胃癌及其有关病变的幽门螺杆菌感染与抑癌基因表达的相关性研究

背景与目的:幽门螺杆菌(Helicobacterpylori,Hp)是确定的胃癌致癌因子,但其致癌的确切机制仍不清楚。p53、p21WAF1、p16为主要的细胞周期负调控基因。本研究旨在探讨胃癌及其有关病变中,上述3种抑癌基因的作用及其与幽门螺杆菌感染的关系。方法:应用HID-AB(pH2.5)-PAS、SP免疫组化染色及Warthin-Starry染色,对65例慢性萎缩性胃炎(chronicatrophicgastritis,CAG),93例肠上皮化生(intestinalmetaplasia,IM),94例胃上皮不典型增生(gastricepithelialdysplasia,GED)及60例胃癌(gastriccarcinoma,GC)中3种抑癌基因表达和幽门螺杆菌感染情况进行检测。结果:在胃癌发生的不同阶段,p53阳性表达率随病变发展而升高,在CAG、IMⅠ～Ⅱ、IMⅢ、GEDⅠ级、GEDⅡ～Ⅲ级及GC中分别为0、1.64%、6.25%、5.45%、23.08%、70.00%。p21WAF1和p16阳性表达率随病变发展而降低,p21WAF1阳性表达率分别为100%、95.08%、100%、100%、71.79%、45.00%,p16阳性表达率分别为83.08%、81.97%、78.13%、89.09%、69.23%、40.00%。三者表达在GEDⅡ～Ⅲ与GEDⅠ组间、GC与GEDⅡ～Ⅲ组间的差异均具有显著性(P<0.05)。同一病变中,Hp感染阳性组p53、p21WAF1及p16阳性表达率虽然高于Hp阴性组,但差异无显著性(P>0.05)。结论:p53突变及p21WAF1。

Detection of Serum Aberrant CDKN2/P16 DNA in Colorectal Cancer

Objective： To search for a biomarker for colorectal cancer. Methods： The MSP, SSCP and deletion tests with serum have been taken simultaneously in 100 cases of colorectal cancer and 2 groups of controls, as well as the specimens of 26 cancer tissues and 22 paracancerous tissues and 29 cases of benign disease tissues for a contrast. Results： The aberrant methylation rate of P16 in the serum was 69.00%, deletion rate 4.00% and suspicious point mutation rate 15.00% in colorectal cancer patients. The data of cancer tissues were the same as those of the serum, but in paracancerous tissue those were significantly lower. In 10 cases, sequencing analysis revealed that there were 3 cases of missense, one case of frameshift and one case of nonsense. Among them, four cases had P16 protein deletion. As a tumor marker, the sensitivity of combined use of three methods was 88.00%, specificity 96.87% and accuracy 90.15%. The combined use of MSP and SSCP could obtain the same results. Conclusion： The content of DNA in serum is minimal, but it reflects the tumor burden of patients. The 10^-3 fragments of DNA could be detected in the serum by MSP. It can be used in the clinical diagnosis or popular investigation, and long-term postoperative follow-up.

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.

鼻咽癌组织中P16基因缺失突变的分析

目的探讨p16基因缺失突变在鼻咽癌中的发生情况与临床意义。方法采用多重PCR分析法,对157例鼻咽癌标本及47例治疗后病理阴性组织标本P16基因突变情况进行了检测。结果鼻咽癌组织中P16基因第2外显子的缺失率高于疗后病理阴性组织,统计学上有显著差异性(P<0.000),P16基因第2外显子的缺失与鼻咽癌的临床分期、性别、年龄统计学上无显著差异(χ2值分别为1.726、0.582、0.196,P>0.05),P16基因第2外显子的缺失率不足3年生存组高于超过3年生存组,统计学上有显著差异性(χ2=14.12,P<0.000)。结论P16基因第2外显子缺失可能与鼻咽癌的发生发展预后有关,与临床分期、性别、年龄无关,可作为预后评价指标。

乳癌组织端粒酶活性表达及其与p16基因的关系

目的 :探讨乳房恶性肿瘤组织中端粒酶活性的表达情况及p16抑癌基因的影响。方法 :应用端粒重复放大程序 酶标法 (TRAP ELISA)及TRAP 银染法检测 35例乳癌组织及 2 4例良性肿瘤组织中的端粒酶活性 ,并采用多重PCR技术对乳癌组织的p16基因的缺失突变进行了分析。结果 :35例乳癌标本中有 2 8例端粒酶活性表达为阳性 (80 % ) ,而 2 4例乳房良性肿瘤标本中仅 1例端粒酶活性表达阳性 (4 1% )。此外 35例乳癌标本中的 15例存在有 p16基因的外显子 2及外显子 1的缺失 (4 2 8% ) ,而该 15例中有 14例被检测为端粒酶活性阳性 (93.3% ) ,但 2 0例未缺失标本的端粒酶阳性率仅为 70 % (14/2 0 )。结论 :端粒酶活性与乳房肿瘤组织的恶性程度密切相关 ,提示端粒酶参与了肿瘤的形成过程 ,而端粒酶活性的增高与p16抑癌基因的缺失突变之间可能存在有一定的关系。

P16基因产物在人类肝细胞癌中的表达及临床意义

目的探讨P16蛋白在肝细胞癌中的表达,并推测P16基因在肝细胞癌发生、发展过程中的作用及临床意义。方法采用免疫组化SP法测定26例肝细胞癌及癌旁肝细胞中P16蛋白表达进行对比研究。结果26例肝细胞癌P16蛋白表达量明显低于癌旁肝组织。P16蛋白表达量随病理分级增高而降低。部分肝细胞癌组织存在P16蛋白的表达缺失现象。结论P16蛋白表达障碍可能与肝细胞癌的发生和组织分化有关。可考虑将P16蛋白作为本病预后分析的参考指标值。

苯乙酸对胶质瘤C6细胞p16基因的激活作用及意义

目的 观察诱导分化剂苯乙酸对C6胶质瘤细胞增殖分化过程中抑癌基因p16的蛋白水平表达变化的影响。方法 3 H -TdR掺入法测细胞增殖率 ;免疫组化SP法检测p16基因蛋白水平的表达。结果 应用苯乙酸后 ,细胞cpm值降低 ;p16基因胞浆阳性表达显著升高。结论 苯乙酸对胶质瘤细胞存在剂量依赖性抑制 ,增强了抑癌基因p16表达活性 。

p16抑癌基因产物在原发性肝细胞癌中的表达及临床意义

目的 分析p16蛋白测定在肝癌临床中的地位 ,并推测p16基因在肝细胞癌发生发展过程中的作用。方法 采用免疫组化SP法测定34例原发性肝细胞癌、20例癌旁肝细胞中p16蛋白表达 ,分析与肝细胞癌组织学分级、TNM分期、生存预后等关系。结果 64.7%的原发性肝细胞癌p16蛋白表达阳性 ,35.3 %阴性 ,癌旁肝组织表达100%阳性。两者差异显著 (P<0.05)。组织学分化Ⅳ级的肝细胞癌表达阳性率为41.7 %、Ⅰ～Ⅲ级分化的阳性率75%～77.8% ,对比有差异显著性 (P<0.05)。TNMⅠ～Ⅳ期各组间p16蛋白的表达无差异。术后1、3年生存率在p16蛋白阳性者分别为93.7%和72.7% ,阴性者分别为77.8%和60 %。结论 部分肝细胞癌组织存在p16蛋白的表达缺失现象。p16蛋白表达障碍可能与肝细胞癌的发生和组织分化有关。p16蛋白是否可作为本病恶性行为判断、预后分析的参考指标值得进一步研究。

METHYLATION OF p16 AND p15 GENES IN MULTIPLE MYELOMA

Objective.To investigate the frequency of p16a nd p15gene methylation in multiple myeloma (MM),and its relationship with bone marrow ce ll apoptosis and clinical outcome.Methods.Twenty-two patients with MM were stu died to detect p16and p15gene methylation.Methyla-tion-specific polymerase chain rea ction(MSP)was used to detect gene methylation,and terminal trans-ferase-mediated dUTP nick end-labeling(TUNEL)was used to detect cell apoptosis.Results.p16and /or p15gene methylatoin was d etected in 10of 22patients(45.4%).There were 3pa-tients with p16gene methylation,9p atients with p15gene methylation,a nd 2patients with both genes methyla-tion.The incidence of methylation o f p15gene was higher than that of p16g ene(P<0.05).The patients with p16and /or p15gene methylation had a delayed cell apoptosis,poor respon se to chemotherapy,and a short over-all survival(OS).Conclusion.The methylation of p16and /or p15gen e plays a key role in MM apoptosis path ogenesis.The patients with both p16and p15gene me thylation had a poor prognosis.

口腔颌面部鳞癌组织中p16基因变异的初步研究

目的检测口腔颌面部鳞癌中p16基因的缺失和突变,以了解p16基因在口腔颌面部恶性肿瘤发生发展过程中的作用。方法应用聚合酶链反应-单链构象多态性分析(PCR—SSCP)技术,检测33例原发性口腔颌面部鳞癌的石蜡标本,将p16基因的变异频率与口腔颌面部鳞癌的临床分期、组织学分级及淋巴结转移进行统计学分析。结果p16基因的缺失率为21%,点突变率为6%,变异频率为27%。临床I、II、III、IV期每两期之间无显著性差异(P>0.05);而临床I、II期和III、IV期之间有显著性差异(P<0.05)。组织学分级和有、无淋巴结转移之间p16基因变异无显著性差异(P>0.05)。结论p16基因的缺失和突变是口腔颌面部鳞癌中常见的分子事件,它的失活在口腔颌面部鳞癌的发生发展中起着重要作用;说明其变异与肿瘤的临床分期密切相关,随着肿瘤临床进程的发展,其变异频率增高。研究未发现其变异与肿瘤组织学分级和淋巴结转移之间存在相关关系。

Expression of p16 gene and Rb protein in gastric carcinoma and their clinicopathological significance

AIM:To analyze the correlation between the protein expression of p16 and Rb genes in gastric carcinoma (GC), to investigate the role of p16 gene in invasion and lymph node metastasis of GC, and to examine the deletion and mutation in exon 2 of p16 gene in GC. METHODS: The protein expression of p16 and Rb genes was examined by streptavidin-peroxidase conjugated method (S-P) in normal gastric mucosa, dysplastic gastric mucosa and GC. The deletion and mutation of p16 gene were examined by polymerase chain reaction (PCR) and polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) respectively in normal gastric mucosa and GC. RESULTS: The positive rates of P16 and Rb protein expression respectively were 96% (77/80) and 99% (79/80) in normal gastric mucosa, 92% (45/50) and 80% (40/50) in dysplastic gastric mucosa, 48% (58/122) and 60% (73/122) in GC. The positive rates of P16 and Rb protein expression in GC were significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P<0.05). The positive rate of P16 protein expression in mucoid carcinoma (10%, 1/10) was significantly lower than that in poorly differentiated carcinoma (51%, 21/41), undifferentiated carcinoma (58%, 15/26) and signet ring cell carcinoma (62%, 10/16) (P<0.05). The positive rates of P16 protein in 30 cases of paired primary and lymph node metastatic GC were 47% (14/30) and 17% (5/30) respectively, being significantly lower in the later than in the former (P<0.05). There was no mutation in exon 2 of p16 gene in the 25 freshly resected primary GCs. But five cases in the 25 freshly resected primary GCs displayed deletion in exon 2 of p16 gene. The positive rate of both P16 and Rb proteins was 16% (14/90), and the negative rate of both P16 and Rb proteins was 8% (7/90) in 90 GCs. The rate of positive P16 protein with negative Rb protein was 33% (30/90). The rate of negative P16 protein with positive Rb protein was 43% (39/90). There was reverse correlation between P16 and Rb expression in 90 GCs CONCLUSION: The loss protein expression of p16 and Rb genes is related to GC. The loss expression of P16 protein is related to the histopathologic subtypes and lymph node metastasis of GC. Expression of P16 and Rb proteins in GC is reversely correlated. The deletion but not mutation in exon 2 of p16 gene may be involved in GC.

Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

Objective To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared wkh that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases （P〈0.01） and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in pl6 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

A Quantitative DNA Methylation Assay Using Mismatch Hybridization and Chemiluminescence

To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. Methods Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. Results The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. Conclusion Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

DELETIONS AND POINT MUTATIONS OF p16,p15 GENE IN PRIMARY TUMORS AND TUMOR CELL LINES

Aberrations of chromosome 9 p21 22 are involved in the genesis of many forms of cancer.The gene p16 and p15 have been assigned to this region.Both p16 and p15 are an inhibitor of cyclin D cdk4,cyclin D cdk6 complex and have been implicated in a wide variety of cancer types,including the germline of patients with familial melanoma.In order to investigate and compare the status of p16,p15 gene in primary tumors and cell lines,we examined 357 primary tumors and 29 cell lines derived from diverse tumor types.In addition to analysis of these primary tumors and cell lines,blood specimens from 91 patients either with sporadic multiple cancers or from cancer prone families were also analyzed.The data showed the following:1)Homozygous deletions of p16,p15 were comparatively rare and far less common than previously reported,although hemizygous deletions were observed in a significant fraction of many tumor types;2)the incidence of p16,p15 deletions(either homozygous deletions or heterozygous deletions)varied significantly among different tumor types;3)most deletions involved in both p16 and p15 genes;4)sequence variations in the coding sequence of p16,p15 were comparatively rare among these tumor types,though mutations and polymorphisms were identified;5)some tumors which showed LOH at 9p,containing p16 and p15 gene,did not show deletions or point mutations in the p16,p15 gene.6)In a subset of retinoblastoma and osteosarcoma where no Rb gene mutations were present a significant fraction was found to contain p16,p15 gene deletions.

The effects of CYP1A1 gene polymorphism and p16 gene methylation on the risk of lung cancer in a Chinese population

Objective： To investigate the relationship between the genetic polymorphism of CYP1A1 and the genetic susceptibility to lung cancer as well as to study the effects of the methyiation in p16 gene on the risk of lung cancer in a Chinese population. Methods： A case control study was conducted among 47 cases of lung cancer and 94 controls. The genetic polymorphism of CYP1A1 was tested with method of PCR-RFLP, and a methylation-specific PCR （MSP） was performed to detect p16 methylation. Results： It showed that there was no significant difference in frequencies of the genotypes of CYP1A1 between the two groups （P 〉 0.05）. Synergistic effects were not found between smoking and CYP1AI. Methylated p16 gene was found in 44.7% （21/47） of lung cancer tissues and in 17.0% （8/47） of normal lung tissues with significant difference （P 〈 0.05）. Conclusion： The genetic polymorphism of CYP1A1 does not increase the risk of lung cancer in a Chinese population. The methylation in p16 gene may be the most common mechanism to inactivate p16 gene in lung cancer, and is not significantly associated with genotype of CYP1A1,

PROMOTER HYPERMETHYLATION OF p16 GENE AND DAPK GENE IN SERA FROM HEPATOCELLULAR CARCINOMA (HCC) PATIENTS

Objective： To analyze the aberrant methylation of p16 gene and DAPK gene in sera from primary liver cancer patients ad to evaluate the clinical significance. Methods： A methylation-specific PCR was performed for the detection of promoter hypermethylation of p16 gene and DAPK gene in blood DNA from 64 cases of HCC patients, and to analyze the relation of the aberrant methylation of p16 gene and KAPK gene and the clinical pathological data. Results： 76.6%（49/64） of the sera from 64 cases of HCC patients showed hypermethylation for p16 promoter and 40.6% （26/64） for KAPK promoter, whereas no methylated p16 gene promoter and DAPK gene promoter were found in sera from benign liver diseases patients and normal control. Methylated p16 gene and KAPK gene promoters in sera did not strongly correlated with HBsAg, stage, metastasis and differentiation in HCC; but strongly correlated with AFP. Conclusion： Detection of the aberrant methylation of p16 gene and KAPK gene in blood DNA from HCC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.

Quantitative Study on Expression of P16 Multiple Tumor Suppressor Gene in Salivary Gland Neoplasm

The expression of P16 gene were found in all 3 groups. The positive unit (PU) was higher in tumor group and cancer group than that in normal group ( P <0.01). Furthermore, the PU of P16 was stronger in cytoplasm than in nucleus. Malignant tumors and acini surrounding the tumor revealed strong positives and week positives respectively. The PU of P16 gene was higher in deep lobe of recurrent parotid neoplasm with incomplete capsule than that in shallow lobe of primary parotid neoplasm with complete capsule. Our findings suggests that P16 gene plays equally important role in the salivary gland tumors and tumors in other part of the body.

Tumor suppressor gene p16 and Rb expression in gastric cardia precancerouslesions from subjects at a high incidence area in northern China

AIM:To further understand the molecular basis for gastric cardia carcinogenesis and to provide etiological clues. METHODS: Endoscopic mucosa biopsy and histopathological examinations were made on 37 subjects from a high incidence area for both esophageal and gastric cardia carcinomas in northern China. All the biopsy samples were fixed in 850 ml. (-1)L alcohol and embedded in paraffin. Each block contained one piece of tissue and was serially section at 5 microm. Immunohistochemistry (ABC) was carried out on these gastric cardia samples to determine the alterations of p16 and Rb. RESULTS: Based on the histopathlogical examination there were 11 cases of chronic superficial gastritis, 12 cases of chronic atrophic gastritis and 14 cases of dysplasia. The immunostaining demonstrated different levels of unclear immunostaining of p16 and Rb in normal gastric cardia tissue and the tissues with different severity of lesions. With the lesions progressing, the positive immunostaining rates for p16 protein had a decreasing tendency. In contrast, the positive immunostaining rate for Rb protein had an increasing tendency. There was a significant negative relationship between the two parameters. Changes of p16 was CSG 11(100%), CAG 7(58%), DYS 4(29%) and changes of Rb was CSG 2(18%), CAG 8(67%) and DYS 12(86%), (P【0.05). CONCLUSION: The alterations of p16 and Rb protein may play a role in the early stages of gastric cardia carcinogenesis.

p16 gene methylation in colorectal cancers associated with Duke's staging

AIM: To explore the association of methylation of the CpG island in the promotor of the p16 tumor suppressor gene with the clinicopathological characteristics of the colorectal cancers. METHODS: Methylation-specific PCR (MSP) was used to detect p16 methylation of 62 sporadic colorectal cancer specimens. RESULTS: p16 methylation was detected in 42% of the tumors.Dukes'staging was associated with p16 methylation status.p16 methylation occurred more frequently in Dukes'C and D patients (75.9%) than in Dukes'A and B patients (12.1%). CONCLUSION: p16 methylation plays a role in the carcinogenesis of a subset of colorectal cancer, and it might be linked to poor prognosis.

Effects of Exogenous p16^(ink4a) Gene on Biological Behaviors of Human Lung Cancer Cells

The effects of exogenous p16^ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16^ink4a gene were investigated. Exogenous p16^ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16^ink4a gene was homozygously deleted. The expression of p16^ink4a mRNA and protein was detected by RT-PCR and immunocytochemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16^ink4a gene could be stably expressed. The growth of A549 cells transfected with p16^ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16^ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhibited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exogenous p16^ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16^ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.