DAPK1 Research Articles

Study Value of Promoter Methylation of DAPK Gene in Hep-2 Cell Line

Study Value of Promoter Methylation of DAPK Gene in Hep-2 Cell Line

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.

Tumor Progression Through Epigenetic Gene Silencing of O6−Methylguanine-DNA Methyltransferase in Human Biliary Tract Cancers

We previously demonstrated in an immunohistochemical study that reduced expression of O(6)-methylguanine-DNA methyltransferase (MGMT) correlated with a poorer prognosis in patients with biliary tract cancers. The purpose of this study was to clarify how MGMT deficiency leads to a poor outcome in biliary tract cancer. Thus, we examined epigenetic (promoter methylation) and genetic (gene mutation) alterations in biliary tract cancer. We examined 37 biliary tract cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by one-step or two-step methylation-specific polymerase chain reaction. Gene mutation was identified by direct sequencing. The expression of MGMT protein in paraffin-embedded tissue was examined by immunohistochemistry. Frequencies of promoter methylation were 70% for p16/INK4a, 49% for MGMT, 46% for hMLH1, 41% for E-cadherin, and 32% for DAPK genes. MGMT methylation status was closely correlated with the MGMT protein expression determined by immunohistochemistry (P < .001). Although this was not statistically significant, biliary tract cancer tumors with MGMT methylation expressed multigene methylation more frequently than tumors without MGMT methylation (P = .071). A total of 33 mutations were identified in 4 cancer-related genes: p53, K-ras, beta-catenin, and p16/INK4a genes. The most common mutation was GC to AT transitions (58%), which were significantly associated with MGMT promoter methylation (P = .011). These findings suggest that loss of MGMT expression by promoter methylation results in accumulation of GC to AT gene mutations. Reduced MGMT expression may increase the malignant potential of biliary tract cancer through both epigenetic and genetic mechanisms.

Methylation analysis of the cell cycle control genes in myelofibrosis with myeloid metaplasia

Promoter hypermethylation represents a primary mechanism in the inactivation of tumor suppressor genes during tumorigenesis. We analyzed the promoter methylation status of eight tumor-associated genes (p14 ARF, p15 INK4B, p16 INK4A, Rb, hMLH1, hMSH2, APC, and DAPK) in 30 patients with myelofibrosis with myeloid metaplasia (MMM) by methylation specific PCR. The study showed no hypermethylation of the promoters of p16 INK4A, Rb, hMLH1, hMSH2, APC, and DAPK genes. The p14 ARF, p15 INK4B promoters were hypermethylated in only one patient each. This study indicates that, although methylation of these genes is important in other cancers, it is rare in MMM and causation of this disease should be focused elsewhere.

PD.64 Promoter hypermethylation, mRNA andprotein expression of p16, DAPK, MGMT and GSTP1 genes in chewing-tobacco induced oral cancers

PD.64 Promoter hypermethylation, mRNA andprotein expression of p16, DAPK, MGMT and GSTP1 genes in chewing-tobacco induced oral cancers

Multiplicity of abnormal promoter methylation in lung adenocarcinomas from smokers and never smokers

The prevalence of methylation of the p16, DAPK and RASSF1A genes was investigated in lung adenocarcinoma from smokers, former uranium miners and never smokers. The association between a common genetic alteration in adenocarcinoma, mutation of the K-ras gene and methylation of these genes, as well as survival was examined. Adenocarcinomas from 157 smokers, 46 never smokers and 34 former uranium miners were evaluated for methylation of the p16, DAPK and RASSF1A genes using the methylation-specific PCR assay. Comparisons were also made to prevalences of methylation of the MGMT gene and mutation of the K-ras gene previously examined in these tumors. The prevalence of methylation for all genes was similar between adenocarcinomas from smokers and never smokers, although the prevalence for methylation of the p16 gene tended to be higher in smokers compared to never smokers. A significantly higher prevalence for p16 methylation was seen in central vs. peripheral lung tumors. At least 1 gene was methylated in 35% of stage I tumors, whereas 2 and >/=3 genes were methylated in 40% and 16% of tumors, respectively. Methylation of all genes was independent of K-ras mutation, whereas methylation of the DAPK and RASSF1A genes was positively associated. Environmental tobacco smoke, the strongest lung cancer risk factor among never smokers, induces adenocarcinoma in part through inactivation of the p16, DAPK and RASSF1A genes. Adenocarcinomas may develop through 2 distinct processes: multiple gene inactivations through promoter hypermethylation and activation of the K-ras gene.

Promoter hypermethylation of cancer-related genes: a strong independent prognostic factor in acute lymphoblastic leukemia

AbstractPromoter hypermethylation plays an important role in the inactivation of cancer-related genes. This abnormality occurs early in leukemogenesis and seems to be associated with poor prognosis in acute lymphoblastic leukemia (ALL). To determine the extent of hypermethylation in ALL, we analyzed the methylation status of the CDH1, p73, p16, p15, p57, NES-1, DKK-3, CDH13, p14, TMS-1, APAF-1, DAPK, PARKIN, LATS-1, and PTEN genes in 251 consecutive ALL patients. A total of 77.3% of samples had at least 1 gene methylated, whereas 35.9% of cases had 4 or more genes methylated. Clinical features and complete remission rate did not differ among patients without methylated genes, patients with 1 to 3 methylated genes (methylated group A), or patients with more than 3 methylated genes (methylated group B). Estimated disease-free survival (DFS) and overall survival (OS) at 11 years were 75.5% and 66.1%, respectively, for the nonmethylated group; 37.2% and 45.5% for methylated group A; and 9.4% and 7.8% for methylated group B (P < .0001 and P = .0004, respectively). Multivariate analysis demonstrated that the methylation profile was an independent prognostic factor in predicting DFS (P < .0001) and OS (P = .003). Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in ALL.

Aberrant methylation in promoter-associated CpG islands of multiple genes in relapsed childhood acute lymphoblastic leukemia

Methylation profile was analyzed in nine cases of relapsed childhood acute lymphoblastic leukemia (ALL) for p14, p15, p16, Rb, MGMT, APC, hMLH1, RARbeta, RIZ, DAPK, and FHIT genes by using methylation specific polymerase chain reaction (MSP) analysis. Frequency of methylation in each gene was: MGMT, 56%; RARbeta, 44%; and p16, 22%, respectively. None of the p14, p15, Rb, APC, hMLH1, RIZ, DAPK, and FHIT genes were hypermethylated. Five (56%) of 9 cases showed methylation of at least one gene. All of the samples with hypermethylation in p16 and MGMT gene at relapse, had already acquired the change at the time of initial diagnosis. Interestingly, three of 4 cases with RARbeta gene methylation at relapse did not have methylation of this gene at the time of initial presentation. These results suggest that hypermethylation might be involved in the relapse of childhood ALL.

Real-time PCR analysis of the apoptosis related genes in ATRA treated APL t(15;17) patients.

All-trans retinoic acid (ATRA) treatment of the acute promyelocytic leukemia (APL) have subsequently resulted in cell apoptosis, but the molecular mechanism of this effect remains elusive. In order to understand a possible involvement of genes regulating apoptotic signal pathways, expression levels of bcl2, bax, dapk1, myc, bad, wt1, and mcl genes were analyzed during ATRA treatment in five APL patients with t (15;17) using Real- time PCR (LightCycler). Two samples from each patient were compared to each other: primary diagnostic sample and a sample taken at remission. Effect of the ATRA treatment was demonstrated by the concomitant induction of cd14 and il1beta genes in four patients. Also other apoptosis related genes were found down-regulated in general but especially the down regulated levels of wt1 and bax attract attention. Result suggested that ATRA dependent apoptosis of APL was under the control of both internal and external pathways without relationships to the amount of the blast populations. Ratio of bcl2 to bax may be more important for this regulation than the ratio of bcl2 to bad. Either bcl2 family or less known apoptosis related genes as wt1 will still be required to further studies in this setting.

Aberrant methylation in promoter-associated CpG islands of multiple genes in therapy-related leukemia

Methylation profile was analyzed in eleven cases of therapy-related leukemia (t-leukemia) for p14, p15, p16, Rb, hMLH1, hMSH2, MGMT, APC, RAR beta, DAPK, RIZ1, FHIT, and SOCS-1 genes by using methylation specific polymerase chain reaction (MSP) analysis. Six (55%) of eleven cases showed methylation of at least one gene. The average time to the development of t-leukemia after the treatment of the primary tumor was significantly shorter in patients with methylation than those without methylation (49.3 months vs. 133.2 months, P=0.044). These results suggest that hypermethylation might be involved in the development of t-leukemia.

Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues

The methylation pattern in the promoter region of p16, DAPK, MGMT and GSTP1 genes was investigated in oral cancer tissues and tumor associated adjacent tissues, using methylation specific PCR assay. The samples constituted 60 primary oral tumors and corresponding adjacent clinically and histopathologically normal mucosa, and buccal epithelial scrapings from 20 normal healthy individuals without any tobacco habits. The incidence of hypermethylation in oral tumor and adjacent mucosa for p16 gene was 66.7 and 50%, for DAPK was 68.3 and 60%, and MGMT gene was 51.7 and 26.7%, respectively. The overall hypermethylation in the three genes in the primary tumor was 86.7%, and corresponding adjacent normal mucosa tissues 76.7%. Hypermethylation was not observed in the promoter region of GSTP1 gene in either the primary tumors or the corresponding adjacent normal mucosa. Absence of aberrant methylation in the four genes was noted in buccal scrapings from normal healthy individuals with no tobacco habits. Thus, a high frequency of promoter region hypermethylation was observed in p16, DAPK and MGMT genes in oral cancer tissues as well as in corresponding adjacent normal mucosa. Our results indicate that epigenetic alteration of these genes is a frequent event in oral cancer, and is an early event observed in normal oral mucosa of the patients, indicating the critical importance of the epigenetic alteration in chewing tobacco associated oral carcinogenesis.

Frequent Promoter Methylation of CDH1, DAPK, RARB, and HIC1 Genes in Carcinoma of Cervix Uteri: Its Relationship to Clinical Outcome

BackgroundCervical cancer (CC), a leading cause of cancer-related deaths in women worldwide, has been causally linked to genital human papillomavirus (HPV) infection. Although a host of genetic alterations have been identified, molecular basis of CC development is still poorly understood.ResultsWe examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 16 gene promoters in 90 CC cases. We found a high frequency of promoter methylation in CDH1, DAPK, RARB, and HIC1 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed the following: a) overall promoter methylation was higher in more advanced stage of the disease, b) promoter methylation of RARB and BRCA1 predicted worse prognosis, and c) the HIC1 promoter methylation was frequently seen in association with microsatellite instability. Promoter methylation was associated with gene silencing in CC cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.ConclusionsThese results may have implications in understanding the underlying epigenetic mechanisms in CC development, provide prognostic indicators, and identify important gene targets for treatment.

Assignment of DAP1 and DAPK—Genes That Positively Mediate Programmed Cell Death Triggered by IFN-γ—to Chromosome Regions 5p12.2 and 9q34.1, Respectively

Stress adaptation and virulence of various bacterial pathogens require stringent response pathways involving guanosine pentaphosphate and inorganic polyphosphate (PolyP). In M. tuberculosis, intracellular PolyP levels are maintained by the activities of polyphosphate kinase (PPK-1, PPK-2) and exopolyphosphatases (PPX-1, PPX-2). We demonstrate that these exopolyphosphatases cumulatively contribute to biofilm formation and survival of M. tuberculosis in nutrient limiting, low oxygen growth conditions and in macrophages. Characterization of single (Δppx2) and double knock out strain (dkppx) of M. tuberculosis demonstrated that these exopolyphosphatases are essential for establishing infection in guinea pigs and mice. Transcriptional profiling revealed that relative to the parental strain the expression of genes belonging to DosR regulon were significantly reduced in mid-log phase cultures of dkppx strain. We also show that PolyP inhibited the autophosphorylation activities associated with DosT and DosS sensor kinases. Host RNA-seq analysis revealed that transcripts involved in various antimicrobial pathways such as apoptosis, autophagy, macrophage activation, calcium signalling, innate and T-cell response were differentially expressed in lung tissues of dkppx strain infected mice. Taken together, we demonstrate that enzymes involved in PolyP homeostasis play a critical role in physiology and virulence of M. tuberculosis. These enzymes are attractive targets for developing novel interventions that might be active against drug-sensitive and drug-resistant M. tuberculosis.