Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2

INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecular mechanisms of hepatocarcinogenesisremain poorly understood, an increasing number ofgenetic abnormalities have been recognized[7-10],for example, the p16 gene[11,12] the p53gene[13-18], the E-cadherin gene[19], and the c-mycgene[20].

Expression and significance of tumor suppressor gene p16 in human ovarian neoplasm

To observe the relationship between tumor suppressor gene p16 expression and ovarian cancer occurrence and development. Metbods: Using ABC immunohistochemistry method, we investigated the expression of p16 in 72 cases of ovarian neoplasm. Results: The positive rates of p16 in malignant, benign, borderline tumors and normal ovarian tissue were 7. 89%, 60.00%, 66. 67% and 83. 33%, respectively (P<0.01). In the cases whose tumors were more malignant and poorly differentiated, and who relapsed and died, the positive stainings were not discovered. Conclusiou: p16 is well related with the occurrence and development of malignant ovarian tumor.

Overexpression and mutations of tumor suppressor gene p53 in hepatocellular carcinoma

AIMS To examine the prevalance of p53 mutations in hepatocellular carcinoma (HCC) from Chongqing area and the relationship between the p53 mutations and clinicopathological features of HCC,as well as the risk factors. METHODS The overexpression and point mutations of tumor suppressor gene p53 in 38 cases of HCC were detected by a sensitive antigen retrieval fluid (ARF) immunohistochemical method and polymerase chain re- action(PCR)-restriction fragment length polymorphism (RFLP),and single strand conformation polymorphism (SSCP)-silver staining analysis. RESULTS The results showed that 16 of 38 HCCs had positive p53 protein (42.1%),7 HCCs had p53 mutation at 249 (18.4 % ) and 2 HCCS had point muta- tion within exon 7 other than 249. Among 9 cases of HCC with mutations,8 cases demonstrated positive p53 protein,its coincidental rate was 88.9%. The overexpression and mutations of p53 were significantly related to the differentiation and metastasis of HCCs. The frequency of p53 mutations was consistent with high prevalence of HBV and a moderate aflatoxin B1 (AFB1) exposure in our area. CONCLUSIONS The results suggest that AFB1 acts synergistically with HBV in the generation of p53 mutations. Furthermore,dietary exposure to AFB1 may mainly contribute to the tumor specific mutation at codon 249,while HBV may account for other scattered mutations in HCC.

Relationship between hepatitis B virus associated primary hepatocellular carcinoma and alteration of tumor suppressor gene p53

Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.

Effects of multiple tumor suppressor 1 on the proliferation of human ovarian cancer HO-8910 cells

objective: To investigate the effects of multiple tumor suppressor 1 on the proliferation of ovarian cancer cell lines. Methods: Growth characteristics of HO-8910 (without p16 gene expression ), 8910-p16 (trans fected with p16 gene) and 8910-pcDNA3 (transfected with the vector pcDNA3) cells were studied by comparison of the cell growth curves. DNA synthesis was also compared among the 3 kinds of cells. Results: After trans fected with p16 gene, the 8910-p16 cells were markedly inhibited in both the proliferation and DNA synthesis. There was no significant difference between the 8910-pcDNA3 cells and the HO-8910 cells. Conclusion: Multiple tumor suppressor 1 can inhibit the proliferation and DNA synthesis of human ovarian cancer HO-8910 cells.

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.

Are there tumor suppressor genes on chromosome 4p in sporadic colorectal carcinoma?

AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients.METHODS: Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test.RESULTS: Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency (< 30%) by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus (0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively). On D4S403 locus, LOH was significantly associated with tumor gross pattern (11.11%, 0, 33.33%, P = 0.030). No relationship was detected on other loci compared with clinicopathologial features.CONCLUSION: By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 (4p15.2) and D4S405 (4p14) locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm).

Loss of chromosome 9p21 and decreased p16 expression correlate with malignant gastrointestinal stromal tumor

AIM: To investigate loss of heterozygosity (LOH) of chromosome 9p21 and the prognostic relevance of p16 expression in gastrointestinal stromal tumor (GIST). METHODS: Fifty-one GIST patients (30 men and 21 women; median age 59 years; range 29-80 years) treated surgically within a 10-year period were grouped by aggressive behavior risk (17 with very low and low, 14 intermediate, and 20 high risk). GISTs were characterized immunohistochemically and evaluated for LOH of 9p21 by microsatellite analysis at D9S1751, D9S1846, D9S942, and D9S1748. LOH of 9p21 and immunohistochemicalexpression of p16 protein encoded at 9p21 were correlated with clinicopathological parameters, and the prognostic significance of p16 alterations was evaluated. RESULTS: Thirty-one (63.3%) cases showed LOH with at least one microsatellite marker. LOH frequency was 37.0% at D9S1751, 37.5% at D9S1846, 42.1% at D9S942, and 24.2% at D9S1748. There was a higher LOH frequency of D9S942 in high-risk than in non-highrisk tumors (P < 0.05, χ 2 = 4.47). Gender, age, tumor size and site were not correlated with allelic loss. Ninety percent (18/20) of the GIST patients in the high risk group showed LOH with at least one of the 9p21 markers, while 57.1% (8/14) in the intermediate risk group and 33.3% (5/15) in the very low and low risk groups, respectively (P < 0.05, χ 2 = 12.16). Eight (28.5%) of 31 patients with LOH and 1 (5.6%) of 18 patients without LOH died of the disease during the follow-up period. Loss of p16 protein expression occurred in 41.2%, but in 60% of the high risk group and 23.5% of the very low and low risk groups (P < 0.05, χ 2 = 4.98). p16 loss was associated with poor prognosis (P < 0.05, χ 2 = 4.18): the 3and 5-year overall survival rates were 84.8% and 70.8% for p16-negative and 100% and 92.0% for p16-positive patients, respectively. CONCLUSION: LOH at 9p21 appears to play an important role in GIST progression; decreased p16 expression in GIST is highly predictive of poor outcome.

Exogenous p16 gene therapy combined with X-ray irradiation suppresses the growth of human glioma cells

In this study, we infected human glioma U251 cells with a replication-defective recombinant adenovirus carrying the p16 gene. This adenovirus constructed was able to transfect exogenous p16 into the human glioma cells efficiently, and direct a high level of p16 protein expression. Tumor-inhibition experiments demonstrated that treatment with the adenovirus-p16 significantly inhibited the growth of glioma cells in vitro as well as the in vivo development of tumors in nude mice bearing a brain glioma. The combination of adenovirus-p16 gene treatment and X-ray irradiation resulted in a greater inhibition of tumor growth. Adenovirus-mediated p16 gene therapy conferred a significant antitumor effect against human glioma cells both in vitro and in vivo, and that there was a synergistic effect when X-ray irradiation was also used.

采用组织芯片技术检测大肠癌组织中p16和cyclinD1的表达

目的 :研究大肠癌组织中多肿瘤抑制蛋白 (P16 )和细胞周期蛋白 (cyclinD1)的表达及其意义。 方法 :采用组织芯片技术制作 80例大肠癌组织芯片 ,同时用S P免疫组织化学方法检测大肠癌组织芯片中P16和cyclinD1的表达。 结果 :80例大肠癌中P16和cyclinD1的阳性率分别为 4 0 0 %、5 7 5 %。P16低表达、cyclinD1高表达与大肠癌的浸润程度密切相关 ,大肠癌中P16低表达与cyclinD1的高表达密切相关。 结论 :P16和cyclinD1的表达水平可以作为评估大肠癌预后的参考指标 ,P16低表达影响cyclinD1的高表达。应用组织芯片大规模高效检测临床组织样本是可行的 ,具有快速、方便、经济、准确的特点。

P16、Ki-67表达与直肠癌患者预后的关系

目的研究多肿瘤抑制基因P16、细胞增殖核抗原Ki-67表达与直肠癌患者预后的关系。方法选择2016年8月—2018年12月浙江省杭州市萧山区第一人民医院60例直肠癌患者术后的肿瘤及癌旁正常组织,以免疫组织法检测P16、Ki-67的表达情况,观察P16、Ki-67在不同年龄、病理类型、TNM分期、分化程度及淋巴结有无转移中的阳性率,比较P16、Ki-67阳性患者与阴性患者术后1年的生存情况,通过Spearman系数分析P16和Ki-67的相关性。结果直肠癌癌组织中P16及Ki-67的阳性率显著高于癌旁正常组织(P<0.05)。TNM分期Ⅲ期、高分化程度患者P16、Ki-67阳性率显著高于TNM分期Ⅱ期、低-中分化患者(P<0.05);有淋巴结转移患者Ki-67阳性率显著高于无淋巴结转移患者,有淋巴结转移患者P16阳性率显著高于无淋巴结转移患者(均P<0.05)。P16阴性患者生存率显著高于阳性患者(P<0.05)。Ki-67阳性患者1年生存率显著低于Ki-67阴性患者(P<0.05)。经相关性分析P16和Ki-67直肠癌组织中的表达无相关性(r=0.157,P=0.461)。结论直肠癌癌组织内P16和Ki-67水平表达与直肠癌临床病理特征密切相关,可能为直肠癌的临床治疗及预后评估提供参照。

采用组织芯片技术探讨p16和cyclin D1在胆囊癌中的表达及临床意义

目的探讨多肿瘤抑制蛋白(p16)和细胞周期蛋白(cyclin D1)的表达在胆囊癌发生发展中的作用及其对预后的影响,研究应用组织芯片在大规模高效检测临床组织样本的可行性。方法取40例胆囊癌、24例胆囊良性病变和16例癌旁黏膜标本,采用组织芯片技术制作成组织芯片,同时用S-P免疫组织化学方法检测组织芯片中p16和cyclinD1的表达。结果p16和cyclin D1在胆囊癌中的阳性表达率分别为40.0(和57.5(,与胆囊良性病变(75.0(,29.2()和癌旁黏膜(93.8(,12.5()相比有显著性差异(P<0.005)。p16的低表达和cyclin D1的高表达与胆囊癌的分化程度(P(0.025,P<0.01)、浸润深度(P<0.01,P<0.025)、淋巴结转移(P<0.01,P<0.01)以及患者的5年生存率(P<0.025,P<0.05)密切相关。p16和cyclinD1在胆囊癌中的表达呈负相关(r=-0.54)。结论p16和cyclin D1的异常表达在胆囊癌的发生、发展过程中起重要作用。p16和cyclin D1的表达水平可以作为评估胆囊癌预后的参考指标。应用组织芯片大规模高效检测临床组织样本是可行的,具有快速、方便、经济、准确的特点。

采用组织芯片技术检测大肠癌组织中p16、cyclinD1和CDK4的表达

目的研究大肠癌组织中多肿瘤抑制蛋白(p16)、细胞周期蛋白(cyclinD1)及细胞周期蛋白依赖激酶(CDK4)的表达及其意义。方法采用组织芯片技术制作80例大肠癌组织芯片,同时用S-P免疫组织化学方法检测大肠癌组织芯片中p16、cyclinD1和CDK4的表达。结果80例大肠癌中p16、cyclinD1和CDK4的阳性率分别为40.0%、57.5%、47.5%。不同组织学类型、淋巴结转移状况的大肠癌中,p16、cyclinD1的表达无明显差异(P>0.05)。不同组织学类型、浸润程度及淋巴结转移状况的大肠癌中CDK4的表达均无明显差异(P>0.05)。结论p16表达、cyclinD1高表达与大肠癌的浸润程度密切相关,大肠癌中p16低表达与cyclinD1的高表达密切相关。p16低表达影响cyclinD1的高表达。应用组织芯片大规模高效检测临床组织样本是可行的,具有快速、方便经济、准确的特点。

胃癌组织中P16、P53蛋白表达的相关性和临床意义

目的 探讨 P16、 P5 3基因在胃癌发生发展及预后判断上的作用 ,以及可能的基因调节机制。方法 应用免疫组化方法检测 61例胃癌标本和 4 9例癌旁正常胃粘膜的 P16和 P5 3蛋白表达。结果 胃癌组织 P16蛋白的阳性率为 70 .5 % ( 4 3 / 61) ,而癌旁正常胃粘膜的阳性率为 10 0 % ( 4 9/ 4 9) ,10例早期胃癌均有 P16蛋白的表达 ,而且在同一切片中可以观察到分化较好或浸润浅层的癌细胞表达 P16蛋白 ,而分化较差、浸润深层的癌细胞则没有表达。P16蛋白表达强度与胃癌的分化程度、浸润深度、有无淋巴结转移及病人的生存期密切相关 ( P<0 .0 1) ;P5 3蛋白表达与 P16蛋白的失表达无显著相关 ,但对于 P16有表达的病例 ,P5 3阳性时 ,其表达强度较强 ( P<0 .0 5 )。结论 确有部份的胃癌组织存在 P16基因的失表达 ,且失表达发生在胃癌发生发展的晚期阶段 ;胃癌细胞中可能存在 Rb- P16的负反馈调节。

Changes of p53 and Waf1p21 and cell proliferation in esophageal carcinogenesis

AIM To study the correlationship between the changes of p53, Waf1p21 and the cell proliferation determined by PCNA at different stages of human esophageal carcinogenesis. METHODS Biopsied and resected esophageal tissues from a high risk population for esophageal cancer in northern China were used in this study. All the specimens were fixed with 85% alcohol and further processed with routine histology. The avidin biotin peroxidase complex (ABC) method was used for the detection of p53, Waf1p21 and PCNA. RESULTS The strong nuclear staining for p53, Waf1p21 and PCNA was observed in the normal esophageal epithelium and the epithelia with different severities of lesions. As the lesions progressed to dysplasia (DYS) and to esophageal squamons cell carcinoma (SCC), the percentage of Waf1p21 immunoreactivity decreased. The number of Waf1p21 immunostaining positive cells increased slightly from normal to basal cell hyperplasia (BCH), but there was no further increase in DYS and in SCC. The total number of positive cells for Waf1p21 stain appeared to be lower than that of p53 in normal and BCH esophageal epithelia and much lower in DYS and SCC. The Waf1p21 positive immunostaining cells were located at the third and forth cell layers in half of the samples examined, which was 2～4 cell layers higher than that of PCNA and p53 in the same histological categories of normal, BCH and DYS. CLNCLUSION The low levels of Waf1p21 at the stage of DYS may be related to a functional loss of p53. Other mechanisms may also be responsible to the lack of Waf1p21 expression in DYS and SCC.

DNA methylation and carcinogenesis in digestive neoplasms

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HYPERMETHYLATION OF p14^(ARF) PROMOTER REGION AND EXPRESION OF p14^(ARF) GENE PRODUCT IN NON-SMALL CELL LUNG CANCER

Objective： This study was designed to investigate promoter methylation status and protein expression of p14^ARF gene in non-small cell lung cancer, and value the role of p14^ARF promoter methylation in carcinogenesis of non-small cell lung cancer. Methods： Promoter methylation status and protein expression of p14^ARF gene in 40 cases of non-small cell lung cancer were analyzed by methylation specific polymerase china reaction （MSP）, restriction enzyme-related polymerase chain reaction （RE-PCR） and immunohistochemistry （IHC）. Results： The positive rates of p14^ARF promoter methylation in tumor tissues and normal tissues adjacent to cancer were 17.5% （7/40） and 2.5% （1/40） respectively. There were statistically significant differences between them, P〈0.05. The results of RE-PCR were consistent with that of MSP. The expression rate of p14^ARF protein in tumor tissues was significantly lower than that in normal tissues adjacent to cancer, p〈0.01. Promoter methylation status and protein expression of p14^ARF gene in non-small cell lung cancer showed significantly an inverse correlation （r=-0.56, P〈0.01）, and both of them did not relate statistically with the clinicopathologic characteristics of patients such as histological classification, clinical stage, differentiation grade and lymph node involvement. Conclusion： Promoter methylation is a crucial mechanism of inactivation of p14^ARF gene. Promoter methylation of p14^ARF gene might he involved in carcinogenesis of non-small cell lung cancer, and is an early event in development process of non-small cell lung cancer. It might be used as a new target in gene treatments in the future.

组织中P16、VEGF表达与结肠癌患者临床分期的相关性

目的探讨组织中多肿瘤抑制基因P16、血管内皮生长因子(VEGF)表达与结肠癌患者临床分期的相关性。方法收集2020年5月至2021年5月潢川县人民医院收治的80例结肠癌患者临床资料,所有患者均接受腹腔镜手术治疗,取病理组织,采用免疫组织化学方法检测患者组织中P16、VEGF表达情况,查阅资料,记录患者临床分期情况,分析组织中P16、VEGF表达与结肠癌患者临床分期的相关性。结果80例结直肠癌患者中,0~Ⅰ期19例(23.75%),Ⅱ期24例(30.00%),Ⅲ期26例(32.50%),Ⅳ期11例(13.75%);中、晚期组组织中P16阴性表达、VEGF阳性表达患者占比高于早期组,差异有统计学意义(P<0.05);中期组、晚期组组织中P16、VEGF表达比较,差异无统计学意义(P>0.05);经logistic回归分析,结果显示,组织中P16阴性表达、VEGF阳性表达与结肠癌患者临床高分期相关(OR>1,P<0.05)。结论组织中P16阴性表达、VEGF阳性表达与结肠癌患者临床高分期有关。

MUTATION AND ABNORMAL EXPRESSION OF P16^(INK4a) INHEPATOCELLULAR CARCINOMA

Objective:To investigate the relationship betweenp16INK4a and primary hepatocellular carcinoma (HCC),especially hepatitis B-related HCC. Methods: p16INK4a andits protein in HCC were analyzed with PCR-SSCP and theimmunohistochemistry methods respectively. Results: Thepositive incidence of p16INK4 protein expressing in HCC waslower than that of normal liver tissue (P<0.05), and theabsence of p16INK4 protein was associated with HCCmetastasis (P<0.05). The low frequency of mutation ofp16INK4 exonl and exon2 upstream fragment was found inHCC. Conclusion: Absence of p16INK4 protein in HCC wasnot associated with HBV-infection.

B淋巴细胞瘤-2关联永生基因3蛋白、多重肿瘤抑制基因1在宫颈癌癌前病变中表达及联合液基细胞学检查的临床意义

目的研究B淋巴细胞瘤-2关联永生基因3(BAG3)蛋白、多重肿瘤抑制基因1(P16)在宫颈癌癌前病变中表达及联合液基细胞学检查的临床意义。方法选择绵阳市中心医院2015年6月至2017年6月在妇科门诊筛查并确诊的120例宫颈病变病人为研究对象,根据其病变情况分为两组,其中癌前病变组病人69例,宫颈癌组病人51例。对比两组病人的BAG3、P16水平,将不同严重程度宫颈癌癌前病变病人的BAG3、P16水平进行比较,分析联合检测效能。结果宫颈癌组病人的BAG3(2.74±0.37)水平、P16(1.45±0.38)水平显著高于癌前病变组BAG3(1.70±0.51)、P16(0.53±0.16),差异有统计学意义(P<0.05)。不同严重程度宫颈癌癌前病变病人的BAG3和P16水平差异有统计学意义(P<0.05),其中宫颈上皮内瘤变Ⅰ级(CINⅠ)组的BAG3(1.01±0.24)水平低于CINⅡ组BAG3(1.78±0.79)和CINⅢ组的BAG3(2.33±0.88),且CINⅡ组低于CINⅢ组,差异有统计学意义(P<0.05);CINⅠ组的P16(0.21±0.06)水平低于CINⅡ组P16(0.45±0.10)和CINⅢ组的P16(0.72±0.17)水平,且CINⅡ组低于CINⅢ组,差异有统计学意义(P<0.05)。联合诊断对于宫颈癌的诊断特异度显著高于单独检测,差异有统计学意义(P<0.05)。通过ROC曲线分析结果发现联合检测对于宫颈癌癌前病变的诊断ROC曲线下面积显著高于单独检测(P<0.05)。结论随着宫颈癌前病变的进展,P16、BAG3表达增加,BAG3蛋白、P16联合液基细胞学检查对于病人的诊断具有积极的意义。