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].

Mutations in the p16 gene in DMBA-induced pancreatic intraepithelial neoplasia and pancreatic cancer in rats

BACKGROUND：7,12-dimethylbenzanthracene（DMBA）-induced pancreatic intraepithelial neoplasia（PanIN）and pancreatic cancer in rats provide a classic model for uncovering the molecular mechanisms underlying pancreatic cancer.However,this model has not been characterized genetically,and in particular,the major genetic alterations in the p16 gene are unknown.METHODS： Lesions of PanlN and pancreatic cancer were induced with DMBA implantation in 40 rats, and control pancreatic tissue was obtained from 10 age-matched rats without exposure to DMBA. Pancreatic tissue was harvested three months after DMBA implantation and DNA was extracted. Homozy- gous deletions and point mutations of the pl6 （exons 1 and 2） gene were detected by PCR amplification and direct sequencing. RESULTS： DMBA implantation in the 40 rats induced 26 Pan- INs and 9 carcinomas. The overall frequency of p 16 alterations in the pancreatic tissue of these rats was 42.86% （15/35）, and the changes were point mutations, not homozygous deletions. p16 mutations were present in 30.77% （8/26） of the rats with PanIN and 77.78% （7/9） of the rats with carcinoma （P〈0.05）. The increasing incidence of p16 alterations was detected in 20.00% （1/5） of PanIN-1, 28.57% （2/7） of PanIN-2 and 35.71% （5/14） of PanIN-3 lesions. CONCLUSION： Our findings indicated that p16 alteration is a common event in the carcinogenesis of this model and that the mutation pattern is analogous to that of human lesions.

Mutations and altered expression of p16^(INK4a) in human pancreatic carcinoma cell lines with different potential of metastasis

Objective:To analyze the p16INK4a genomic alteration and expression status in 3 human pancreatic carcinoma cell lines with different potential of metastasis. Methods:Using PCR-SSCP, Dot-blot and immunohistochemistry, the p16INK4a genomic mutation and expression were analyzed on DNA, mRNA and protein levels in 3 human pancreatic carcinoma cell lines Patu8902, Patu8988 and SW1990, which had different potential of metastasis. Results: (1) On DNA level: there was no deletion of p16INK4a Exon Ⅰ in 3cell lines; p16INK4a Exon Ⅱ was only deleted in Patu8902 while no deletion in Patu8988 and SW1990. No insertion, microdeletion and point mutation were found in the 3 cell lines. (2) On RNA level: the expression of p16INK4a protein was negative in Patu8902, low expressed in SW1990, but highly expressed in Patu8988.(3) On protein level: P16 protein was strongly stained in Patu8988, much lower in SW1990, but not stained in Patu8902. Conclusion:The genomic type and expression of p16INK4a are quite different in 3 pancreatic carcinoma cell lines which have different potential of metastasis. It is suggested that genomic homozygous deletion and low expression of mRNA might relate to the potential of metastasis of pancreatic cell lines. In other words, dysfunction of p16INK4a might be an important mechanism in the metastasis of pancreatic carcinoma.

FREQUENT DELETION OF MTS1/p16 GENE AND CORRELATION WITH CLINICOPATHOLOGICAL PARAMETERS IN ENDOMETRIAL CARCINOMA

Objective: To investigate the possible relationship between deletion of MTS/p16 gene and progression of endometrial carcinoma Methods: Forty six primary endometrial carcinoma, 7 tumor adjacent endometrial tissue, 10 normal endometrial tissue specimen and 5 xenografts from patients with endometrial carcinoma were examined for homozygous deletion of MTS/p16 gene by polymerase chain reaction based analysis Results: Of 46 endometrial cancer specimens, 9 showed homozygous deletion, no deletion was detected in the tumor adjacent and normal endometial tissues Nor was it detected in well differentiated endometrial carcinoma and all xenografts Conclusions: Deletion of MTS1/p16 gene might contribute to the progression of endometrial carcinoma and could be served as indicator for predicting prognosis

Expression,deleton and mnutation of ρ16 gene in human gastric cancer

AIM To investigate the relationship between the expression of p16 gene and the gastric carcinogenesis,depth of invasion and lymph node metastases, and to evaluate the deletion and mutation of exon 2 in p16 gene in gastric carcinoma.METHODS The expression of P16 protein was examined by streptavidin-peroxidase conjugated method (S-P); the deletion and mutation of p16 gene were respectively examined by polymerase chain reaction (PCR) and polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) in gastric carcinoma.RESULTS Expression of P16 protein was detected in 96.25% (77/80) of the normal gastric mucosa, in 92.00% (45/50) of the dysplastic gastric mucosa and in 47.54% (58/122) of the gastric carcinoma. The positive rate of P16 protein expression in gastric carcinoma was 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 10.00% (1/ 10) was significantly lower than that in poorly differentiated carcinoma 51.22% ( 21/ 41 ),undifferentiated carcinoma 57.69% (15/26) and signet ring cell carcinoma 62.50% (10/ 16) (P＜0.05). The positive rate of p16 protein in 30 cases paired primary and lymph node metastatic gastric carcinoma: There was 46.67% (14/30) in primary gastric carcinoma, 16.67% (5/30) in lymph node metastatic gastric carcinoma. The positive rate of lymph node metastatic carcinoma was significantly lower than that of primary carcinoma (P＜0.05). There was of p16 gene mutation in exon 2, but 5 cases displayed deletion of p16 gene in exon 2 in the 25 primary gastric carcinomas.CONCLUSIONS The expression loss of P16 protein related to the gastric carcinogenesis, gastric carcinoma histopathological subtypes and lymph metastasis. The mutation of p16 gene in exon 2 may not be involved in gastric carcinogenesis. But the deletion of p16 gene in exon 2 may be involved in gastric carcinogenesis.

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

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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.

IN SITU PCR AND IMMUNOHISTOCHEMICAL STUDIES ON p16 GENE IN PITUITARY ADENOMAS

Objective: To examine the occurrence of p16 gene deletion and to analyze p16 expression on paraffin-embedded human pituitary adenoma specimens. Efforts were made to optimize the technical conditions forin situ PCR. Methods:In situ PCR techniques and inimuno-histochemistry were used. Results: Immunohistochemically, p16-positive tumor cells were observed in all cases with various proportions. The majority of the stromal cells and part of tumor cells was devoid of p16 immunostaining, but signal ofin situ PCR for p16 gene, exon 2, was displayed in these cells. Conclusion: The results implied that p16 gene might not be deleted in these pituitary adenomas. It also indicated thatin situ PCR, both direct and indirect methods, proved feasible and informative to the aim of DNA detection. It is critical to overcome non-specific amplification in directin situ PCR by means of higher annealing temperature, fewer cycle, lower magnesium concentration and stringent washing. A target DNA-deleted sample as the negative control is extremely necessary. For the indirect method, the way to improve the sensitivity is to loosen the conditions for amplification and washing, so that more amplification products are subject to hybridization, and signal detection is facilitated.

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.

THE EXPRESSION OF p16 AND CYCLIN D_1 IN PROLIFERATIVE ENDOMETRIUM AND ENDOMETRIAL CARCINOMA

Objective To study the role of p16 and cyclin D 1 in the genesis and development of endometrial carcinoma.Methods 12 cases of normal endometrium,22 cases of proliferative endometrium and 41 cases of endometrial carcinoma were detected for the expression of p16 and cyclin D 1 by means of immunohistochemical S P. Results In normal endometrium p16 was expressed while cyclin D 1 was almost negative in the proliferative phase,but both of them were negative in the secretory phase.Among the groups of the simple and compound hyperplasia, the atypical hyperplasia and the endometrial carcinoma,the expression of p16 showed a descending tendency, while the expression of cyclin D 1 showed an ascending tendency.In endometrial carcinomas the expression of p16 was significantly lower than that of normal endometrium and proliferative endometrium( P <0.01, P <0.05).However, the expression of cyclin D 1 in proliferate endometrium and endometrial carcinoma was significantly higher than that in normal endometrium ( P<0.05,P<0.01) .The overexpression of cyclin D 1 in the atypical hyperplasia group was obviously different from that in the simple and compound hyperplasia group ( P <0.01).In endometrial carcinoma,the expression of p16 was decreasing with the descending of cell differentiate degree, on the opposite, the expression of cyclin D 1 was increased and there existed a negative correlation between them.It was also observed that the overexpression of cyclin D 1 was significant different between G 1 and G 2,G 3(P<0.01).Conclusion p16 is a negative regulating factor of cell cycle in endometrial carcinoma, while cyclin D 1 is a positive one.Both of them are important in the genesis and development of endometrial carcinoma.The low expression of p16 and the overexpression of cyclin D 1 are related with the malicious biological behaviors of endometrial carcinoma and maybe play an important role in the judgement of prognosis.Overexpression of cyclin D 1 may be an earlier molecular event in the genesis of endometrial carcinoma.

p16、CyclinD1、Cdk4、Rb基因在胰腺癌中的表达及其意义

目的：探讨ｐ１６ 、 Ｃｙｃｌｉｎ Ｄ１、 Ｃｄｋ４ 、 Ｒｂ 在胰腺癌中的作用及其相互关系。方法：免疫组织化学检测ｐ１６ 、 Ｃｙｃｌｉｎ Ｄ１、 Ｃｄｋ４ 、 Ｒｂ 蛋白在胰腺癌中的表达，原位杂交检测ｐ１６ 和 Ｃｙｃｌｉｎ Ｄ１ 在胰腺癌中的存在状况。结果：ｐ１６ 在胰腺癌中低表达， Ｃｙｃｌｉｎ Ｄ１ 和 Ｃｄｋ４ 过度表达，未发现 Ｒｂ 在胰腺癌标本中缺失；ｐ１６ 与 Ｃｙｃｌｉｎ Ｄ１，ｐ１６ 与 Ｒｂ 呈负相关关系（ Ｐ＜ ０．０５） ， Ｃｄｋ４ 与 Ｃｙｃｌｉｎ Ｄ１ 呈正相关关系（ Ｐ＜ ０ ．０５） 。结论：胰腺癌发生机制涉及ｐ１６ 、 Ｃｙｃｌｉｎ Ｄ１、 Ｃｄｋ４ 和 Ｒｂ 调节环路中多个基因的异常，ｐ１６ 低表达和 Ｃｙｃｌｉｎ Ｄ１ 过度表达可能起在胰腺癌发生中起协同作用。

胰腺癌中p16基因甲基化改变及其蛋白表达分析

目的探讨胰腺癌与癌旁组织中p16基因启动子区异常甲基化的改变及其蛋白表达的特点,以及其与胰腺癌发生发展的关系。方法分别采用免疫组化SP法及甲基化特异PCR(MSP)检测46例人胰腺癌和癌旁组织中p16基因表达及其甲基化的水平,并结合临床资料进行分析。结果胰腺癌中p16蛋白表达率为41.3%(19/46),而癌旁组织表达率为95.7%(44/46),两者有差异显著性(P<0.01)。p16蛋白阳性的19例胰腺癌标本中均未检出基因甲基化;p16蛋白缺失的27例标本中有18例检出基因甲基化,甲基化率为39.1%。p16基因甲基化与蛋白缺失有明显关系(P<0.05)。p16基因表达及其启动子区甲基化的发生率与胰腺癌的大小,患者性别?年龄的差异无显著意义(P>0.05),但与组织分化程度?淋巴结转移?PTNM分期有显著意义(P<0.05)。结论p16基因启动子的异常甲基化可影响p16蛋白的表达,它们与胰腺癌的发生发展有关;p16甲基化和蛋白表达可能成为胰腺癌诊断及预后的候选标志物之一。

p16与PCNA表达与脑胶质瘤分级的相关性及意义

目的： 研究ｐ１６ 与增殖性细胞核抗原（ Ｐ Ｃ Ｎ Ａ）蛋白在各级人脑胶质瘤中的表达率及与胶质瘤病理分级的关系．方法： 利用免疫组化方法检测５２ 例胶质瘤手术标本中ｐ１６ 与 Ｐ Ｃ Ｎ Ａ 蛋白的表达水平，并比较二者的相关性，及其与胶质瘤病理分级的关系． 结果： 随着胶质瘤病理级别的升高ｐ１６ 蛋白的表达率逐渐降低， Ⅰ～Ⅳ级阳性率分别为 ７５． ０％ ，６４７％ ，４２．８％ 和３３．３％ ，低度恶性组（Ⅰ～Ⅱ级）与高度恶性组（Ⅲ～Ⅳ级）有显著差异（ Ｐ＜ ０．０５）． Ｐ Ｃ Ｎ Ａ 表达强度随胶质瘤病理级别的升高而增加（ Ｐ＜ ０．０５）． 所选病例的平均 Ｐ Ｃ Ｎ Ａ 指数（ Ｐ Ｃ Ｎ Ａ Ｌ Ｉ）为 ２７．１， ｐ１６ 与 Ｐ Ｃ Ｎ Ａ 表达存在负相关（ｒ＝ ０．４８５， Ｐ＜ ０．０５）． 结论： ｐ１６ 及 Ｐ Ｃ Ｎ Ａ 蛋白的表达对分析判断胶质瘤病理分级的适用性及恶性转化具有一定意义．

P^(16)和CyclinD_1在不同胃病患者胃粘膜中表达的临床意义

为探讨多肿瘤抑制基因 P1 6 和细胞周期素 D1 (Cyclin D1 )在正常胃、慢性胃炎、不典型增生及胃癌中的表达 ,以及其与胃癌发生、发展及临床、病理参数的关系 ,采用 m RNA原位杂交技术检测了 P1 6 Cyclin D1 在不同胃粘膜病变中的表达情况。结果显示 ,正常胃粘膜 P1 6 阳性率为 90 .9% ,Cyclin D1 为阴性 ;浅表性胃炎、萎缩性胃炎、不典型增生、胃癌的 P1 6 阳性率分别为 87.1%、78.6 %、37.5 %、2 6 .2 5 % ,Cyclin D1 的阳性率依次为 16 %、2 1%、5 6 %、73.75 % ,癌组织与正常胃粘膜比较有显著性差异。 P1 6 的阳性率与肿瘤恶性程度呈负相关 ,Cyclin D1 与癌分化无明显相关性。胃癌有淋巴结转移者的 P1 6阳性率低于无淋巴结转移者 (P<0 .0 1)。 Cyclin D1 明显高于无淋巴结转移者。随访 2年 ,80例胃癌患者中 13例死亡 ,均为 P1 6阴性、Cyclin D1 阳性者。胃癌中 P1 6与 Cyclin D1 有交叉共存现象者约占 70 %。相关分析显示 ,胃癌中 P1 6与 Cyclin D1 的阳性率呈显著负相关。认为 P1 6与 Cyclin D1 两种基因变化与胃癌的形成关系密切 ;随病变加重 ,P1 6缺失率增加 ,Cyclin D1 阳性率增加 ,两者可单独或协同促进胃癌的发生、发展。其反向表达趋势提示两者可能存在相互抑制机制。多基因异常病例易发生淋?

胃癌患者P^(16)和P^(53)蛋白的表达及其相互关系

目的 探讨P16、P53 基因产物在胃癌中表达的生物学意义及其相互关系。方法 用辣根酶标记链霉卵白素免疫组化法对 33例胃癌的临床病理特征进行分析 ,同时与 17例癌旁和 13例正常胃粘膜中P16和P53 蛋白的表达进行了比较。结果 胃癌患者P16蛋白阳性表达率明显高于正常胃粘膜 (P <0 .0 5 ) ,伴淋巴结转移的胃癌阳性细胞密度高于未转移者 (P <0 .0 5 )。P53 蛋白在正常胃粘膜未见表达 ,胃癌中P53蛋白阳性表达显著高于癌旁组织 (P <0 .0 5 )。P53 与P16在胃癌中共同表达 10例 (30 .30 % ) ,P53 和P16蛋白表达与胃癌的病理学分期、分化程度无关。结论 P16基因异常有促进胃癌转移的效应 ,这对判定胃癌患者的预后有一定的指导意义。P53 与P16在胃癌中有协同作用 ,二者异常表达参与胃癌的发生与发展。

p^(16)基因在原发性肝癌中丢失的研究

为了解抗癌基因ｐ１６在原发性肝癌中的变异情况，采用差别ＰＣＲ技术研究３４例原发性肝癌及其癌周组织。结果显示：１例原发性肝癌存在ｐ１６Ｅｘｏｎ１纯合子缺失，１５例原发性肝癌存在ｐ１６Ｅｘｏｎ２纯合子缺失，其余的１９例肝癌及所有的癌周组织无ｐ１６基因纯合子缺失。研究结果表明ｐ１６基因的缺失与原发性肝癌的发生发展密切相关。

鼻咽癌P^(16)基因的异常表达及其临床意义的探讨

目的 :研究鼻咽癌抑癌基因P16存在的状态及其与鼻咽癌发生、发展的关系。方法 :采用免疫组织化学S P法对 6 0例鼻咽癌、38例鼻咽癌旁组织和 2 0例正常或炎性鼻咽组织P16基因的表达进行检测。结果 :鼻咽癌、癌旁、正常或炎性组织中P16基因总阳性率为 2 3 33%、73 6 8%和 80 0 0 % ,前者与后两者阳性之间比较均有显著性差异 (P <0 0 5 ,r=- 0 5 114 ) ,而后两者阳性率比较无显著性差异 (P >0 0 5 )。在 6 0例鼻咽癌中 ,有淋巴结转移 4 4例 ,P16基因阳性率 18 88% ,无淋巴结转移 16例 ,P16基因阳性率 5 0 0 0 % ,两者阳性率比较有显著性差异 (P <0 0 5 ,r =- 0 376 4 )。结论 :P16基因低表达或缺失与鼻咽癌的发生。

P^(16)蛋白在喉鳞癌中的表达与临床病理和吸烟的关系

目的：应用免疫组化技术SP法结合高压锅抗原修复对36例喉鳞癌P16蛋白的表达进行比较，观察P16蛋白的表述情况。探讨肿瘤抑制基因P16蛋白的表达与临床喉癌病理和吸烟的关系。应用X2检验及精确概率法分析P16蛋白表达与鳞癌分级、浸润深度及转移的相关性。结果：P16蛋白在喉鳞癌中的表达率为63．89％（23／36）；其中，高分化鳞癌P16蛋白的表达率为100％（6／6）；中分化鳞癌P16蛋白的表达率为66．67％（12／18）；低分化鳞癌P16蛋白的表达率为41．67％（5／12），显著低于高中分化组，差异有显著性意义（P＜0．05），结论：P16蛋白的表达与喉鳞癌的分期、浸润深度及转移密切相关。与病人的吸烟程度密切相关。

p^(16)和bcl-2基因与结核分枝杆菌L型感染在前列腺癌中的表达及意义

目的探讨p16抑癌基因和bcl-2凋亡抑制基因及结核分枝杆菌L型感染与前列腺癌的生物学行为。方法采用抗酸染色、免疫组化和核酸原位杂交等方法检测了58例前列腺癌患者中的结核分枝杆菌L型的检出率及p16和bcl-2基因的表达。结果58例前列腺癌中结核分枝杆菌L型检出率为37.9%(22/58),结核分枝杆菌抗原阳性表达为39.7%(23/58);p16蛋白表达阳性率51.7%(30/58),失表达率48.3%(28/58),其中Ⅰ级、Ⅱ级、Ⅲ级的p16蛋白失表达率各为21.1%、56.3%、85.7%(P<0.05),p16mRNA失表达率分别为20%、40%、80%(P<0.05),而bcl-2蛋白表达阳性率60.3%(35/58),Ⅰ级、Ⅱ及、Ⅲ及分别为31.6%、68.8%、100%,bcl-2mRNA阳性表达率各为20%、60%、100%(P<0.05)。结论细菌L型感染可能和bcl-2、p16基因协同在前列腺癌发生和发展中起重要作用。p16基因的缺失、突变和高甲基化,bcl-2基因的异常表达在前列腺癌的发生和发展中起重要的作用。

抑癌基因P^(16)表达与胰腺癌临床病理的关系

应用免疫组织化学 ABC法检测 5 6例胰腺癌组织中抑癌基因 P1 6 的表达 ,并和良性组织作对照研究。结果显示 :胰腺癌中 P1 6 的表达显著低于良性组织 ,且与临床病理分期及预后有关。提示 P1 6