Frequent Silencing Research Articles

Decreased expression and aberrant methylation of Gadd45G is associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma

The growth arrest DNA damage-inducible gene (Gadd45) family, which is composed of Gadd45A, Gadd45B, and Gadd45G, is involved in DNA damage response and cell growth arrest. The present study was to detect the role of Gadd45 gene family in esophageal cancer and the relationship of Gadd45G methylation to a series of pathological parameters in a large esophageal squamous cell carcinoma (ESCC) sample, in order to elucidate more information on the role of Gadd45 gene family with regard to the pathogenesis of ESCC. Frequent silencing of Gadd45G but not Gadd45A and Gadd45B were found in esophageal cancer cell lines and the silencing of Gadd45G may be reversed by 5-Aza-dC or TSA treatment in Eca109 cell line. The aberrant proximal promoter methylation of Gadd45G induces silencing of Gadd45G expression in Eca109 cell line. Gadd45A mRNA and protein expression in ESCC tumor tissues was significantly different compared to corresponding normal tissues. Decreased mRNA and protein expression of Gadd45G was observed in ESCC tumor tissues and was associated with Gadd45G proximal promoter methylation. Gadd45A or Gadd45B expression was not correlated with ESCC patients survival, while Gadd45G methylation status and protein expression were independently associated with ESCC patients' survival. These data indicated that Gadd45G may be a functional tumor suppressor and its inactivation through proximal promoter methylation may play an important role in ESCC carcinogenesis and reactivation of Gadd45G gene may has therapeutic potential and may be used as a prognostic marker for ESCC patients.

Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer

PURPOSE: There is accumulating evidence that galanin receptors (GALRs) may be tumor suppressors in head and neck squamous cell carcinoma (HNSCC). Promoter methylation status and gene expression were assessed in a large panel of primary tumors, based on the hypothesis that CpG hypermethylation might silence the galanin gene. EXPERIMENTAL DESIGN: Galanin expression was examined using reverse transcription-polymerase chain reaction (PCR). The methylation status of the galanin promoter was studied using bisulfate sequencing and methylation-specific PCR. UM-SCC-54 was stably transfected to express galanin. RESULTS: Galanin expression was absent in 3/12 (25.0%) UM-SCC cell lines, whereas three nonmalignant cell lines had stable expression. Galanin methylation was found in 24/100 (24.0%) cases. HNSCC tumor specimens was significantly correlated with the GALR1 methylation status (P = 1.88E-06). The presence of galanin promoter hypermethylation was statistically correlated with a decrease in disease-free survival (log-rank test, P = 6.02E-05). A multivariate logistic regression analysis showed that methylation of galanin and methylation of the gene pair galanin and GALR1 had an odds ratio for recurrence of 8.95 [95% confidence interval (CI), 2.29–35.03] and 23.84 (95% CI, 2.74–207.17), respectively. UM-SCC-54 cells that are GALR1-proficient but have hypermethylated galanin exhibited suppressed cell proliferation following exogenous expression of galanin. CONCLUSIONS: Association of frequent promoter hypermethylation and gene silencing with poor survival, combined with growth suppression of HNSCC cells after forced gene expression, supports the hypothesis that galanin acts as a tumor suppressor. These data suggest that galanin and GALR1 are potential therapeutic targets and prognostic factors.

Molecular Pathways: Current Role and Future Directions of the Retinoic Acid Pathway in Cancer Prevention and Treatment

Retinoids and their naturally metabolized and synthetic products (e.g., all-trans retinoic acid, 13-cis retinoic acid, bexarotene) induce differentiation in various cell types. Retinoids exert their actions mainly through binding to the nuclear retinoic acid receptors (α, β, γ), which are transcriptional and homeostatic regulators with functions that are often compromised early in neoplastic transformation. The retinoids have been investigated extensively for their use in cancer prevention and treatment. Success has been achieved with their use in the treatment of subtypes of leukemia harboring chromosomal translocations. Promising results have been observed in the breast cancer prevention setting, where fenretinide prevention trials have provided a strong rationale for further investigation in young women at high risk for breast cancer. Ongoing phase III randomized trials investigating retinoids in combination with chemotherapy in non-small cell lung cancer aim to definitively characterize the role of retinoids in this tumor type. The limited treatment success observed to date in the prevention and treatment of solid tumors may relate to the frequent epigenetic silencing of RARβ. Robust evaluation of RARβ and downstream genes may permit optimized use of retinoids in the solid tumor arena.

Frequent concomitant epigenetic silencing of SOX1 and secreted frizzled‐related proteins (SFRPs) in human hepatocellular carcinoma

Except for genetic mutations, epigenetic changes are also involved in the development of human cancers. Recently, we have identified SOX1, SRY (sex determining region Y)-box 1, is hypermethylated in cervical cancer and ovarian cancer. Therefore, we investigated whether promoter hypermethylation of SOX1 is common in hepatocellular carcinoma (HCC). We used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to analyze the methyaltion level of the SOX1 promoter in seven HCC cell lines, 54 clinical HCCs, 42 cirrhotic livers, 21 livers with chronic hepatitis, and 15 control livers. Then, we employed quantitative MS-PCR (QMSP) to validate in an independent set of samples (60 paired HCCs and 30 control livers). Finally, we used luciferase reporter and colony formation assay to check the effect of SOX1 in HCC. Promoter methylation of SOX1 was significantly frequent in HCC cell lines and clinical HCCs, cirrhotic livers, but not in control livers (P < 0.0001). There is a significant correlation between downregulation of SOX1 expression and promoter methylation. QMSP results confirmed that promoter hypermethylation of SOX1 is significantly more frequent in HCCs than control livers (P < 0.0001). The frequency of SOX1 methylation in patients with secreted frizzled-related proteins (SFRPs) methylation is significantly higher than in patients without SFRPs methylation (P < 0.0001). Furthermore, ectopic expression of SOX1 could suppress T-cell factor-dependent transcriptional activity and colony formation number in HCCs. Concomitant epigenetic silencing of SOX1 and SFRPs through promoter hypermethylation is frequent in HCCs, and this might contribute to abnormal activation of canonical Wnt signal pathway.

MLH1‐silenced and non‐silenced subgroups of hypermutated colorectal carcinomas have distinct mutational landscapes

Approximately 15% of colorectal carcinomas (CRCs) exhibit a hypermutated genotype accompanied by high levels of microsatellite instability (MSI-H) and defects in DNA mismatch repair. These tumours, unlike the majority of colorectal carcinomas, are often diploid, exhibit frequent epigenetic silencing of the MLH1 DNA mismatch repair gene, and have a better clinical prognosis. As an adjunct study to The Cancer Genome Atlas consortium that recently analysed 224 colorectal cancers by whole exome sequencing, we compared the 35 CRCs (15.6%) with a hypermutated genotype to those with a non-hypermutated genotype. We found that 22 (63%) of the hypermutated CRCs exhibited transcriptional silencing of the MLH1 gene, a high frequency of BRAF V600E gene mutations, and infrequent APC and KRAS mutations, a mutational pattern significantly different from their non-hypermutated counterparts. However, the remaining 13 (37%) hypermutated CRCs lacked MLH1 silencing, contained tumours with the highest mutation rates ('ultramutated' CRCs), and exhibited higher incidences of APC and KRAS mutations, but infrequent BRAF mutations. These patterns were confirmed in an independent validation set of 250 exome-sequenced CRCs. Analysis of mRNA and microRNA expression signatures revealed that hypermutated CRCs with MLH1 silencing had greatly reduced levels of WNT signalling and increased BRAF signalling relative to non-hypermutated CRCs. Our findings suggest that hypermutated CRCs include one subgroup with fundamentally different pathways to malignancy than the majority of CRCs. Examination of MLH1 expression status and frequencies of APC, KRAS, and BRAF mutation in CRC may provide a useful diagnostic tool that could supplement the standard microsatellite instability assays and influence therapeutic decisions.

Frequent epigenetic silencing of PCDH10 by methylation in human colorectal cancer

Aberrant DNA methylation is common in cancer cells. Epigenetic alterations resulting in the loss of tumor suppression gene functions are frequently involved in tumor development and progression. Recently, methylation of PCDH10 was reported to be associated with multiple hematologic malignancies as well as some solid tumors. Whether the down-regulation of PCDH10 happens in CRC remains unknown. Methylation status of PCDH10 was evaluated by methylation-specific PCR analysis. The effects of PCDH10 re-expression were determined in growth, colony formation, cell cycle, and invasion assays. In this study, we found that 100 % (8 of 8) of colorectal cancer cell lines were silenced for PCDH10, but not normal colorectal epithelial cells. Demethylation treatment confirmed that the reduced expression is associated closely with promoter methylation. Hyper-methylation of PCDH10 was also detected in 85 % of primary colorectal tumors, but not in adjacent normal colorectal tissues. Our results suggest that PCDH10 is an important tumor suppression gene with key roles of suppressing cell proliferation, clonogenicity, and inhibiting cell invasion in the development of colorectal cancer. Thus, PCDH10 methylation may constitute a useful biomarker of colorectal cancer patients.

Frequent silencing of protocadherin 8 by promoter methylation, a candidate tumor suppressor for human gastric cancer

The cadherins are a family of cell surface glycoproteins responsible for cell adhesion which play an important role in cell morphology, contact inhibition and signal transduction during tumorigenesis. Protocadherin 8 (PCDH8), a member of the cadherin family, has been reported to act as a tumor suppressor involved in oncogenesis in breast cancer. In this study, we aimed to investigate the epigenetic inactivation of PCDH8 and its tumor suppressor function in gastric cancer. The expression of PCDH8 was markedly reduced or silenced in gastric cancer cell lines compared with normal gastric cells or tissues. Methylation of the PCDH8 gene promoter was observed in 100% (4/4) of cell lines and 55.38% (36/65) of the primary gastric cancer by methylation-specific PCR, but not in normal gastric mucosa (0/10). Methylated PCDH8 was significantly associated with lymph node metastasis in a logistic regression analysis. The demethylation reagent 5-aza-2'-deoxycytidine was able to restore or upregulate PCDH8 expression in gastric cancer cell lines. Ectopic expression of PCDH8 in silenced gastric cancer cells significantly inhibited cell migration and induced apoptosis. For the first time, our study demonstrates the epigenetic inactivation of PCDH8 by promoter methylation and its tumor suppressor function in human gastric cancer. Thus, PCDH8 could be identified as a candidate tumor suppressor in human gastric cancer.

Platelet factor 4 induces cell apoptosis by inhibition of STAT3 via up-regulation of SOCS3 expression in multiple myeloma

Platelet factor 4 (PF4) is an angiostatic chemokine that suppresses tumor growth and metastasis. We previously revealed frequent transcriptional silencing of PF4 in multiple myeloma, but the functional roles of this chemokine are still unknown. We studied the apoptotic effects of PF4 on myeloma cell lines and primary myeloma in vitro, and investigated the involved signaling pathway. The in vivo effects were also studied using a mouse model. PF4 not only suppressed myeloma-associated angiogenesis, but also inhibited growth and induced apoptosis in myeloma cells. We found that PF4 negatively regulated STAT3 and concordantly inhibited constitutive and interleukin-6-induced phosphorylation of STAT3, and down-regulated the expression of STAT3 target genes (Mcl-1, survivin and VEGF). Overexpression of constitutively activated STAT3 could rescue PF4-induced apoptotic effects. Furthermore, we found that PF4 induced the expression of SOCS3, a STAT3 inhibitor, and gene silencing of SOCS3 abolished its ability to inhibit STAT3 activation, suggesting a critical role of SOCS3 in PF4-induced STAT3 inhibition. Knockdown of LRP1, a putative PF4 receptor, could also abolish PF4-induced apoptosis and STAT3 inhibition. Finally, the tumor growth inhibitory effect of PF4 was confirmed by in vivo mouse models. Immunostaining of rabbit bone xenografts from PF4-treated mice showed induction of apoptosis of myeloma cells and inhibition of angiogenesis, which was associated with suppression of STAT3 activity. Together, our preclinical data indicate that PF4 may be a potential new targeting agent for the treatment of myeloma.

Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1in tumors of epithelial, NK- and B-cell origins

BackgroundEpstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of BZLF1 and BRLF1 promoters (Zp and Rp) in tumor samples.MethodsWe evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as BZLF1 and BRLF1 expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins.ResultsWe found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing BZLF1 and BRLF1. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1.ConclusionsHypermethylation of Zp and Rp mediates the frequent silencing of BZLF1 and BRLF1 in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.

The Ubiquitin Peptidase UCHL1 Induces G0/G1 Cell Cycle Arrest and Apoptosis Through Stabilizing p53 and Is Frequently Silenced in Breast Cancer

BackgroundBreast cancer (BrCa) is a complex disease driven by aberrant gene alterations and environmental factors. Recent studies reveal that abnormal epigenetic gene regulation also plays an important role in its pathogenesis. Ubiquitin carboxyl- terminal esterase L1 (UCHL1) is a tumor suppressor silenced by promoter methylation in multiple cancers, but its role and alterations in breast tumorigenesis remain unclear.Methodology/Principal FindingsWe found that UCHL1 was frequently downregulated or silenced in breast cancer cell lines and tumor tissues, but readily expressed in normal breast tissues and mammary epithelial cells. Promoter methylation of UCHL1 was detected in 9 of 10 breast cancer cell lines (90%) and 53 of 66 (80%) primary tumors, but rarely in normal breast tissues, which was statistically correlated with advanced clinical stage and progesterone receptor status. Pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. Ectopic expression of UCHL1 significantly suppressed the colony formation and proliferation of breast tumor cells, through inducing G0/G1 cell cycle arrest and apoptosis. Subcellular localization study showed that UCHL1 increased cytoplasmic abundance of p53. We further found that UCHL1 induced p53 accumulation and reduced MDM2 protein level, and subsequently upregulated the expression of p21, as well as cleavage of caspase3 and PARP, but not in catalytic mutant UCHL1 C90S-expressed cells.Conclusions/SignificanceUCHL1 exerts its tumor suppressive functions by inducing G0/G1cell cycle arrest and apoptosis in breast tumorigenesis, requiring its deubiquitinase activity. Its frequent silencing by promoter CpG methylation may serve as a potential tumor marker for breast cancer.

悪性胸膜中皮腫の新しい分子生物学的異常と治療応用の可能性

悪性胸膜中皮腫の新しい分子生物学的異常と治療応用の可能性

The epigenetic modifier PRDM5 functions as a tumor suppressor through modulating WNT/β-catenin signaling and is frequently silenced in multiple tumors.

BackgroundPRDM (PRDI-BF1 and RIZ domain containing) proteins are zinc finger proteins involved in multiple cellular regulations by acting as epigenetic modifiers. We studied a recently identified PRDM member PRDM5 for its epigenetic abnormality and tumor suppressive functions in multiple tumorigeneses.Methodology/Principal FindingsSemi-quantitative RT-PCR showed that PRDM5 was broadly expressed in human normal tissues, but frequently silenced or downregulated in multiple carcinoma cell lines due to promoter CpG methylation, including 80% (4/5) nasopharyngeal, 44% (8/18) esophageal, 76% (13/17) gastric, 50% (2/4) cervical, and 25% (3/12) hepatocellular carcinoma cell lines, but not in any immortalized normal epithelial cell lines. PRDM5 expression could be restored by 5-aza-2′-deoxycytidine demethylation treatment in silenced cell lines. PRDM5 methylation was frequently detected by methylation-specific PCR (MSP) in multiple primary tumors, including 93% (43/46) nasopharyngeal, 58% (25/43) esophageal, 88% (37/42) gastric and 63% (29/46) hepatocellular tumors. PRDM5 was further found a stress-responsive gene, but its response was impaired when the promoter was methylated. Ectopic PRDM5 expression significantly inhibited tumor cell clonogenicity, accompanied by the inhibition of TCF/β-catenin-dependent transcription and downregulation of CDK4, TWIST1 and MDM2 oncogenes, while knocking down of PRDM5 expression lead to increased cell proliferation. ChIP assay showed that PRDM5 bound to its target gene promoters and suppressed their transcription. An inverse correlation between the expression of PRDM5 and activated β-catenin was also observed in cell lines.Conclusions/SignificancePRDM5 functions as a tumor suppressor at least partially through antagonizing aberrant WNT/β-catenin signaling and oncogene expression. Frequent epigenetic silencing of PRDM5 is involved in multiple tumorigeneses, which could serve as a tumor biomarker.

DCL3 and DCL4 are likely involved in the light intensity - RNA silencing cross talk in Nicotiana benthamiana

Plants have substantially invested in RNA silencing as the central defense mechanism to combat nucleotide ‘invaders’ such as viruses, trasposable elements and transgenes. The quantity and quality of light perceived by a plant is a constant environmental stimulus refining cell homeostasis and RNA silencing mechanism seems not to be an exception In our recent paper in BMC Plant Biology we documented that light intensity, in physiological ranges, positively affects silencing initiation and spread.1 Here, we show that virus induced gene silecing under high light conditions results in more frequent systemic silencing events of a transgene and is acompanied by elevated DCL3 and DCL4 mRNA levels. In addition, our results show that DCL3 holds a vital role in systemic silencing spread and the positive effect of light intensity on RNA silencing requires DCL4 function.

Location-Specific Epigenetic Regulation of the Metallothionein 3 Gene in Esophageal Adenocarcinomas

BackgroundMetallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs).Methods and ResultsUsing quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from −372 to −306 from the transcription start site (TSS) were highly methylated in tumor (n = 64) and normal samples (n = 51), whereas CpG nucleotides closest to the TSS (−4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from −139 to −49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, −log10(FDR)>3.0]. The DNA methylation levels from −127 to −8 CpG sites showed the strongest correlation with MT3 gene expression (r = −0.4, P<0.0001). Moreover, the DNA hypermethylation from −127 to −8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks.ConclusionIn summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

Frequent epigenetic inactivation of KIBRA, an upstream member of the Salvador/Warts/Hippo (SWH) tumor suppressor network, is associated with specific genetic event in B-cell acute lymphocytic leukemia

The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5' CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2'-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in < 20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 {t(12;21) (p13;q22)} chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.

Frequent silencing of popeye domain-containing genes, BVES and POPDC3 , is associated with promoter hypermethylation in gastric cancer

The Popeye domain-containing (POPDC) genes BVES, POPDC2 and POPDC3 encode proteins that regulate cell-cell adhesion and cell migration during development. Herein, we report the frequent downregulation of BVES and POPDC3 by promoter hypermethylation in gastric cancer. POPDC expression in 11 gastric cancer cell lines and 96 paired gastric tumor and normal adjacent tissues was analyzed with quantitative reverse transcription-polymerase chain reaction. The methylation status of BVES and POPDC3 was analyzed with methylated DNA immunoprecipitation sequencing, bisulfite sequencing and pyrosequencing. Expression of BVES and POPDC3 was downregulated in 73% of the gastric cancer cell lines and in 69% (BVES) and 87% (POPDC3) of the gastric cancer tissues. The BVES and POPDC3 promoter regions were hypermethylated in the gastric cancer cell lines in which they were silenced. Combined treatment with a DNA methylation inhibitor and a histone deacetylase inhibitor strongly induced BVES and POPDC3 expression. BVES and POPDC3 were hypermethylated in 69% (BVES) and 64% (POPDC3) of the gastric cancer tissues. We knocked down POPDC3 expression with short hairpin RNAs and examined the consequences on cell migration and invasion. Knockdown of POPDC3 in SNU-216 cells caused increased cell migration and invasion. Thus, epigenetic inactivation of BVES and POPDC3 occurs frequently in gastric tumors and may promote gastric cancer cell migration and invasion.

T1711 Dynamic Epigenetic Changes of MT3 Promoter Regulate Its Expression in Esophageal Adenocarcinomas

Background: Chronic gastro-esophageal reflux, in which accumulation of reactive oxygen species (ROS) and subsequent oxidative DNA damage occur, is the main risk factor for the development of Barrett's esophagus and progression to esophageal adenocarcinoma (EAC). Metallothionein 3 (MT3) is involved in maintaining intracellular metal homeostasis and was recently reported to be involved in the protection against ROS-induced DNA damage. The MT3 expression and epigenetic alterations have not been reported in EACs. Method and Results: We designed several assays and quantified the changes of the DNA methylation levels of the MT3 promoter CpG island region from -370 to +340 of the transcription start site (TSS) by using the Pyrosequencing technology. The results demonstrated unique dynamic DNA methylation profiles in the MT3 CpG island region. Interestingly, the CpG island region between -370 to -306 from the TSS demonstrated a 48-55% methylation level in all EAC and normal samples. On the other hand, the region from -161 to -130 of TSS showed high levels of DNA methylation in EACs (35.3%) but low methylation levels in NS (mean, 11.2%) (P<.01). Analysis of the CpG island region from -127 to +340 demonstrated less than 10% methylation levels in all normal samples. The methylation level of this region in EAC showed more than 25% methylation. However, the CpG nucleotides adjacent to the TSS (4 to +3) had an unexpected drop in their methylation levels to 10% in EAC samples. Using 10% as the cutoff for DNA hypermethylation, the average methylation level of CpG sites from -127 to -26 was significantly higher in 62% (49/79) of EACs (mean ± SD, 22.6 ± 21.2) than in normals (4.9 ± 2.5). Of note, the methylation level of these sites showed inverse correlations with MT3 expression, confirming that this is a critical region in regulating MT3 transcription. We have also determined the histone code along the CpG island using quantitative chromatin immunoprecipitation (CHIP) assay on two cells lines with different methylation and expression levels of MT3. The results demonstrated more repressive histone markers (H3K9me2, H3K27me2) and a less active histone marker (H3K9ace) on this region of MT3 in OE33 cells, as compared to Flo-1 cells. The results were concordant with the DNA methylation and mRNA expression levels of these cell lines. Treatment of OE33 cells with 5-Aza restored MT3 expression with demethylation of its promoter region and reversal of the repressive histone code to an active code. Conclusions: Our results suggest 1) frequent epigenetic silencing of MT3 in EACs; 2) selecting the CpG sites for functional analysis of DNA methylation is necessary.

Abstract 174: Characterization of methylation-controlled DnaJD1 in Hep3B hepatoma cell line

Abstract The development of hepatocllular carcinoma (HCC) is a multistep process with accumulation of genetic and epigenetic alternations in regulatory genes, leading to activation of oncogenes and inactivation of tumor suppressor genes. For genome-wide DNA methylation study, we used homemade MCGI (mouse CpG islands) microarray to identify many genes that were hypermethylation in the liver tumor tissues of HBx-overexpressd transgenic mice. These selected hypermethylation genes were also confirmed in human hepatocellular carcinoma cell (HCC) lines. In this study, we focused on a methylation-controlled DNAJ family (MCJ) gene, DnaJD1. It has been observed highly frequent DnaJD1 gene methylation and silencing in ovarian cancer, and associated with increased resistance to chemotherapeutic. The DnaJD1 was found hypermethylation in the promoter region and followed reduced mRNA expression in Hep3B hepatoma cells, which contain HBV genome. After treated by 5-aza-2’-deoxycytidine (DAC) in Hep3B cells, the methylation level of DnaJD1 could be reduced and mRNA re-expressed. We over-expressed DnaJD1 in Hep3B cells to study the physiologic effects of DnaJD1 in cell proliferation, cell migration, and invasion ability in vitro. The tumorgenesis in xenotransplanted nude mice were also examined. Data suggested that DnaJD1 re-expressed in Hep3B cells that could affect the cell morphology and lost cell contact insensitive. In these results, we concluded that DnaJD1 could be the one of main role of HCC progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 174.

Abstract 2081: Frequent silencing of tumor suppressive MicroRNA-34b/c by aberrant methylation in malignant mesothelioma

Abstract Background: Altered expression of microRNA (miRNA) is strongly implicated in human malignancies. Some miRNAs are classified as oncogenic or tumor suppressive miRNAs according to their function in cellular transformation. Similar to encoding genes, tumor suppressive miRNAs are silenced with DNA methylation. The miR-34a and b/c are direct transcriptional targets of p53 and sometimes down-regulated in malignancies. Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with a dismal prognosis. Unlike other malignant tumors, mutation of P53 gene is rare and p53 expression is generally intact in MPM. In this study, we examined methylation and expression status of miR-34a and b/c in MPM. We also investigated the effect of miR-34b/c induction into MPM cells. Methods: We examined 6 MPM cell lines (H28, H290, 211H, H2052, H2452, and HP1), 20 primary MPMs and 7 non-malignant mesotheial tissues. Methylation and expression status of miR-34a and b/c were determined with methylation-specific PCR and quantitative PCR, respectively. Colony formation assay was applied to examine the anti-proliferative effect by induction of miR-34b/c and scramble sequence control with plasmid vector. Protein expression and cell cycle were analyzed with western blotting and flow cytometry, respectively. Results: Aberrant methylation was found in 2 (33.3%) of 6 MPM cell lines and 6 (33.3%) of 20 primary MPMs in miR-34a and in all 6 MPM cell lines (100%) and 18 (90%) of 20 primary MPMs in miR-34b/c. No methylation was found in 7 non-malignant mesothelial tissues. Expression of miR-34a and b/c in all methylated cell lines was silenced and restored with 5-Aza-CdR treatment, indicating that methylation caused gene silencing. Because the frequency of methylation was particularly high in miR-34b/c, we focused on miR-34b/c in following study. Ectopic induction of miR-34b/c gene in examined 3 MPM cell lines (H28, H290, and 211H) showed significant inhibition of colony formation compared with induction of control vector, demonstrating anti-proliferative effect of miR-34b/c on MPM. Western blotting analysis for all 3 MPM cell lines after induction of miR-34b/c showed suppression of total and phospho-c-met, cdk4, cdk6, and cyclin E2 collated genes of cell proliferation, suggesting that these genes were interfering targets of miR-34b/c. Cell cycle analysis showed that G2-M fractions were reduced with flow cytometry in all 3 MPM cell lines with miR-34b/c induction. Conclusion: Our results demonstrated that miR-34b/c methylation is frequent in MPM and resulting miR-34b/c silencing plays an important role in pathogenesis of MPM. The miR-34b/c can be a promising candidate as a therapeutic miRNA for MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2081.

MICROSATELLITE AND RDNA ANALYSIS REVEAL UNIQUE REPRODUCTION IN DOGROSES

The taxonomically complex dogroses (Rosa sect. Caninae) are mostly pentaploid (2N=Sx=35) but only seven bivalents are formed in the first meiotic division. The remaining chromosomes form univalents and occur only in eggcells where they are transmitted through the seed parent. An allopolyploid origin of dogroses was suggested many years ago but genomic configuration remained a mystery until the Microsatellite Allele Counting - based on Peak Ratios (MAC-PR) technique was developed. Using this approach for determination of microsatellite allele copy number in polyploids, analyses of different dogrose species and offspring from controlled crosses, showed that bivalent formation involves one biparentally inherited, highly homozygous diploid genome. The maternally transmitted univalent genomes are instead highly differentiated within the genotype and also between taxa. Later, several rRNA gene (rDNA) families were identified from extensive cloning analysis of the ITS-1 region. The beta family occurs in each of the investigated dogrose species, whereas the others occur only in some species. Samples of pollen DNA contain proportionally more of the beta family, suggesting that this resides mainly on the bivalents. Expression analyses have shown that the beta family is considerably upregulated compared to other rRNA families, suggesting that the univalent genomes may undergo frequent epigenetic silencing.