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.

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.

Gene Product Expression of Cyclin D_2 and p16 During the Transition from Cardiac Myocyte Hyperplasia to Hypertrophy

The current study was to investigate mRNA expression of cyclin D 2 and p16 during the transition from cardiac myocyte hyperplasia to hypertrophy. Cultured cardiac myocytes (CM) and fibroblasts (FC) obtained from 1 day old Sparague Dawley rats were used in this study. We have determined (1) hyperplasia by cell growth curve and fluorescence activated cell sorting (FACS); and (2) ultrastructure by electron microscope observation; and (3) expressions of cyclin D 2 mRNA and p16 mRNA by using in situ hybridization and image analysis. The results were shown (1) Results of cell growth curve and FACS analysis showed CM could proliferate in the first 3 cultured days (4 days in postnatal development). But the ability decreased quickly, concomitant with the differentiation. (2) The ultrastructure of CM showed the large amount of myofilaments and mitochondrion and FC showed moderate amount of rough endoplasmic reticulum. (3) The expression of cyclin D 2 mRNA in 3 , 4 , 5 day CM group was 0.89 times(p<0.05), 0.80 times (p<0.05)and 0.56 times (p<0.01)of that in 1 day group respectively. P16 mRNA in 2 , 3 , 4 , 5 day CM group were 1.63 times(p<0.01),1.72 times(p<0.01),1.99 times (p<0.01)and 2.84 times (p<0.01) of that in 1 day group respectively. It can be concluded that cultured neonatal rat cardiac myocytes could proliferate during the first 3 cultured days, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D 2 and p16 have the key roles during the transition from myocyte hyperplasia to hypertrophy.

PROMOTER HYPERMETHYLATION OF p16 GENE IN PRE- AND POST-OPERATIVE PLASMA OF PATIENTS WITH GASTRIC ADENOCARCINOMA

Objective: To detect promoter hypermethylation of p16 gene in matched pre- and post-operative plasma of patients with gastric adenocarcinoma for evaluating the effectiveness of therapeutic intervention. Methods: Tissue samples, pre- and post-operative plasma of 84 patients were collected. Plasma of 15 healthy people was collected as control. After sodium-bisulfite treatment, extracted DNA was amplified for p16 promoter by methylation-specific polymerase chain reaction (MSP). The PCR products were detected by both gel-ethidium bromide electrophoresis and high performance liquid chromatogram (HPLC). Results: Among 84 patients, p16 hypermethylation was detected in 26 (31.0%) cancer tissues and 2 (0.02%) tumor-adjacent tissues and 12 (14.3%) pre-operative plasma, while negative in plasma of healthy people. For positive plasma cases, the paired tumor tissues were confirmed to be methylated. Within available 30 pairs of matched pre- and post-operative plasma, 6 pre-operative plasma was positive, and only 1 of 6 plasma remained hypermethylated after surgery. The results detected by HPLC exactly matched those by gel-electrophoresis. Conclusion: The alteration of status of p16 hypermethylation in post-operative plasma is considered the consequences of surgical intervention. Although p16 hypermethylation has no role in pre-operative staging of gastric cancer, detecting hypermethylated p16 in plasma could be utilized in monitoring patients after surgery.

EFFECTS OF p16^(INK4) GENE ON CHEMOSENSITIVITY OF HUMAN GLIOMA U251 CELL LINE TO TENIPOSIDE

Objective: To determine the effects on the cell growth, tumorigenicity and chemosensitivity of p16/CDK4I in human glioma. Methods: p16 gene was transfected into U251 cells by lipofectin. Expression of exogenous p16 gene was confirmed by immunohistochemistry and Northern blot. The effects of exogenous p16 gene on the growth and chemosensitivity to teniposide were examined. Results: Expression of exogenous p16 gene inhibited the growth dramatically in vitro. G1 arrest of tumor cells was observed. However, wt p16-positive U251 was less sensitive than control cell lines and the number of apoptotic cells after chemotherapy was reduced. Conclusion: The expression of exogenous p16 gene could inhibit the growth of glioma. On the other hand, the chemosensitivity to teniposide of p16-positive U251 was decreased.

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.

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

Study on P16 Gene in Acute Leukemia

To stddy the chang of suppressing cancer gene P16 in acute leukemia, the P16 antigen expression of leukemia cell surfaces in 61 cases were investigated with ABC assay and gene structural defects in 51 cases of acute leukemia were examined with multiple comparative PCR method. It was found that antigen expression of P16 in leukemia was obviously lower than that innormal subjects ( P <0 001). At the same time, antigen expression in All was lower than that AML ( P <0 05). No significant difference was found betwee the complete reission (CR) and non remission (NR) subjects from AML and ALL groups ( P >0 05). THe exon 2 of P16 gene showed homozygous deletion only inn4 cases out of 30 cases in ALL. No stuctural defect was revealed in 21 cases of AML. It was suggested that expression defect of P16 gene was a main cause in development and progression of acute leukemia, and structural defect of exon 2 was not a primary molecular event.

左归丸与六味地黄丸对衰老大鼠抗自由基能力及P_(16)蛋白表达影响的比较研究

目的比较具有补泻不同配伍特点的两补阴方左归丸和六味地黄丸及两方共有"三补"药物组抗衰老作用机制。方法观察左归丸、六味地黄丸及两方共有"三补"药物组对D-半乳糖(D-gal)致亚急性衰老大鼠抗自由基能力和P16蛋白表达的影响。结果亚急性衰老大鼠总抗氧化能力(T-AOC)和过氧化氢酶(CAT)水平及抑制超氧阴离子自由基能力下降,过氧化氢(H2O2)含量升高,P16蛋白表达水平上调;左归丸对上述改变具有逆转作用,六味地黄丸除对H2O2含量的降低作用无统计学意义外,对上述其他改变均有逆转作用;"三补"药物组可以提高T-AOC水平和下调P16蛋白表达水平,但对CAT活力和抑制超氧阴离子自由基能力的提高以及降低H2O2含量作用差异无统计学意义。结论左归丸和六味地黄丸具有抗衰作用,与两全方相比,恰当的中药配伍对疗效的强度和稳定性具有重要作用。

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．

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。

抑癌基因p^(16)在肝细胞癌及癌旁组织中的分布、表达及意义

目的 :探讨抑癌基因 p16与原发肝癌 (primaryhepatocellularcarcinoma ,HCC)发生及恶性程度的关系。方法 :p16是一种新的抑癌基因 ,应用免疫组织化学法检测了 6 9例HCC和 6 3例癌旁组织的 p16表达。结果 :p16蛋白免疫组织化学阳性信号为棕黄色颗粒 ,主要分布在胞浆 ,少数细胞核内也可见阳性显色 ,p16蛋白在癌组织及癌旁组织中的阳性率分别为 2 6 5 % (18/6 9)和 92 % (5 8/6 3) ,两者比较差异显著 (P <0 0 1)。 6 9例HCC标本 ,Ⅰ级、Ⅱ级、Ⅲ级中 p16蛋白的检出率分别为 5 2 6 % (10 /19)、 2 3 3 % (7/30 )、5 % (1/2 0 ) ,p16蛋白在高分化HCC中的表达率明显高于低分化HCC组 (P <0 0 5 )。结论 :抑癌基因 p16的表达和分布与HCC发生、发展及恶性程度有密切的关系 。

早期喉癌人乳头状瘤病毒感染和P53、P16、nm23蛋白表达及端粒酶激活相关性分析

目的:初步探讨早期喉癌发生与人乳头状瘤病毒(HPV)16/18感染及P16、P53、nm 23基因表达、端粒酶启动的相关性。方法:采用免疫组织化学方法、原位杂交及TRAP法分别检测了62例早期喉癌手术标本、14例癌前病变标本及12例正常声带粘膜/声带息肉标本的P53、P16、nm 23-H1蛋白的表达,HPV 16/18 DNA、端粒酶活性的检出率,并进行了统计学分析。结果:(1)早期喉癌中P16表达检出率低于正常声带粘膜/声带息肉及癌前病变的检出率(P<0.01);早期喉癌中P53表达检出率高于正常声带粘膜/声带息肉(小结)及癌前病变的检出率(P<0.05),癌前病变的检出率高于正常声带粘膜/声带息肉(小结)检出率(P<0.05)。nm 23H1表达在上述不同标本中无显著性差异。癌前病变及癌组织标本中有70％有端粒酶激活,对照组为0％。(2)癌前病变及癌组织63％(48/76)有HPVl6/18感染,对照组为0％。(3)与HPV阴性标本比较,HPV阳性标本中P16表达检出率减少,P53表达检出率及端粒酶启动增多。结论:喉癌的发生与喉粘膜P16表达下调、P53表达上调及端粒酶启动相关;P53表达上调可能是癌变的早期事件;HPV感染可能介导并促进P16、P53的表达失调及端粒酶启动,在喉癌发生中起重要作用。

p16基因异常甲基化检测对周围型肺癌的诊断价值

目的:了解周围型肺癌患者外周血血浆中p16基因异常甲基化发生情况及其在周围型肺癌临床辅助诊断中的应用价值。方法:选取56例肺周围型结节手术患者的血浆和组织标本,其中31例为周围型肺癌,25例为肺部良性结节,采用甲基化特异性聚合酶链反应方法检测p16基因启动子的异常甲基化情况。结果:在周围型肺癌患者的血浆和肿瘤组织中,p16基因甲基化检出率分别为64.5%(20/31)和70.9%(22/31);周围型肺癌患者血清、肿瘤组织中p16基因的甲基化异常事件与肿瘤分期、分型无明显相关性。在25例良性肺部疾病患者中,3例血浆和手术切除标本检测出p16基因启动子的异常甲基化存在;良、恶性肺周围型结节的p16基因异常甲基化发生情况差异存在显著性。结论:检测周围型肺癌患者血浆中p16基因异常甲基化情况有可能成为有价值的临床辅助诊断手段。

p16基因甲基化对肺癌早期诊断的临床研究

目的检测肺癌患者支气管灌洗液及全DNA中p16基因甲基化,并探讨其在肺癌早期诊断中的作用。方法利用半巢式甲基化特异性PCR技术,检测26例肺癌患者支气管灌洗液及相应全血中DNA p16基因的异常甲基化状态。结果34.6%(9/26)肺癌组织出现p16基因甲基化,出现甲基化组织对应的血液标本中88.9%(8/9)出现p16基因甲基化。支气管灌洗液中p16基因的甲基化状况与全血中是否出现p16基因甲基化关系密切(P<0.01)。结论肺癌患者支气管灌洗液及血液标本中p16基因甲基化的检测可能有助于肺癌的早期诊断。

低剂量螺旋CT扫描联合血清p16基因甲基化检测在肺癌早期诊断中的应用

目的研究在无症状的肺癌高危人群中利用低剂量CT（LDCT）联合血清p16基因甲基化检测进行肺癌早期诊断的可行性。方法肺癌高危人群入组标准：男性，年龄55～75岁；吸烟指数≥400支／年，目前仍在吸烟或戒烟不超过10年。共893例受检者被随机分为两组。一组为447例（LDCT—p16组），平均年龄66岁，进行LDCT联合血清p16基因甲基化检测；另一组为446例（CXR组），平均年龄67岁，接受后前位胸片检查。两组检查阳性病例将接受进一步组织病理学检查。并分别统计两组阳性结节检出率及肺癌检出率，并行X2检验。结果LDCT—p16组与CXR组分别有96．8％和92．8％的受检者完成了检查。LDCT—p16组中1113％病人可疑肺癌，明显高于CXR组的6．5％（P〈0．05）。其中LDCT—p16组中有7例，CXR组中有2例确诊为肺癌。LDCT—p16组肺癌检出率高于CXR组，但无统计学意义（P〉0．05）。结论低剂量CT联合血清p16基因甲基化检测是一种敏感、安全、可行的筛查早期肺癌的方法，能够取代胸片筛查早期肺癌。

巢式PCR法在燃煤型砷中毒患者p16基因启动区的异常甲基化检测中的应用

目的检测燃煤型砷中毒(地砷病)患者p16基因启动区的异常甲基化情况,探讨p16基因启动区的异常甲基化在地砷病癌变中的作用。方法采用巢式甲基化特异性PCR法(nMSP)对117例地砷病患者(参考地方性砷中毒诊断标准将其分为轻度中毒组、中度中毒组、重度中毒组)、63例内对照人群和70例外对照人群p16基因启动区的甲基化情况进行了检测。结果砷中毒病例组甲基化阳性率为39.32%,内对照组阳性率为12.70%,外对照组阳性率为4.29%;其中病例组中轻度、中度和重度组阳性率分别为24.00%,48.89%和54.55%。病例组与外对照组和内对照组比较,差异有统计学意义(P<0.01)。外对照组、内对照组、轻度、中度和重度砷中毒组甲基化阳性率经趋势χ2检验,差异有统计学意义(P<0.01)。结论p16基因启动区的异常甲基化在地砷病人的癌变过程可能起重要作用。

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?

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。