INK4系列抑癌基因在白血病中纯合子缺失的实验研究

目的：探讨p16基因家族失活与白血病发生、发展及预后的关系。方法：PCR检测p16、p15、p18、p19基因在白血病中纯合子缺失。结果：p16、p15基因外显子1在AL组纯合子缺失率分别为22．37％、17．05％,在ALL组为45．95％、32．43％,在ANLL组均为5．88％;在CML的慢性期均为0。p16、p15基因外显子2在AL组纯合子缺失率分别为12．5％、5．68％;在ALL组为24．32％、10．81％;在ANLL组为3．92％、1．96％;在CML和对照组二者均无缺失。p18、p19基因外显子1在AL组纯合子缺失率分别为1．14％、0;在ALL组分别为2．70％、0;在ANLL和对照组均无缺失。结论：p16、p15基因纯合子缺失在AL的发生频率较高,ALL缺失率明显高于ANLL,复发一LLL组基因失活率最高。p16、p15基因纯合子缺失是AL,尤其是ALL的发病重要因素之一。p18、p19基因在AL组中几乎未见纯合子缺失。在ALL中,p16纯合子缺失率高于p15纯合子缺失率。两个基因的纯合子缺失常伴随存在。在ANLL中,p16、p15基因的纯合子缺失均少见。     

脑胶质瘤 p16基因的纯合性缺失、高甲基化、突变及表达

目的：研究p16基因在脑胶质瘤中的纯合性缺失、高甲基化和突变等结构变化及其与表达之间的关系。方法：应用聚合酶链反应（polymerase chain reaction,PCR)和PCR甲基化银染分析技术（PCR-methylation assay with silver staining,PCR-MASS)对50例脑胶质瘤标本进行了p16基因纯合性缺失和甲基化检测；用PCR－SSCP和DNA测序技术进行了p16基因突变分析，用免疫组化检测了p16蛋白的表达。结果：50例脑胶质瘤中23例p16蛋白表达阳性，27例表达阴性，表达缺失率54％；9例（18％）纯合性缺失、7例（14％）高甲基化、2例（4％）突变。结论：p16基因在脑胶质瘤中有多种形式的结构异常，其结构的变化使p16蛋白表达障碍。p16基因纯合性缺失和高甲基化是基因失活的重要机制，在脑胶质瘤的发生、发展中起重要作用。     

白血病p16基因失活及其临床意义

目的：探讨ｐ１６基因失活对白血病发病及预后的关系。方法：应用聚合酶链反应（ＰＣＲ）与ＤＮＡ单链构象多态性分析（ＳＳＣＰ）及ＤＮＡ测序技术,甲基化检测技术。结果：ｐ１６基因的失活形式主要以缺失和甲基化为主。ｐ１６基因缺失与急性淋巴细胞白血病的发生密切相关,甲基化则主要发生在髓系白血病。结论：ｐ１６基因结构发生变化在高危白血病患者中比较多,ｐ１６基因的检测对于疾病的治疗及判断预后有着重要的意义。     

子宫内膜癌中p16基因状态对端粒酶活性的影响

目的探讨子宫内膜癌中p16基因状态对端粒酶活性的影响。方法用PCR技术检测癌组织中p16基因的纯合性缺失及第一外显子异常甲基化,用原位杂交法检测端粒酶的表达。结果36例癌组织中,9例发生p16基因缺失,12例发生甲基化,未发现基因缺失与甲基化同时存在的病例;29例内膜癌端粒酶表达阳性,阳性率为80.56%。9例基因缺失标本中均有端粒酶的阳性表达,p16甲基化与否对端粒酶的表达影响无差异,但端粒酶阳性率在p16基因失活组明显高于p16基因未失活组。结论p16基因失活可以导致激活端粒酶端粒,p16基因失活的不同状态端粒酶激活的影响存在差异。     

RASSF1A和p16 转录本在非小细胞肺癌中的表达及启动子区甲基化

 目的 研究RASSF1A和p16基因在国人非小细胞肺癌（NSCLC）组织中的转录及启动子区甲基化情况，探讨其转录失活的机制，为NSCLC的诊断和治疗寻找新的途径。方法 应用半定量RTPCR和甲基化特异性PCR法分析96例NSCLC及远癌正常肺组织中RASSF1A和p16基因mRNA的表达和启动子区甲基化情况。结果 （1）53．12％（51／96）的NSCLC中RASSF1A表达明显下调或缺失；36．46％（35／96）的p16表达下调或缺失，而远癌正常肺组织均表达良好。（2）96例NSCLC中RASSF1A甲基化率48．96％（47／96），该基因表达明显下调或缺失的51例中39例（76．5％）出现甲基化，表达正常的45例中8例（17．8％）出现甲基化，两组对比差异有统计学意义（P〈0．05）；96例NSCLC中33例（34．38％）检测到p16启动予区甲基化，p16基因表达明显下调的35例中20例（57．1％）出现该基因CPG岛的甲基化，而表达正常的61例中13例（21．3％）出现甲基化，两组比较差异显著（P〈0．05）。96例远癌正常肺组织均未检测到此两基因启动子有甲基化。结论 RASSF1A和p16基因mRNA在国人NSCLC中较高比例的表达下调或缺失；甲基化可能是两基因表达失活的主要原因。     

急性白血病P16基因缺失的意义

目的 探讨周期素依赖性激酶4抑制因子基因（p16）缺失在急性白血病中的意义。方法 采用PCR方法对43例急性淋巴细胞白血病（ALL）,36例急性非淋巴细胞白血病（ANLL）中p16基因的纯合缺失进行了研究。结果 21例ALL存在p16基因纯合缺失,3例ANLL存在p16基因纯合缺失。ALL p16基因缺失率明显高于ANLL（P＜0．05）,p15基因缺失的ALL病人有较高的复发率。结论 ALLp16基因缺失率较高,p16基因缺失之ALL预后不良。     

非小细胞肺癌p16基因甲基化及缺失的研究

目的 研究p16基因在非小细胞肺癌组织中的甲基化和缺失情况，探讨其在肺癌诊断中的价值。方法 应用甲基化相关的PCR及双重PCR，检测50例肺癌组织和54例正常肺组织中p16基因第1外显子5′端启动子区域CpG岛甲基化及第2外显子缺失情况。结果 p16基因在非小细胞肺癌组织中甲基化率为32.0%(16/50)。缺失率为28.0%(14/50);54例正常肺组织甲基化率为3.7%(2/54)，缺失率为0，二组比较，甲基化率（Fisher's exact=0.000)及缺失率（Fisher's exact=0.000)均有显著性差异。结论 甲基化和缺失是非小细胞肺癌p16基因失活的主要形式，检测p16基因甲基化和缺失状态可能有助于肺癌的诊断。     

多发性骨髓瘤患者p15、p16基因甲基化及其与预后的相关性

 目的　探讨p15、p16 基因的高甲基化与多发性骨髓瘤（MM）的发病和预后之间的关系。方法　采用巢式甲基特异性PCR法（nMSP）检测47例MM患者p15、p16基因的甲基化状态,并对患者的临床资料及预后因素进行分析。结果　47例MM患者p15、p16 基因的甲基化比例分别为59.57 %（28／47）、 57. 45 %（27／47）,两者同时存在甲基化者23 例（48.94 %）。Ⅱ、Ⅲ期MM患者p15 、p16基因甲基化率明显高于Ⅰ期患者。结论　p16 、p15 基因的高甲基化与MM的发病及预后相关。     

成人急性白血病p16基因的纯合缺失及甲基化研究

目的观察成人急性白血病p16基因的纯合缺失及甲基化的情况。方法用PCR法对成人急性白血病患者骨髓标本进行p16基因第1、第2外显子纯合缺失及甲基化检测。结果1)71例成人急性白血病患者中p16基因纯合缺失2例，均为B-ALL，AML及正常对照组p16基因未见纯合缺失；2)28例ALL中，5例发生异常甲基化；43例ANLL中，有7例发生了异常甲基化。结论p16基因缺失、异常甲基化可能与白血病的发生及预后有关。     

成人急性白血病p16基因的纯合缺失及甲基化研究

目的　观察成人急性白血病p16基因的纯合缺失及甲基化的情况。方法　用PCR法对成人急性白血病患者骨髓标本进行p16基因第 1、第 2外显子纯合缺失及甲基化检测。结果　 1) 71例成人急性白血病患者中p16基因纯合缺失 2例 ,均为B ALL ,AML及正常对照组p16基因未见纯合缺失 ;2 ) 2 8例ALL中 ,5例发生异常甲基化 ;43例ANLL中 ,有 7例发生了异常甲基化。结论　p16基因缺失、异常甲基化可能与白血病的发生及预后有关     

DNA Hypermethylation of Cell Cycle (p15 and p16) and Apoptotic (p14, p53, DAPK and TMS1) Genes in Peripheral Blood of Leukemia Patients

Aberrant DNA methylation of tumor suppressor genes has been reported in all major types of leukemiawith potential involvement in the inactivation of regulatory cell cycle and apoptosis genes. However, most ofthe previous reports did not show the extent of concurrent methylation of multiple genes in the four leukemiatypes. Here, we analyzed six key genes (p14, p15, p16, p53, DAPK and TMS1) for DNA methylation usingmethylation specific PCR to analyze peripheral blood of 78 leukemia patients (24 CML, 25 CLL, 12 AML, and17 ALL) and 24 healthy volunteers. In CML, methylation was detected for p15 (11%), p16 (9%), p53 (23%) andDAPK (23%), in CLL, p14 (25%), p15 (19%), p16 (12%), p53 (17%) and DAPK (36%), in AML, p14 (8%), p15(45%), p53 (9%) and DAPK (17%) and in ALL, p15 (14%), p16 (8%), and p53 (8%). This study highlightedan essential role of DAPK methylation in chronic leukemia in contrast to p15 methylation in the acute cases,whereas TMS1 hypermethylation was absent in all cases. Furthermore, hypermethylation of multiple genesper patient was observed, with obvious selectiveness in the 9p21 chromosomal region genes (p14, p15 and p16).Interestingly, methylation of p15 increased the risk of methylation in p53, and vice versa, by five folds (p=0.03)indicating possible synergistic epigenetic disruption of different phases of the cell cycle or between the cell cycleand apoptosis. The investigation of multiple relationships between methylated genes might shed light on tumorspecific inactivation of the cell cycle and apoptotic pathways.     

Alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia

We investigated the alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia (T-ALL) and T-ALL cell lines by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Mutations of the p53 gene were found in three of 57 (5%) patients at diagnosis, one of 14 (7%) patients at relapse and in 12 of 18 (67%) cell lines. In these 12 cell lines, four had more than two mutations of the p53 gene. The p53 mutations were found in four of five cell lines whose original fresh leukemic cells were simultaneously examined original fresh leukemic cells. However, only one of the four fresh leukemic cells had the same mutation. All patients with p53 mutations in the course of disease died. Mutations of the p21 gene were not identified in 71 fresh samples and in 18 cell lines. N-RAS mutations were found in two of 57 (4%) fresh T-ALL patients at diagnosis, and four of 18 cell lines (22%), whereas no mutations were detected in any samples at relapse. Alterations of the p16 gene were found in 18 of 47 (38%) patients at diagnosis and in seven of 14 (50%) at relapse. These differences were not statistically significant. There were no differences in the frequency of alteration of the p16 and p15 genes between event-free patients and the remaining patients. Furthermore, we found the methylation of p16 gene in three of seven patients lacking homozygous deletions, suggesting higher frequency of p16 inactivation than previous reports in T-ALL. Interestingly, we found that one allele is inactivated by methylation and another allele had nonsense mutation in one cell line (KOPT-KI), resulting in loss of protein expression of p16. This type of p16 inactivation has not been so far reported in leukemia. We conclude that, (1) p53 mutations are infrequent at diagnosis but tend to be associated with poor clinical outcome; (2) RAS and p21 mutations may not be involved in the pathogenesis of T-ALL; (3) not only frequent alterations of p16 and p15 genes but also methylation of p16 gene are involved in initiating the leukemogenesis of T-ALLs, and (4) these 5 genes are independently involved in T-ALL.     

Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia

Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases 4/6 (CDK4/6) and upstream regulators of RB function, and set up the RB/p16 tumor suppressive pathway, which is abrogated frequently in human neoplasms, either through inactivation of the RB or p16 tumor-suppressor protein, or alteration of the cyclin D1 or CDK4 oncoproteins. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (T-ALL). However, in the other subsets of ALL, deletions of p16 and p15 are relatively rare events. To investigate whether these genes are inactivated by methylation of the 5' CpG islands, we examined 35 leukemia cell lines and 29 childhood acute myeloid leukemia (AML) patients by Southern blot, polymerase chain reaction (PCR) and Western blot analyses. We found methylation of p16 in 12 (50%) of 24 ALL cell lines, 5 (50%) of 10 AML cell lines without homozygous deletion of p16, and 11 (38%) of 29 AML patients. Those leukemia cell lines subjected to p16 methylation were found to have lost p16 protein expression. The p15 gene was methylated in 10 (34%) of 29 ALL cell lines, 6 (60%) of 10 AML cell lines without homozygous deletion of p15, and 15 (52%) of 29 AML patients. These results revealed the frequent methylation of p16 and p15 genes in B-ALL and AML despite a low frequency of p16 and p15 deletions and mutations in these leukemias. In the study for expression of RB protein, we found no expression of RB in 4 of 16 leukemia cell lines. Inactivation of the p16 gene was found in all the cell lines with expression of RB. Neither amplification nor rearrangement of cyclin D1 gene was found in any cell lines. These results suggest that inactivation of p16 and p15 genes is one of the most common genetic events in acute leukemia, and plays an important role for the RB/p16 pathway in the pathogenesis of acute leukemia.     

p16、p21基因表达缺失在原发性肝癌中的临床意义

背景与目的:P16,P21蛋白是调控细胞周期的抑制蛋白,在调节细胞增殖中起非常重要的作用,其基因缺失,失活与多种肿瘤的发生,发展有关,本研究观察原发性肝癌中P16、P21蛋白表达及其基因缺失情况,探讨p16、p21基因表达缺失的临床意义.方法:采用回顾性分析方法,收集原发性肝癌42例,正常肝组织(距离癌组织≥2 cm)36例,应用Western印迹试验、逆转录聚合酶链式反应(RT-PCR)检测肝癌和正常肝组织中P16、P21蛋白的表达和相应mRNA扩增情况.结果:在癌组织中,P16蛋白缺失率为24例(24/42,57.1%),正常肝组织中P16蛋白缺失率为13.9%(5/36);而相应组织中P21蛋白缺失率分别为45.2%(19/42)和19.4%(7/36).两者在癌和正常肝组织中缺失差异均有显著性(P＜0.05).14例癌组织(38.9%.14/36)仅p16 mRNA转录而未表达蛋白.P16、P21蛋白的低表达与肝癌大小、分化程度关系密切(P＜0.05),而与是否有包膜、是否肝硬化等无关.结论:P16、P21蛋白的表达缺失与肝癌发生发展密切相关,两者可作为判断肝癌恶性程度的参考指标.     

宫颈癌组织中p16和p15基因产物表达及其生物学意义

目的 :探索p16、p15基因与宫颈癌发生发展的关系。方法 :应用免疫组化ABC方法对 36例宫颈癌组织中p16、p15基因表达产物进行检测。结果 :p16、p15基因产物的阳性表达率分别为 6 3 9%(2 3/ 36 )、55 6 % (2 0 / 36 ) ,二者的表达率与宫颈癌病理类型无显著关系 (P >0 0 5) ,而与宫颈癌的浸润、转移及病人的生存期显著相关 (P <0 0 5)。结论 :p16、p15基因表达产物可以作为判断宫颈癌预后的指标。     

脑肿瘤p16基因缺失，突变及5’CpG岛甲基化研究

目的：探讨不同种类的脑肿瘤P16基因变异及其与脑肿瘤的发生,发展的关系。方法：利用PCR,PCR－SSCP及PCR－based甲基化技术检测了56例胶质瘤,15例脑膜瘤及2例原发性脑淋巴瘤P16基因缺失,突变及5’CpG岛甲基化状况,结果：18例高病理级别的脑胶质瘤及1例原发性脑淋巴瘤发生P16基因缺失;6例脑胶质瘤及1例原发性脑淋巴瘤发生P16基因5’CpG岛甲基化,无1例发生P16基因突变。结论：P16基因失活可能参与脑胶质瘤的发生,恶性进展及原发性脑淋巴瘤的发生,P16基因缺失是P16基因失活的主要机制。     

The prognostic significance of p16INK4a/p14ARF and p15INK4b deletions in adult acute lymphoblastic leukemia.

Cytogenetic/molecular abnormalities significantly influence the prognosis of patients with acute leukemia. Recently, two genes, p16INK4a and p15INK4b, encoding two cyclin-dependent kinase inhibitor proteins of the INK4 family of Mr 15,000 and 16,000, respectively, have been localized to 9p21. Remarkably, the p16INK4a locus has been found to encode a second protein, p14ARF, known as p19ARF in mice, with a distinct reading frame. Like p16INK4a, p14ARF is involved in cell cycle regulation, blocking cells at the G1 restriction point through the activity of MDM-2 and p53. We studied bone marrow samples of 42 newly diagnosed and untreated patients with acute lymphoblastic leukemia for the incidence of deletions of p16INK4a/p14ARF and p15INK4b using Southern blot analysis and determined the clinical outcome with regard to complete remission (CR) duration, event-free survival, and overall survival. We found deletions of p16INK4a/p14ARF in 17 of 42 patients (40%), with homozygous deletions in 11 of 42 patients (26%) and hemizygous deletions in 6 of 42 patients (14%). The gene for p15INK4b was codeleted in most, but not all, cases and was never deleted without deletion of p16INK4a/ p14ARF. No correlation was observed between molecular studies and karyotype abnormalities as determined by conventional cytogenetics. Furthermore, no difference was found in the CR rate, CR duration, event-free survival, and overall survival in patients with homozygous gene deletions compared to patients with no deletions or loss of only one allele.