Comparison of Aberrant Methylation of CpG Islands in the p16/CDKN2A and p14/ARF Promoters in Non-Small Cell Lung Cancer and Acute Lymphoblastic Leukemia

Multiplex methylation-sensitive (MSe-PCR) and methylation-specific (MSp-PCR) PCRs were used to detect aberrant methylation of CpG islands in the promoter regions and first exons of p16/CDKN2A and p14/ARF in non-small cell lung cancer (NSCLC, 54 specimens) and B-cell acute lymphoblastic leukemia (B-ALL, 61 specimens). A difference in CpG methylation was observed for individual specimens and for the two malignancies. A high methylation frequency of the first exon of p16/CDKN2A was detected both in NSCLC (68%) and in B-ALL (55%). The CpG island of the p14/ARF first exon proved to be nonmethylated in both malignancies. Particular CpG-rich fragments were examined in the p16/CDKN2A and p14/ARF promoters. It was shown that methylation frequency can differ between the 5 regions of one promoter. The sensitivity was compared for MSe-PCR and MSp-PCR, which are commonly employed in methylation analysis.     

Abnormal methylation of several tumor suppressor genes in sporadic breast cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, MGMT, HIC1, and N33 promoter regions in breast carcinoma (105 tumors). Methylation was often observed for the two major suppressor genes involved in cell-cycle control through the Cdk-Rb-E2F signaling pathway, RB1 (18/105, 17%) and p16 (59/105, 56%); both genes were methylated in 13 tumors. Methylation involved p15 in two (2%) tumors; CDH1, in 83 (79%) tumors; MGMT, in eight (8%) tumors, and N33, in nine (9%) tumors. The p14 promoter was not methylated in the tumors examined.     

Profile of methylation of certain tumor growth suppressing genes in non-small cell lung cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5' regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk-Rb-E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.     

小鼠骨髓细胞中p16和Ralb基因启动区CpG岛的甲基化位点分析

抑癌基因p16和白血病致癌因子Ralb与白血病的发生密切相关,其启动子区CpG岛的甲基化对基因表达具有重要作用.本文旨在分析p16、Ralb基因启动子区CpG岛甲基化位点信息,并比较这两个基因在小鼠骨髓细胞和原代培养的骨髓细胞中甲基化状态的差异.运用"MethPrimer"软件预测p16、Ralb基因启动子区的CpG岛,设计甲基化特异性引物.利用重亚硫酸盐测序法(BSP)检测甲基化位点信息.结果显示,p16有1个CpG岛,岛上21个CpG位点全部未发生甲基化;Ralb有2个CpG岛,CpG岛1上的5个CpG位点全部呈甲基化状态,而CpG岛2上的17个CpG位点全部呈非甲基化状态,且小鼠骨髓细胞和体外原代培养的骨髓细胞中两基因的甲基化状态一致.表明p16、Ralb基因甲基化状态未受外界培养条件的影响而改变,提示在与两基因甲基化相关的研究中体外试验可替代体内试验.     

Prevalence of aberrant methylation of p14ARF over p16INK4a in some human primary tumors

The INK4a/ARF locus encodes two unrelated tumor suppressor proteins, p16INK4a and p14ARF, which participate in the two main cell-cycle control pathways, p16–Rb and p14–p53. Methylation of CpG promoter islands has been described as a mechanism of gene silencing. Exon 1 of the p16INK4a gene and the p14ARF promoter gene reside within CpG islands. Therefore, both can become methylated de novo and silenced. It has recently been proposed that the methylation changes in certain genes could be used as molecular markers for the detection of almost all forms of human cancer. Here, we analyzed concomitantly in each tumor sample and normal tissue the methylation status of p16INK4a and p14ARF by methylation-specific PCR (MSP) in 100 breast, 95 colon and 27 bladder carcinomas. A series of clinicopathological parameter were obtained from the medical records of the patients, p14ARF showed a higher rate of hypermethylation than p16INK4a in all three tumor types. p16INK4a and p14ARF aberrant methylation was significantly correlated with poor prognosis clinicopathological parameters of the three tumor types. We conclude that both p16INKa and p14ARF hypermethylation may be involved in breast, colon and bladder carcinogenesis, with special emphasis on the role of the lesser studied p14ARF gene, and that tumors with aberrant methylation in the two genes were associated with worse prognosis.     

Diagnostics for epigenetic pathology in hereditary and oncologic diseases

The review considers the epigenetic defects and their diagnostics in several hereditary disorders and tumors. Aberrant methylation of the promoter or regulatory region of a gene results in its functional inactivation, which is phenotypically similar to structural deletion. Screening tests were developed for Prader-Willi, Angelman, Wiedemann-Beckwith, and Martin-Bell syndromes and mental retardation FRAXE. The tests are based on allele methylation analysis by methylation-specific or methylation-sensitive PCR. Carcinogenesis-associated genes (RB1, CDKN2A, ARF14, HIC1, CDI, etc.) are often methylated in tumors. Tumors differ in methylation frequencies, allowing differential diagnostics. Aberrant methylation of tumor suppressor genes occurs in early carcinogenesis, and its detection may be employed in presymptomatic diagnostics of tumors.     

p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Hypermethylation of 5'CpG island of p14ARF flanking exon 1 Beta in human colorectal cancers displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

ABSTRACT: BACKGROUND: It has been suggested that inactivation of p14ARF, a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI), low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs). In this study, we investigated whether p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. RESULTS: Detailed maps of the alterations in the p14ARF gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF mutations was extremely low (1/98; 1%), whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%). Only seven of nine tumors (7%) had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14ARF promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13) versus 0 to 17 (95% CI 0 to 2)) in adjacent colon mucosa (P = 0.004). Hypermethylation of the p14ARF promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03), and MDM2 overexpression (P = 0.02), independently of MSI phenotype. Although no significant correlation between p14ARF methylation and TP53 mutational status was seen (P = 0.23), methylation involving the proximal CpG sites within the 5' CpG flanking exon 1beta was present more frequently in tumors with restricted p53 overexpression than in those with diffuse p53 overexpression (range of methylated clones 17 to 36% (95% CI 24 to 36%) versus range 0 to 3% (95% CI 0 to 3%), P = 0. 0003). CONCLUSION: p14ARF epigenetic silencing may represent an important deregulating mechanism of the p53- MDM2-p14ARF pathway in CRCs exhibiting a restricted p53 overexpression pattern.     

Methylation Profile of Several Tumor Suppressor Genes in Non-Small-Cell Lung Cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5 regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk–Rb–E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.     

RB1 and CDKN2A functional defects resulting in retinoblastoma

Multiplex methylation-sensitive PCR was employed in studying the methylation of the RB1 and CDKN2A/p16 promoter regions in 52 retinoblastomas. Aberrant methylation inactivating RB1 was detected in 14 (27%) tumors. Methylation of p16 was for the first time observed in retinoblastoma (9 tumors, 17%). Both promoters proved to be methylated in two tumors. In four tumors, aberrant methylation was combined with structural defects of both RB1 alleles. Aberrant methylation of the p16 promoter was the second mutation event in two tumors and was not accompanied by RB1 defects in one tumor. Complex testing for RB1 mutations, loss of heterozygosity, and functional inactivation of the two genes revealed a molecular defect in at least one allele in 51 (98%) tumors.     

The CpG island methylator phenotype correlates with long-range epigenetic silencing in colorectal cancer

The CpG island methylator phenotype (CIMP), characterized by an exceptionally high frequency of methylation of discrete CpG islands, is observed in 18% to 25% of sporadic colorectal cancers. Another hypermethylation pattern found in colorectal cancers, termed long-range epigenetic silencing, is associated with DNA/histone methylation in three distinct gene clusters at chromosome 2q14.2, showing that DNA hypermethylation can span larger chromosomal domains and lead to the silencing of flanking, unmethylated genes. We investigated whether these two phenotypes are interrelated in colorectal cancers. The CIMP status of 148 sporadic colorectal cancers was determined by methylation-specific PCR. We determined the BRAF V600E mutation by mutant allele-specific PCR amplification. The methylation status of the MLH1 gene and of three CpG islands (EN1, SCTR, and INHBB), corresponding to three distinct clusters along 2q14.2, was determined by methylation-specific PCR. The average number of sites showing methylation in CIMP+ tumors was 2.21, compared with 1.22 for CIMP- individuals, and this difference was highly significant (P = 3.6 x 10(-8), Mann-Whitney test). Moreover, all CIMP+ tumors showed hypermethylation of at least one of these loci, in contrast to CIMP- tumors, where 18 (16%) samples remained unmethylated. The mean number of simultaneously hypermethylated CpG islands at 2q14.2 differs significantly between CIMP- and CIMP+ tumors, suggesting varying effects of domain silencing in this region. Given that the number of hypermethylated loci at 2q14.2 likely affects the range of silenced flanking genes, high frequency of simultaneous hypermethylation of three CpG islands (EN1, SCTR, and INHBB) may have potential influence on specific characteristics of CIMP+ colorectal cancers.     

Diagnostics of Epigenetic Alterations in Hereditary and Oncological Disorders

The review considers the epigenetic defects and their diagnostics in several hereditary disorders and tumors. Aberrant methylation of the promoter or regulatory region of a gene results in its functional inactivation, which is phenotypically similar to structural deletion. Screening tests were developed for Prader–Willi, Angelman, Wiedemann–Beckwith, and Martin–Bell syndromes and mental retardation FRAXE. The tests are based on allele methylation analysis by methylation-specific or methylation-sensitive PCR. Carcinogenesis-associated genes (RB1, CDKN2A, ARF14, HIC1, CDH1, etc.) are often methylated in tumors. Tumors differ in methylation frequencies, allowing differential diagnostics. Aberrant methylation of tumor suppressor genes occurs in early carcinogenesis, and its detection may be employed in presymptomatic diagnostics of tumors.     

宫颈癌p16基因甲基化及表达的研究

为了探讨p16基因甲基化及异常表达在宫颈癌中的意义,分别采用甲基化特异性PCR（MSP）方法检测不同病理类型和临床分期的60例宫颈癌组织中p16基因启动子区域5´CpG岛甲基化状态;采用PCR方法检测p16基因外显子1（E1)和外显子2(E2)纯合缺失情况;采用免疫组化的方法分析p16蛋白的表达缺失和减弱情况。结果显示正常对照组织及癌旁p16基因无甲基化,且无E1和E2缺失和p16蛋白表达异常。60例宫颈癌标本的甲基化率为21.67%（13/60）;p16 基因缺失率为15.00%（9/60）;p16蛋白表达下降或无表达为51.67%（31/60）。可见p16基因蛋白的阳性表达率随着临床分期升高呈明显下降趋势。结果提示p16基因失活在宫颈癌中多见且与病理分级密切相关。p16 基因甲基化在宫颈癌发生中起着一定作用。Abstract： To detect hypermethylation and aberrant expression of the p16 gene in cervical carcinoma (CC), methylation-specific PCR (MSP) was used to determine the methylation status of 5´CpG islands of the p16 gene, loss or decrease of p16 expression was analyzed by immunohistochemistry (IHC), and homozygous deletion of exon 1 (E1) and/or exon 2 (E2) was determined by PCR. in 60 cases of CC at different pathological grades and clinical stages. The results showed absence of methylation and presence of normal expression of the p16 gene in the control and adjacent tissues of CC. Hypermethylation, loss or decrease of expression and deletion of the p16 gene were detected in 21.67%(13/60), 51.67%(31/60) and 15.00%(9/60) of the tumor tissues, respectively. The rate of p16 expression markedly reduced with the increase of clinical stages. Our data suggested that inactivation of the p16 gene was a frequent event and positively correlated with pathological grades in CC, and that methylation of the p16 gene was an important event in carcinogenesis of CC.     

p16 hypermethylation during gastric carcinogenesis of Wistar rats by N-methyl-N'-nitro-N-nitrosoguanidine

Inactivation of the tumor suppressor gene, p16 by CpG hypermethylation is a common event in various tumors including gastric carcinoma. The aim of this study is to investigate if p16 hypermethylation is an early and frequent event in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The frequency and timing of p16 hypermethylation during the multistep gastric carcinogenesis in Wistar rats were analyzed in various microdissected gastric lesions. The p16 methylation status and the presence of p16 protein were analyzed by methylation-specific PCR and immunohistochemistry, respectively. Results showed that p16 methylation frequency was correlated with the severity of gastric pathologic lesions, positively. For instance, p16 methylation was found in 2.7% of normal gastric epithelium (n = 36), 16.7% of chronic atrophy gastritis (n = 24), 37.5% of dysplasia (n = 24), 67.4% of gastric adenoma (n = 43), and 85.2% of gastric carcinoma (n = 27). The p16 methylation in the distal stomach epithelium was higher than that in the proximal stomach. p16 protein was expressed in all of 15 p16 unmethylated gastric epithelial samples, but not expressed in all of 12 p16 methylated samples. These results suggest that CpG island hypermethylation may account for the silencing of p16 in rat stomach and is an early event whose accumulation will finally lead to gastric carcinogenesis.