant methylation in the promoter of p16 in plasma, sputum, bronchoalveolar lavage fluid (BALF), pleural effusion and biopsy specimens from suspected lung cancer patients and to evaluate the clinical significance in the early diagnosis of lung cancer.METHODS: Using methylation specific PCR (MSP) for the detection of promoter methylation of p16 gene in plasma, sputum, BALF, pleural effusion and biopsy specimens from suspected lung cancer patients.RESULTS: Of the 67 cases of suspected lung cancer patients, 42 were proved by pathology. The positive percentages of p16 gene promoter methylation of the lung cancer patients are as follows: 52.4%（22/42）in plasma, 47.6%（20/42）in sputum, 59.5%（25/42）in BALF, 71.4%（10/14）in pleural effusion and 61.9%（26/42）in biopsy specimens, respectively；while promoter methylation in p16 gene was found only one in plasma and one in pleural effusion in 25 patients with various benign lesions (P<0.05). The positive expression of p16 gene promoter methylation in lung cancer patients was irrelevant to histological type, clinical stage, pathological grade, lymph node metastasis and distant metastasis of the lung carcinomas (P>0.05).CONCLUSION: Detection of the aberrant methylation in the promoter of p16 gene in plasma, sputum, BALF, pleural effusion and biopsy specimens from lung cancer patients by MSP method is a kind of rising technology with development potential for lung cancer early diagnosis.     

Analysis of p16 gene deletion and mutation in gastric carcinoma

AIM:To investigate p16 gene alterations in gastric carcinoma. METHODS:36 fresh tumor specimens taken from gastric cancer patients were analyzed for p16 gene deletion and mutation by polymerase chain reaction (PCR) and DNA sequencing. RESULTS:Homozygous deletion of exon 1 and exon 3 was observed in 2 cases and 3 cases of diffuse carcinoma, respectively. The frequency of homozygous deletion was 13.89%. No p16 gene point mutation was detected. CONCLUSION:The deletion of p16 genes may be related to gastric carcinogenesis.     

Methylation of p15INK4B gene and its mechanism in patients with myelodysplastic syndrome and leukemia

AIM: To explore the relationship between hypermethylation of p15INK4B gene and the pathogenesis of hematopoietic malignances. METHODS: The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in bone marrow cells from 54 cases with hematopoietic malignances were detected by RT-PCR, Western blot, and methylation-specific PCR. RESULTS: The p15INK4B gene was methylated more often in high-risk myelodysplastic syndrome (MDS) patients, patients at blast phase of chronic myeloid leukemia (CML-BP) and acute leukemia patients than that in low-risk MDS patients (P<0.01). The expression levels of DNMT_3A and DNMT_3B in acute leukemia patients, high-risk MDS patients, and CML-BP patients were higher than that in low-risk MDS patients (P<0.05). CONCLUSION: The hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of acute leukemia, high-risk MDS, and blast phase of chronic myeloid leukemia. Furthermore, DNMT_3A and DNMT_3B are substantially over-expressed in the bone marrow cells of these patients.     

Mutation of the transforming growth factor-β typeⅡ receptor gene in sporadic colorectal cancers with microsatellite instability

AIM: To determine the relationship between the mutation of the RII gene and RER status in the tumorigenesis of sporadic colorectal cancer. METHODS: We screened RER status and mutation of the RII gene from 50 sporadic colorectal cancers (19 in the proximal colon, 31 in the distal colorectum). RESULTS: RER was found in 13 cases (8 in the proximal colon, 5 in the distal colorectum), and 5 of them showed mutations of the RII gene. All 5 cancers carrying a TGF-β RII gene mutation showed RER+, but there wasn't any mutation of RII gene in RER(-) cases. Four of 5 RII mutation were located at the cecum. CONCLUSION: These data indicate that the TGF- βRII gene is a major target of microsatellite instability and mutation of the RII gene play an important role in carcinogenesis of sporadic colorectal cancer with microsatellite instability , especially at the cecum.     

Mutations of K-ras oncogene in primary colorectal tumors,related liver metastases and portal vein blood of patients with colorectal cancer

AIM: To evaluate the concordance of K-ras oncogene mutations in primary colorectal tumors, liver metastases and portal vein blood of the patients with colorectal cancer, and to find out the relationship between mutated K-ras oncogene and liver metastases in colorectal cancer.METHODS: Fifty-nine patients with colorectal cancer were screened for the mutations of K-ras oncogene in the tissue samples of primary tumors, portal vein blood and liver metastases (only 15 cases of the 59 patients) by real-time fluorescence quantitative PCR and DNA sequencing. The results were also analyzed with the clinical data of the patients.RESULTS: Point mutations of K-ras were found in the primary tumors in 20 (33.9%) of the 59 patients with colorectal cancer, and 18 (30.5%) of the 59 patients in their portal vein blood. K-ras mutations in 8 (53.3%) of 15 liver metastases were also detected. No significant difference among the rates of K-ras mutation in primary tumor tissues, portal vein blood and related liver metastases was observed (P>0.05). Eighteen cases with mutated K-ras gene in portal vein blood showed the mutations in primary tumor tissues. The patients without mutated K-ras gene in primary tumor tissue also showed negative mutation of K-ras in the portal vein blood. The mutated K-ras gene in both liver metastase and portal vein blood were detected in 8 of the 15 cases with liver metastases, and no mutated K-ras gene was detected in the others with liver metastases. The main types of K-ras mutations found in primary tumors, liver metastases (5 simultaneous, 2 metachronous) and portal vein blood were GGT to GAT and GGT to GTT at codon 12. A K-ras mutation at codon 13 (GGC to GAC) was found in one case with metachronous liver metastases. The rate of concordance of K-ras status between primary tumors and portal vein blood was 96.6%. Detection of K-ras mutations in liver metastases was accordant with that in portal vein blood, but the type of K-ras mutation was partially discordant.CONCLUSION: The K-ras mutations in primary tumors, liver metastases and portal vein blood of patients with colorectal cancer are concordant, and mutated K-ras detected in both cancer tissue and portal vein blood may indicate liver micrometastases from colorectal cancer.     

Study on hyper methylation of p15INK4B gene in multiple myeloma

AIM: To study the characteristics and regulations of p15 (MST INK4B) methylation in multiple myeloma (MM). METHODS: Method of methylation-specific PCR was applied in 23 cases of MM about the methylation rate of p15 gene. RESULTS: Methyaltion rate in MM was 73.5%(17/23). The PCR product was a fragment of 148 bp. In four stageⅠcases of MM with plasma cell-typed bone marrow profile, p15^INK4B gene was non-methylated; In one case of stag ⅡMM and one case of stage Ⅲ MM with mature plasma typed bone marrow profile, p15^INK4B gene was no-methylated, too, while in many cases of stageⅠ、stage Ⅱand stage Ⅲ with naive plasma cell in bone marrow profile which were plasma cell-typed or mixed typed, p15^INK4B gene methylation was frequently detected. The methylation rates for stageⅠ、stage Ⅱand stage Ⅲ MM were respectively 55%(5/9),100%(7/7) and 71.4%(5/7). CONCLUSION: p15 gene methylation was a possible pathogenic factor,and might be related to the progression and prognosis of the disease.     

Relationship between apoptosis,proliferation and expression,mutation of related genes in breast cancer

AIM:To study the relationship between apoptosis, proliferation and expression,mutation of related genes in breast cancer.METHODS:Methods of TUNEL, immunohistochemical S-P and PCR-SSCP were used respectively to study apoptotic index (AI), mitotic index(MI), expression of Bcl-2,p53,c-erbB-2,PCNA,Ki67,TopoⅡ and mutation of p53 in 54 cases of breast cancer.RESULTS:AI and MI were 9.40±3.78 and 5.96±2.36, respectively. There was a significant direct correlation between them(r=0.46.P＜0.01).High expression of Bcl-2,PCNA,Ki67,TopoⅡ coincided with high AI,MI(P＜0.01). High expression of p53,c-erbB-2 and mutation of p53 coincided with high MI(P＜0.01). Type of p53 mutation coincided with AI(P＜0.05).CONCLUSION:Disturbance of gene control between apoptosis and proliferation is related with expression,mutation of related genes in breast cancer.     

Present investigation of PTEN gene in cardiovascular system

PTEN gene, identified in 1997 and named after the separation, was a new candidate tumor suppressor gene. The mutations of PTEN gene, loss of heterozygosity in human tumors are prevalent, such as malignant glioma, endometrial cancer, prostate cancer, breast cancer, etc. The mutation frequency of PTEN is equivalent to p53. As an important functional element and a tumor suppressor gene, most scholars agree that PTEN gene is as important as p53. At present, studies on the PTEN gene mainly concentrated in the tumor area, its functions in the cardiovascular system are few reports. This article reviews the investigation of PTEN gene in the cardiovascular system.     

Methylation and mRNA expression of TIG1 gene in esophageal squamous-cell carcinoma tissues

AIM: To investigate the expression and promoter methylation of tazarotene-induced gene-1 (TIG1) in esophageal squamous-cell carcinoma (ESCC) tissues. METHODS: The methods of methylation-specific PCR and real-time fluorescence quantitative PCR were applied to examine the methylation and mRNA expression of TIG1, respectively, in 43 cases of ESCC tissues, 20 cases of paracancerous tissues and 15 cases of normal tissues. RESULTS: The frequency of promoter methylation of TIG1 gene in ESCC tissues was 25.6% (11/43), which was significantly higher than that in the paracancerous tissues (5.0%, 1/20) and normal tissues (0/20). The hypermethylation of TIG1 gene in these tissues had no correlation with sex, age and clinical stage of the patients. However, it was correlated with the pathological stage (P<0.01) and lymph node metastasis (P<0.05). The mRNA expression of TIG1 in ESCC tissues was significantly lower than that in paracancerous tissues (P<0.05) and normal tissues (P<0.01). However, the expression level of TIG1 mRNA in methylated tissues was significantly lower than that in unmethylated tissues (P<0.01). CONCLUSION: Promoter methylation may be an important mechanism of TIG1 gene inactivation in ESCC, which was related to lymph node metastasis and TNM stage of esophageal carcinoma.     

Relationship of p21WAF1 gene polymorphisms with protein expression in breast carcinomas

AIM: To investigate the relationship between p21^WAF1gene polymorphisms and protein expression in breast carcinoma. METHODS: Polymerase chain reaction single-strand conformation polymorphisms technique (PCR-SSCP) and immunohistochemical assay of S-P immunostaining technique were used to study polymorphisms of p21^WAF1 and protein expression respectively on the specimen of paraffin-embedded tissues in 100 cases of breast carcinomas and 40 benign breast diseases as control. RESULTS: Two p21^WAF1 gene polymorphisms were found in 18% (18/100) of breast carcinomas and 5% (2/40) of control samples. The difference between the two groups was statistically significant (χ²=3.94, P＜0.05). The positive immunohistochemical reaction of p21^WAF1 protein were found in 50% (50/100) of breast carcinomas and 12.5% (5/40) of control samples. The difference between the two groups was statistically significant (χ²=16.84, P＜0.01). The positive immunohistochemical reaction of p21^WAF1 protein were found in 100% (18/18) of breast carcinomas with p21^WAF1 gene polymorphisms and 39% (32/82) of no p21^WAF1 gene polymorphisms. The difference between two groups was statistically significant (χ²=21.95, P＜0.01). The p21^WAF1 gene polymorphisms were correlated with the protein expression in breast carcinomas (r=0.576, P＜0.01). CONCLUSION: p21^WAF1 gene polymorphisms may create the different copies of mRNA and may make relevant protein molecules.     

菊花组织培养继代过程中的DNA甲基化变化

采用MSAP（甲基敏感扩增多态性）方法对菊花（Dendranthema ×grandiflorum）组织培养继代过程中的DNA甲基化情况进行了分析。结果发现,3个单芽系的组培苗较田间材料均有DNA甲基化增加和减少现象,采用7对引物,共扩增出929条带,各单芽系DNA甲基化变异率分别为8.929%、8.902%和8.986%。继代材料较母体均有甲基化变异发生,且变异类型中DNA甲基化减少的比例高于甲基化增加的比例,随着继代次数的增加,两种变异间的差异逐渐减小,比例相近。同时在同一单芽系内的不同次继代个体间有DNA甲基化变化现象,对5次继代的180个个体研究发现,带型变化中18.38%的带型不一致,2.99%为一个或两个个体发生了变化,仅有1.72%在发生变化后趋于稳定,另外10.68%的带型呈现不稳定的随机变化。     

Detection of bcl-2 methylation and the relationship between bcl-2 methylation and expression,prognostic factors in breast cancer

AIM: To establish methylation-specific PCR (MSP) method for detecting bcl-2 gene, and to study the relationship between bcl-2 methylation and expression, prognostic factors in breast cancer. METHODS: The primer of bcl-2 gene for MSP was designed. The methylations in CpG island of bcl-2 gene in 54 cases of breast cancer were detected by using MSP. The expressions of bcl-2, PCNA, ER and PR in 54 cases of breast cancer were detected by using SP immunohistochemical technique. RESULTS: The overall positive rate of bcl-2 methylation was 29.6% in breast cancer. There was a significant negative correlation between the methylation of bcl-2 and the expression of bcl-2 (P＜0.01). The methylation of bcl-2 coincided with those bad prognostic factors such as high PCNA label index (LI), ER-and PR-(P＜0.01). CONCLUSIONS: This study established the MSP method for detecting bcl-2 gene. The results of MSP and sequence analysis testified that the design of the MSP primer of bcl-2 gene in this study was successful. The methylation of bcl-2 would become the marker indicating bad prognosis of breast cancer.