多发性骨髓瘤患者p16基因甲基化及砷剂诱导去甲基化的研究

目的探讨p16基因启动子区CpG岛甲基化在多发性骨髓瘤(MM)患者发病中的作用以及三氧化二砷(As2O3)诱导p16基因的去甲基化作用。方法采用巢式甲基特异性PCR法检测MM患者以及利用As2O3作用前后人MM细胞株U266的p16基因的甲基化状态,RTPCR检测U266细胞用药前后p16基因mRNA的表达变化,生长曲线、MTT法检测As2O3对细胞的生长和增殖抑制。利用流式细胞仪检测DNA含量分析法探讨As2O3对骨髓瘤细胞系U266周期的影响。结果MM患者p16基因的甲基化比例为54.8%,U266细胞存在p16基因甲基化,p16基因不表达,As2O3作用后p16基因甲基化程度明显减弱至消失,与未处理组相比,不同剂量作用72h后p16基因表达阳性条带灰度值与βactin比值分别为0.22±0.10、0.59±0.11、0.68±0.09,阳性对照灰度比值为0.77±0.13,其差异有统计学意义(P<0.01)。结论p16基因甲基化在MM患者中较为常见,这可能为MM的诊断和治疗提供借鉴;As2O3可诱导p16基因去甲基化,使p16基因表达上调,恢复其活性,为去甲基化治疗提供新思路。     

Tumor suppressor p16 methylation in multiple myeloma: biological and clinical implications

The biological and clinical implications of p16 gene methylation in multiple myeloma (MM) are still unclear despite previous studies. In this comprehensive study, using methylation-specific PCR (MS-PCR), we show that p16 methylation is relatively common and occurs in monoclonal gammopathy of undetermined significance (MGUS; n=17), smoldering multiple myeloma (SMM; n=40), and MM (n=522) at a prevalence of 24%, 28%, and 34%, respectively. However, p16 methylation does not appear to affect gene expression level. In a large cohort of patients with long-term follow-up information (n=439), there was no difference in overall survival between patients with or without p16 methylation. We also found no association between p16 methylation and the main cytogenetic categories, although it was more common among patients with 17p13.1 deletions (p53 locus), a genetic progression event in MM. In addition, p16 methylation has no apparent effect on the cycle because there was also no difference in the plasma cell labeling index (a direct measurement of proliferation) between patients with and without p16 methylation. Our results question a major role for p16 methylation in the oncogenesis of the PC neoplasm, and we now believe p16 methylation may be a marker for overall epigenetic changes associated with disease progression, with no obvious direct biological or clinical consequences.     

Methylation is an inactivating mechanism of the p16 gene in multiple myeloma associated with high plasma cell proliferation and short survival

In order to gain further insights into the role of the p16 gene in cell cycle regulation and the prognostic implications of its inactivation, we investigated the methylation status of the p16 gene in 98 untreated patients using a polymerase chain reaction assay based on the inability of some restriction enzymes to digest methylated sequences. Forty-one patients showed a p16 methylated gene (42%). The percentage of S-phase plasma cells (PC) in these patients was almost three times higher than in those with an unmethylated p16 gene (4.16% +/- 3.37%vs 1.5% +/- 1.41%, P < 0.001). The presence of p16 methylation also correlated with both elevated beta2-microglobulin serum levels and high C-reactive protein values. Patients with a p16 methylated gene had shorter overall and progression-free survival than those patients without p16 methylation. However, this feature did not retain independent prognostic influence on multivariate analysis, probably due to its association with the S-phase PC, which had more potent statistical significance in the Cox model. These findings showed methylation of the p16 gene was a frequent event inMM patients at diagnosis, and was associated with an increased proliferative rate of plasma cells and a poor prognosis, indicating an important role for p16 gene in the cell cycle regulation of multiple myeloma tumour cells, and thus in the clinical outcome of the disease.     

Methylation of TIMP3 in esophageal squamous cell carcinoma

AIM： To measure the frequency of DNA methylation of the tissue inhibitor of metalloproteinase 3 （TIMP3） promoter and relate this to any change of gene expression in esophageal squamous cell carcinoma in patients from a region of high incidence in China. METHODS： Cancer cell lines were treated with or without the demethylating reagent 5-aza-2′-deoxycytidine. Methylation of the TIMP3 promoter was assessed in three regions by melt curve analysis and its expression was assessed by real-time RT-PCR. Tumors and proximal resection margins were obtained from 64 patients with esophageal squamous cell carcinoma from a region of high incidence in China. Methylation was assessed by melt curve analysis and expression by immunohistochemistry.
RESULTS： Methylation in one of the three promoter regions assessed correlated with gene silencing in esophageal cell lines. A degree of methylation of TIMP3 was found in only four esophageal squamous cell carcinomas, and partial loss of TIMP3 protein expression in just one.
CONCLUSION： Methylation and loss of expression of TIMP3 occurs infrequently in esophageal squamous cell carcinoma in a region of high incidence in China.     

p16(INK4a) and p15(INK4b) gene methylations in plasma cells from monoclonal gammopathy of undetermined significance.

p15(INK4b) and p16(INK4a) proteins are cell cycle regulators involved in the inhibition of G1 phase progression. High frequency of methylation of both genes has been reported in multiple myeloma (MM), but it remains to be determined how and when these alterations contribute to tumorigenesis. Monoclonal gammopathy of undetermined significance (MGUS) represents an early disease stage in a fraction of MMs. Plasma cells from 33 patients with MGUS and 33 patients with MM were isolated and analyzed for p15(INK4b) and p16(INK4a) methylation by methylation-specific polymerase chain reaction. Selective methylation was found in 19% for p16(INK4a), 36% for p15(INK4b), and 6.5% for both genes in MGUS, and frequencies were similar in MM suggesting that methylation of these genes is an early event, not associated with transition from MGUS to MM. p15(INK4b) and p16(INK4a) gene methylation might contribute to immortalization of plasma cells rather than malignant transformation in the natural history of MM.     

Methylation of the INK4A/ARF locus in blood mononuclear cells

In the detection of DNA hypermethylation as a tumor-specific epigenetic change in blood mononuclear cell fraction in patients with lymphoid and hematopoetic disorders, circulating tumor cells originating from the lymph nodes or bone marrow can be identified. However, it is still not clear whether methylation in mononuclear cells is disease specific. In the present study, we investigated whether methylation of the inhibitor of cyclin-dependent kinase (INK) 4A/alternative reading frame (ARF) locus is present in a disease-specific manner in the blood mononuclear cell fraction of patients with lymphoma, multiple myeloma, or leukemia. To increase the sensitivity of detection, a two-step methylation-specific PCR approach was used to analyze the methylation status of the promoter/exon 1 regions of both p14ARF and p16INK4A genes. Our findings indicate that although INK4A/ARF locus methylation is present in mononuclear cells, this event is not disease-specific since normal subjects also display methylated DNA in their mononuclear cells. In 85.1% of the patients and in 89% of the controls, p16INK4A gene was methylated, while the methylation rates for the p14ARF gene was 32.6 and 36.5%, respectively. The presence of methylated CpG sites in DNA in samples from normal subjects was confirmed by bisulfite genomic sequencing. The difference in the methylation rate between p16INK4A and p14ARF genes among the patients was highly significant (p<0.001). Our results demonstrate that methylation of the INK4A/ARF locus is not a disease-specific molecular change in mononuclear cell fraction and that the p14ARF and p16INK4A genes are differentially methylated.     

CpG island methylation of tumor-related promoters occurs preferentially in undifferentiated carcinoma.

OBJECTIVE: To understand the role of epigenetic inactivation of tumor-related genes in the pathogenesis of thyroid cancer, we investigated the methylation profile of distinct thyroid neoplasms. DESIGN: We analyzed the methylation pattern of 17 gene promoters in nine thyroid cancer cell lines and in 38 primary thyroid carcinomas (13 papillary thyroid carcinoma [PTC], 10 follicular thyroid carcinoma [FTC], 9 undifferentiated thyroid carcinoma [UTC], 6 medullary thyroid carcinoma [MTC]), 12 goiters, and 10 follicular adenomas (FA) by methylation- specific polymerase chain reaction (PCR). Epigenetic inactivation was validated by expression analysis. MAIN OUTCOME: Twelve of these genes (RASSF1A, p16(INK4A), TSHR, MGMT, DAPK, ERalpha, ERbeta, RARbeta, PTEN, CD26, SLC5A8, and UCHL1) were frequently methylated in UTC (15%-86%) and thyroid cancer cell lines (25%-100%). In the more aggressive UTC, the mean methylation index (MI = 0.44) was the highest compared to other thyroid alterations PTC (MI = 0.29, p = 0.123), FTC (MI = 0.15, p = 0.005), MTC (MI = 0.13; p = 0.017), FA (MI = 0.27; p = 0.075) and goiters (MI = 0.23; p = 0.024). Methylation of TSHR, MGMT, UCHL1, and p16 occurred preferentially in UTC and this inactivation was reverted by a demethylating agent. CONCLUSIONS: Our results show that hypermethylation of several tumor-related gene promoters is a frequent event in UTC. The hypermethylation status may be reversed by DNA demethylating agents. Their clinical value remains to be investigated.     

The potential role of epigenetic therapy in multiple myeloma

This review describes the role that epigenetic changes play in the pathogenesis of cancer, concentrating on the plasma cell malignancy multiple myeloma, and highlights recent findings regarding the efficacy of epigenetic therapeutic agents in laboratory studies and clinical trials. DNA methylation is altered in a wide range of cancers with hypermethylation of CpG islands associated with silencing of tumour suppressor genes. Genes found to be silenced by methylation in myeloma samples include VHL, TP53, CDKN2A, and TGFBR2. Myeloma is linked to the overexpression of a histone methylatransferase (MMSET) and inactivating mutations of a histone demethylase (UTX), suggesting that the regulation of histone methylation is a potential therapeutic target. Abnormal expression of histone deacetylases (HDACs) has been widely described in solid tumours and haematological malignancies. In myeloma, histone deacetylase inhibitors show promising results both in laboratory‐based cell culture studies and in clinical trials, where they demonstrate particularly good therapeutic outcome when administered in combination with other standard chemotherapeutic agents. The study of epigenetics shows great promise for understanding the alterations in gene expression that underlie malignancies and provides exciting novel drugable targets.     

Aberrant DNA methylation of p57(KIP2) gene in the promoter region in lymphoid malignancies of B-cell phenotype

The cyclin-dependent kinase inhibitor p57(KIP2) is thought to be a potential tumor suppressor gene (TSG). The present study examines this possibility. We found that the expression of p57(KIP2) gene is absent in various hematological cell lines. Exposing cell lines to the DNA demethylating agent 5-aza-2'-deoxycytidine restored p57(KIP2) gene expression. Bisulfite sequencing analysis of its promoter region showed that p57(KIP2) DNA was completely methylated in cell lines that did not express the p57(KIP2) gene. Thus, DNA methylation of its promoter might lead to inactivation of the p57(KIP2) gene. DNA methylation of this region is thought to be an aberrant alteration, since DNA was not methylated in normal peripheral blood mononuclear cells or in reactive lymphadenitis. Methylation-specific polymerase chain reaction analysis found frequent DNA methylation of the p57(KIP2) gene in primary diffuse large B-cell lymphoma (54.9%) and in follicular lymphoma (44.0%), but methylation was infrequent in myelodysplastic syndrome and adult T-cell leukemia (3.0% and 2.0%, respectively). These findings directly indicate that the profile of the p57(KIP2) gene corresponds to that of a TSG.     

Epigenetic change in pituitary tumorigenesis

Throughout the genome CpG dinucleotides are found at one-fifth of their expected frequency and their rarity is further marked by the fact that 70% are methylated. In contrast, CpG islands (CGI), found associated with the promoters of many genes, have maintained their expected frequency of this dinucleotide, and remain unmethylated. Inappropriate methylation of CGIs is associated with histone deacetylation and gene silencing, while methylation of CpGs outside of CGIs is associated with significantly higher mutation rates. Methylation of CGIs is a frequent event in numerous tumour types including those that arise within the pituitary gland. Several studies now show highly frequent methylation of the p16 gene that is significantly associated with loss of cognate protein and that appears to be an early change in pituitary tumorigenesis. Collectively, studies show that somatotrophinomas are an infrequent target for p16 CGI methylation. However, in this pituitary tumour subtype, loss of pRb is associated with either CGI methylation or micro-deletion within the protein-pocket binding domain. As in other tumour types loss of p16 or RB1 appear to be mutually exclusive events in non-functional adenomas and somatotrophinomas respectively. Investigation of the Death Associated Protein Kinase gene shows that loss of its protein (DAPK), a pro-apoptotic molecule, in pituitary tumours is also associated with either methylation or deletion within its associated CGI. In the case of DAPK, however, these changes segregate with invasive pituitary tumours irrespective of tumour subtype. Methylation represents a positive signal that can be detected with exquisite sensitivity; in addition, this change targets multiple genes that show tumour type specificity. Taken together, the detection of DNA methylation changes, using either a panel of predefined marker-islands, or CGI arrays, provides the opportunity to generate "methylation profiles". This new knowledge will increase our understanding of tumour biology and could ultimately aid medical management in these different tumour types, including those of pituitary origin.     

Methylation status of the p15 and p16 genes in paediatric myelodysplastic syndrome and juvenile myelomonocytic leukaemia

Aberrant DNA methylation is frequently observed in adults with myelodysplastic syndrome (MDS), and is recognized as a critical event in the disease's pathogenesis and progression. This is the first report to investigate the methylation status of p15 and p16, cell cycle regulatory genes, in children with MDS (n = 9) and juvenile myelomonocytic leukaemia (JMML; n = 18) by using a methylation-specific polymerase chain reaction. The frequency of p15 hypermethylation in paediatric MDS was 78% (7/9), which was comparable to that in adult MDS. In contrast, p15 hypermethylation in JMML was a rare event (17%; 3/18). In JMML, clinical and laboratory characteristics including PTPN11 mutations and aberrant colony formation were not different between the three patients with hypermethylated p15 and the others. Aberrant methylation of p16 was not detected in children with either MDS or JMML. Since p15 and p16 genes were unmethylated in two children with JMML, in whom the disease had progressed with an increased number of blasts, a condition referred to as blastic crisis, we infer that the aberrant methylation of these genes is not responsible for the progression of JMML. The results suggest that demethylating agents may be effective in most children with MDS and a few patients with JMML.     

Concurrent p16 methylation pattern as an adverse prognostic factor in multiple myeloma: a methylation-specific polymerase chain reaction study using two different primer sets

Disruption of cell cycle control genes, including p16, is known to contribute to the cancerogenesis of multiple myeloma (MM). We investigated the methylation status of p16 and its association with common cytogenetic changes, clinicolaboratory findings, and survival in MM. Methylation-specific polymerase chain reaction was performed in 99 newly diagnosed MM patients using two different sets of primers (p16M1 and p16M2). Four patterns of p16 promoter methylation were observed: (1) concurrent methylation of p16M1 and p16M2 (P1P2), 27.3%; (2) methylation of p16M1 alone (P1N2), 7.1%; (3) methylation of p16M2 alone (N1P2), 26.3%; and (4) no methylation (N1N2), 39.4%. Patients with p16P1P1 showed shorter survivals than those with the other methylation patterns (P1N2, N1P2, or N1N2; median survival, 12 vs. 43?months; P?<?0.001), regardless of the treatment protocol. In a multivariate analysis, p16P1P2 was an independent prognostic factor of adverse outcome in MM. According to International Staging System (ISS), the study population could be divided into 21.2% (20/94) for stage I, 22.3% (21/94) for stage II, and 56.4% (53/94) for stage III (P?=?0.003). ISS can divide patients into prognostic groups. Of note, in patients older than 60?years, ISS was not reflective of disease stage (P?=?0.114). If p16P1P2 sets up as stage 4 of ISS, modified ISS could be a more reliable staging system irrespective of age in Korean MM patients (P?=?0.003 and P?=?0.004 in patients younger than 60?years and in patients older than 60?years, respectively). Our study suggests the potential use of p16 methylation status in predicting the outcome of MM patients and the applicability of demethylating agents in MM.