5-Aza Treatment Research Articles

Use of integrative epigenetic and cytogenetic analyses to identify novel tumor-suppressor genes in malignant melanoma

The objective of this study was to identify novel tumor-suppressor genes in melanoma, using an integrative genomic approach. Data from: (i) earlier reports of DNA loss and gain in malignant melanoma accompanied by comparative genomic hybridization high-definition array data of the entire human genome; (ii) microarray expression data from melanoma-derived cell lines identifying genes with significantly increased expression due to methylation using a pharmacologic demethylating strategy; and (iii) publicly available RNA expression microarray data of primary tumors and benign nevi were integrated using statistical tools to define a population of candidate tumor-suppressor genes. Twenty-seven genes were identified in areas of deletion that demonstrated diminished expression in primary melanomas relative to benign nevi and were significantly increased in expression by 5-Aza treatment. Seven genes of these 27 genes demonstrated methylation and deletion in a validation cohort of 14 separate primary tumors. These were: CHRDL1, SFRP1, TMEM47, LPL, RARRES1, PLCXD1, and KOX15. All of these genes demonstrated growth-suppressive properties with transfection into melanoma-derived cell lines. Seven putative tumor-suppressor genes in malignant melanoma were identified using a novel integrative technique.

CHFR Suppression by Hypermethylation Sensitizes Endometrial Cancer Cells to Paclitaxel

Impairment of a cell cycle checkpoint is often associated with sensitivity to chemotherapeutic drugs. Here, we studied the correlations between the checkpoint with forkhead-associated and ring finger (CHFR) gene expression and responses to paclitaxel in endometrial cancer cells. We cultured 6 endometrial cancer cell lines exposed to paclitaxel, studied the cell cytotoxicity, cell cycle distribution, CHFR expression, and methylation status before and after a demethylation agent (5-aza) treatment. CHFR was silenced by small interfering RNA (siRNA). Then we examined tumor growth and CHFR expression with paclitaxel alone or combined with 5-aza pretreatment in vivo. We found that HEC-1B, RL-952, and AN3CA cells were sensitive to paclitaxel. Moreover, CHFR was weakly expressed in these cells, whereas paclitaxel-resistant cells (ISH, HEC-1A, and KLE) had high CHFR expression. Then we found that restored expression of CHFR by demethylation decreased the sensitivity to paclitaxel in AN3CA cells. In addition, cells with CHFR demethylation resulted in G2/M phase arrest that induced to paclitaxel resistance. These results were confirmed again in small interfering RNA-transfected HEC-1A cells. Furthermore, in nude mice model, restored expression of CHFR by demethylation inhibited tumor growth and decreased sensitivity to paclitaxel. Our data suggest that CHFR suppression regulated by hypermethylation may sensitize endometrial cancer cells to paclitaxel, and CHFR may be a promising marker to predict the response of endometrial cancer to paclitaxel.

Minimal residual disease-directed preemptive treatment with azacitidine in patients with NPM1-mutant acute myeloid leukemia and molecular relapse

Minimal residual disease-directed preemptive treatment with azacitidine in patients with NPM1-mutant acute myeloid leukemia and molecular relapse

Transformation of Human Mesenchymal Cells and Skin Fibroblasts into Hematopoietic Cells

Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

5-Azacytidine Induces Cardiac Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells by Activating Extracellular Regulated Kinase

5-Azacytidine (5-Aza) induces differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. However, the underlying mechanisms are not well understood. Our previous work showed that 5-Aza induces human bone marrow-derived MSCs to differentiate into cardiomyocytes. Here, we demonstrated that 5-Aza induced cardiac differentiation of human umbilical cord-derived MSCs (hucMSCs) and explored the potential signaling pathway. Our results showed that hucMSCs had cardiomyocyte phenotypes after 5-Aza treatment. In addition, myogenic cells differentiated from hucMSCs were positive for mRNA and protein of desmin, β-myosin heavy chain, cardiac troponin T, A-type natriuretic peptide, and Nkx2.5. Human diploid lung fibroblasts treated with 5-Aza expressed no cardiac-specific genes. 5-Aza did not induce hucMSCs to differentiate into osteoblasts. Further study revealed that 5-Aza treatment activated extracellular signal related kinases (ERK) in hucMSCs, but protein kinase C showed no response to 5-Aza administration. U0126, a specific inhibitor of ERK, could inhibit 5-Aza-induced expression of cardiac-specific genes and proteins in hucMSCs. Increased phosphorylation of signal transducers and activators of transcription 3, and up-regulation of myocyte enhancer-binding factor-2c and myogenic differentiation antigen in 5-Aza-treated hucMSCs were also suppressed by U0126. Taken together, these results suggested that sustained activation of ERK by 5-Aza contributed to the induction of the differentiation of hucMSCs into cardiomyocytes in vitro.

279 Methylation of Wnt antagonists and effects of AZA treatment on Wnt pathway in MDS cells

279 Methylation of Wnt antagonists and effects of AZA treatment on Wnt pathway in MDS cells

Abstract 4800: Epigenetic changes in normal and cancer prostate cells grown as prostatospheres

Abstract Background: Normal and cancer prostate cells grown in suspension cultures or low attachment media grow as spheroids and are called Prostatospheres. These cultures when maintained as self-renewing spheres express many of the stem cell markers and are useful models to understand stem cell differentiation and pathways that could be responsible for cancer cell growth and/or resistance to therapy. We are interested in studying the epigenetic signatures that may be different from monolayer cultures to cells grown as sphere cultures such that we can identify prostate specific genes in prostate stem cell differentiation and growth. Methods: We have established successful culture systems to grow and maintain Prostatospheres (PSs) from normal (RWPE1/2) and prostate cancer (DU-145, PC3, LnCaP) cell lines. After several generations in sphere cultures, the cells were collected and subjected to analysis for promoter DNA methylation. We used a 24-gene, 96 well Methyl ProfilerTM DNA methylation PCR platform array from SABiosciences which provides a fast and reliable quantitative DNA methylation analysis without bisulfite conversion. The percent of methylated, unmethylated and intermediate methylated genes were determined in both monolayer and sphere cultures. The array comprises of prostate cancer related genes that belong to the family of tumor suppressors, apoptosis, cell cycle, and genes involved with growth, differentiation and development. Finally, we confirmed the methylation status of candidate genes from the array analysis using Methylated Specific Primers (MSP) corresponding to the candidate gene. Next we examined the effect of demethylating agent AZA (5-aza-2’-deoxycytidine) and Histone deacytlase inhibitor TSA (Trichostatin A) to ascertain the reversal of methylation of candidate genes and their function in monolayer and sphere cultures. Results: Methylation specific array data show a significant shift toward increase in methylation of several genes in PSs compared to monolayer cultures. Of the 24 genes examined, more are hypermethylated in DU145 PSs (82%) than they are in DU145 monolayers (52%). Of the 10 hypermethylated genes, 7 genes (BRCA1, CDH1, CDKN1C, CDKN2A, PRDM2, RASSF1, and TP73) are tumor suppressor genes, 1 gene (PYCARD) is an apoptotic gene, 1 gene (CCND2) is a cell cycle regulation gene and 1 receptor/transcription factor (ERS1). MSP-PCR confirmed the reversal of methylation upon AZA and TSA treatment. RT-PCR data showed that several genes tested in PSs showed significant increase in their mRNA expression upon AZA or TSA treatment as single agents. Conclusion: Our preliminary data confirm that several genes are hypermethylated in PSs compared to monolayer cultures and more predominantly are those that belong to the family of tumor suppressor and apoptotic genes. Suggesting that silencing of these genes may be an important adaptation for prostate cancer stem cells during differentiation and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4800. doi:10.1158/1538-7445.AM2011-4800

Abstract 3862: Functional characterization of sFRP1 expression loss in human papillary bladder cancer

Abstract Introduction: Secreted frizzled-related protein 1 (sFRP1) is an inhibitor of the Wnt-signaling pathway and expression loss is associated with e.g. colon and breast cancer. Our previous studies revealed an involvement of sFRP1 loss in progression of papillary bladder cancer. We therefore performed functional studies to characterise the role of sFRP1 in papillary bladder cancer cell lines. Material and methods: Human papillary bladder cancer cell lines RT112 and BFTC 905 were used for the analyses. mRNA-expression status of sfrp1 and Wnt-target genes c-myc and cyclin D1 was determined using qRT-PCR. Methylation status of sfrp1 promoter was analysed with MS-PCR. sFRP1 knockdown was performed using sFRP1-specific siRNAs. 5’ Aza-2’deoxycytidine (5’Aza) treatment was used for promoter demethylation and restoration of sFRP1 expression. To determine Wnt pathway activity a luminescence-based TOP flash/FOP flash assay was used. Proliferation level was investigated with ELISA-based BrdU Cell Proliferation assay. Results: RT112 is strongly expressing sfrp1 on mRNA level, whereas BFTC905 showed no sfrp1 expression. MS-PCR revealed sfrp1-promoter methylation in BFTC905. This methylation could be reversed after 5’-Aza-treatment and led to re-expression of sfrp1 on mRNA level. sfrp1 siRNA transfection in RT112 led to ∼80% gene silencing. sfrp1 knockdown resulted in an upregulation of c-myc (515%) and cyclin D1 (51,5%) on mRNA level, however TCF/LEF-presence could not be detected, neither in untreated nor in siRNA transfected RT112 cells. Untreated BFTC905 cells showed a very weak Wnt signalling activitiy, compared to positive control cell line SW480, but 7-fold higher than in untreated and siRNA treated RT112. sfrp1 knowdown in RT112 had no influence on cell proliferation. Conclusion: sfrp1 knockdown in RT112 cells is associated with an up-regulation of proliferation-associated genes c-myc and cyclin D1, whereas this increased expression showed no effects on proliferation behaviour of the cells. Knockdown of sFRP1 did not result in significant Wnt-signaling activity in RT112 cells. Reduced sFRP1 expression might increase the proliferative potential of the cells; however additional genetic events might have to occur to induce increased proliferation of the cells. Ongoing analyses of BFTC905 with transient restoration of sFRP1 expression will give further insights into the role of sFRP1 in papillary bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3862. doi:10.1158/1538-7445.AM2011-3862

A demethylating agent enhances chemosensitivity to vinblastine in a xenograft model of renal cell carcinoma

Renal cell carcinoma (RCC) is resistant to chemo-therapy partly due to the overexpression of the P-glycoprotein. Several tumor suppressor genes have been reported to be silenced by hypermethylation of the promoter region in RCC. We recently reported that the in vitro cytotoxicity of vinblastine (VBL) was enhanced by pre-treatment with the demethylating agent, 5-aza-2'-deoxycytidine (Aza), in the RCC cell line, Caki-1. In this study, we investigated the combined effect of Aza and VBL in a Caki-1 xenograft model and in other RCC cell lines in vitro. In the xenograft model, tumor volume and weight were significantly suppressed in the co-treatment group, compared to the control, and the expressions of P-glycoprotein, Bcl-2 and cyclin B1 were reduced. Thus, this combined effect could be mediated by the accumulation of intracellular VBL and the enhancement of apoptosis and cell cycle arrest. More-over, the cytotoxicity of VBL was enhanced in vitro in three RCC cell lines by Aza treatment. These findings suggest that the combination treatment with Aza and VBL is effective against RCC.

MicroRNA-193a represses c-kit expression and functions as a methylation-silenced tumor suppressor in acute myeloid leukemia

Aberrant activation of c-kit proto-oncogene contributes to abnormal cell proliferation by altering the tyrosine kinase signaling and constitutes a crucial impetus for leukemogenesis. Epigenetic silencing of tumor-suppressive microRNAs (miRNAs) is a key oncogenic mechanism for the activation of oncogenes in tumors. In this study, several miRNAs potentially binding to the 3'-untranslated region of human c-kit mRNA were screened by luciferase reporter assays. Among these miRNAs, miR-193a was embedded in a CpG island and epigenetically repressed by promoter hypermethylation in acute myeloid leukemia (AML) cell lines and primary AML blasts, but not in normal bone marrow cells. Importantly, miR-193a levels were inversely correlated with c-kit levels measured in 9 leukemia cell lines and 27 primary AML samples. Restoring miR-193a expression in AML cells harboring c-kit mutation and/or overexpression, either by synthetic miR-193a transfection or by DNA hypomethylating agent 5-azacytidine (5-aza) treatment, resulted in a significant reduction in c-kit expression at both RNA and protein levels and inhibition of cell growth. The growth-inhibitory activity of miR-193a was associated with apoptosis and granulocytic differentiation. Moreover, 5-aza-induced c-kit reduction could be partially blocked by miR-193a inhibitor, leading to a reversal of antiproliferative and proapoptotic effects of 5-aza. These data reveal a critical role for methylation-repressed miR-193a in myeloid leukemogenesis and the therapeutic promise of upregulating miR-193a expression for c-kit-positive AML.

Impact of maintenance immunosuppressive regimens - balance between graft protective suppression of immune functions and a near physiological immune response

The Symphony study showed superior 1-year kidney graft outcome in patients on immunosuppression with tacrolimus/mycophenolate mofetil (Tacr/MMF). To analyze whether differences in clinical outcome between maintenance regimens may be explained by their impact on clinically relevant immune parameters, we assessed CD4 helper activity, immunoglobulin-secreting cell (ISC) formation, neopterin, sCD30, and intracellular cytokine production in a prospective study in 77 renal transplant recipients treated with cyclosporine A/azathioprine (CsA/Aza), CsA/MMF, Tacr/Aza or Tacr/MMF at 2 years post-transplant. Tacr- compared with CsA-based immunosuppression was independently associated with increased IL-2 (P<0.0001, CD4 cells; P=0.014, CD8 cells) and CD4 cell IL-4 responses (P=0.046; stepwise logistic regression) resulting in physiological responses in Tacr/Aza patients as compared with 25 healthy controls. MMF versus Aza treatment was proven to be an independent variable associated with suppression of CD4 cell IL-10 responses (P=0.008), B-cell IL-6R expression (P<0.0001) and ISC formation [P=0.020, staphylococcus cowan strain I (SAC I); P=0.021, pokeweed mitogen (PWM)]. Our data suggest that Tacr/MMF had the most effective impact on graft protective Th2 responses (enhanced CD4 cell IL-4 by Tacr, decreased CD4 cell IL-10 responses by MMF) and suppression of B-cell functions (MMF), whereas Tacr/Aza was associated with physiological IL-2 and IL-4 and stronger humoral responses which may reduce the risk of infectious disease complications.

A Potential Biochemical Mechanism Underlying the Influence of Sterol Deprivation Stress on Caenorhabditis elegans Longevity

To investigate the biochemical mechanism underlying the effect of sterol deprivation on longevity in Caenorhabditis elegans, we treated parent worms (P0) with 25-azacoprostane (Aza), which inhibits sitosterol-to-cholesterol conversion, and measured mean lifespan (MLS) in F2 worms. At 25 μM (∼EC(50)), Aza reduced total body sterol by 82.5%, confirming sterol depletion. Aza (25 μM) treatment of wild-type (N2) C. elegans grown in sitosterol (5 μg/ml) reduced MLS by 35%. Similar results were obtained for the stress-related mutants daf-16(mu86) and gas-1(fc21). Unexpectedly, Aza had essentially no effect on MLS in the stress-resistant daf-2(e1370) or mitochondrial complex II mutant mev-1(kn1) strains, indicating that Aza may target both insulin/IGF-1 signaling (IIS) and mitochondrial complex II. Aza increased reactive oxygen species (ROS) levels 2.7-fold in N2 worms, but did not affect ROS production by mev-1(kn1), suggesting a direct link between Aza treatment and mitochondrial ROS production. Moreover, expression of the stress-response transcription factor SKN-1 was decreased in amphid neurons by Aza and that of DAF-28 was increased when DAF-6 was involved, contributing to lifespan reduction.

Enhancement of chemotherapeutic efficacy in hypermethylator breast cancer cells through targeted and pharmacologic inhibition of DNMT3b

A subset of primary breast cancers and breast cancer cell lines express a hypermethylation defect (characterized by DNMT hyperactivity and DNMT3b overexpression) which contributes to chemotherapy resistance and provides a target for development of new treatment strategies. The objective of the current study was to determine if targeting the epigenome enhances the sensitivity of breast cancer cells to cytotoxic chemotherapy. Hypermethylator breast cancer cell lines (MDA-MB-453, BT549, and Hs578T) were treated with 250 or 500 nM 5-aza-2'-deoxycytidine (5-aza) and/or were subjected to RNAi-mediated DNMT3b knockdown (KD), and then tested for sensitivity to doxorubicin hydrochloride (DOX), paclitaxel (PAX), and 5-fluorouracil (5-FU). In MDA-MB-453 cells, DNMT3b KD reduces the IC(50) for DOX from 0.086 to 0.048 μM (44% reduction), for PAX from 0.497 to 0.376 nM (24%), and for 5-FU from 0.817 to 0.145 mM (82%). Treatment with 250 nM 5-aza for 7 days did not increase the efficacy of DOX, PAX, or 5-FU, but 7-day treatment with 500 nM 5-aza sensitized cells, reducing the IC(50) for DOX to 0.035 μM (60%), PAX to 0.311 nM (37%), and 5-FU to 0.065 mM (92%). 5-aza treatment of DNMT3b KD cells reduced the IC(50) for DOX to 0.036 μM (59%), for PAX to 0.313 nM (37%) and for 5-FU to 0.067 (92%). Similar trends of enhancement of cell kill were seen in BT549 (13-60%) and Hs578T (29-70%) cells after RNAi-mediated DNMT3b KD and/or treatment with 5-aza. The effectiveness of DOX, PAX, and 5-FU is enhanced through targeted and/or pharmacological inhibition of DNMT3b, strongly suggesting that combined epigenetic and cytotoxic treatment will improve the efficacy of breast cancer chemotherapy.

Reactivation of Syk gene by AZA suppresses metastasis but not proliferation of breast cancer cells

Spleen tyrosine kinase (Syk) is reported to be involved in the suppression of proliferation and invasion of breast cancer. Methylation-mediated Syk gene silencing is found in a subset of breast cancer. In this study, we used a DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (AZA), to restore Syk expression of breast cancer cells. Surprisingly, we found that AZA treatment could reestablish the expression of Syk, but not affect the proliferation of breast cancer cells. Moreover, tumor formation in situ by MDA-MB-435s treated with (+) or without (-) AZA in a nude mice MFP (Mammary fat pad) model did not show significant difference, too. Interestingly, pulmonary metastasis was still significantly suppressed in MDA-MB-435s(+) group (1/9 vs. 7/9). Our findings suggested Syk may be more correlated to metastasis rather than proliferation. This study implied a potential use of Syk methylation as a valuable biomarker to detect high metastatic potential cancerous lesions and the prospect of AZA to join the arsenal of drug candidates to be developed as a new reagent for management of advanced breast cancer.

Pretreatment of rat bone marrow mesenchymal stem cells with a combination of hypergravity and 5‐azacytidine enhances therapeutic efficacy for myocardial infarction

The in vivo cardiac differentiation and functional effects of unmodified adult bone marrow mesenchymal stem cells (BMSCs) after myocardial infarction (MI) is controversial. Our previous results suggested that hypergravity promoted the cardiomyogenic differentiation of BMSCs, and thus we postulated that ex vivo pretreatment of BMSCs using hypergravity and 5-azacytidine (5-Aza) would lead to cardiomyogenic differentiation and result in superior biological and functional effects on cardiac regeneration of infarcted myocardium. We used a rat MI model generated by ligation of the coronary artery. Homogeneous rat BMSCs were isolated, culture expanded, and differentiated into a cardiac lineage by adding hypergravity (2G) for 3 days and 5-Aza (50 lmol/L, 24 h). Rats underwent BMSCs (labeled with DAPI) injection after the infarction and were randomized into five groups. Group A rats received the control medium, Group B rats received unmodified BMSCs, Group C rats received BMSCs treated with hypergravity, Group D rats received BMSCs treated with 5-Aza, and Group E rats received BMSCs treated with 5-Aza and hypergravity (n = 6). After hypergravity and 5-Aza treatment, BMSCs showed positive for the early muscle and cardiac markers GATA-4, MEF-2, and Nkx2-5 with RT-PCR. We also found that hypergravity could enhance the activities of MEF-2 via promoting the nuclear export of HDAC5. The frozen section showed that the implanted BMSCs labeled with DAPI survived and angiogenesis was identified at the implantation site. In Groups B, C, D, and E rats, pre-treated BMSCs colocalized with α-actinin, and Group E rats showed a significantly larger increase in left ventricular function. The biological ex vivo cardiomyogenic differentiation of adult BMSCs with hypergravity and 5-Aza prior to their transplantation is feasible and appears to improve their in vivo cardiac differentiation as well as the functional recovery in a rat model of the infarcted myocardium.

Spatial distribution of AT- and GC-rich DNA within interphase cell nuclei of Triatoma infestans Klug

Heterochromatin bodies in single- and multichromocentered interphase cell nuclei of Triatoma infestans, a vector of Chagas disease, have been suggested to contain AT-rich DNA, based on their positive response to Q-banding and Hoechst 33248 treatment. No information exists on whether GC-rich DNA is also present in these nuclei and whether it plays a role on chromatin condensation. Considering that methodologies more precise than those previously used to determine DNA base composition in situ are currently available, and that the spatial distribution of chromatin areas differing in composition in interphase cell nuclei of different species is a matter of interest, the localization of AT- and GC-rich DNA in T. infestans nuclei is revisited here. The methodologies used included DAPI/AMD and CMA 3/Distamycin differential staining, Feulgen-DNA image analysis following Msp I and Hpa II enzymatic digestion, 5-methylcytidine immunodetection, AgNOR response, confocal microscopy, and the 5-aza-2′-deoxycytidine (5-AZA) demethylation assay. The results identified the presence of AT-rich/GC-poor DNA in chromocenters and evenly distributed AT and GC sequences in euchromatin. A GC-rich DNA zone encircling the chromocenters was also found but it could not be associated with NOR regions. To corroborate the DNA AT-richness in T. infestans nuclei, bioinformatic analyses were also performed. Methylated cytosine was evident at some points of the chromocenters’ edge in single- and multichromocentered nuclei and at the euchromatin of multichromocentered nuclei and could be transiently affected by the 5-AZA treatment. The present results suggest that in the particular case of chromocenters of the hemipteran T. infestans, cytosine methylation is not a relevant factor involved in chromatin condensation.

Epigenetic Silencing of Erythropoietin in Human Cancers

The glycoprotein hormone erythropoietin (EPO) is a key regulator in the production of red blood cells. EPO is produced mainly in the embryonic liver and kidney of adults. Other organs are also known to express varying amounts of EPO. In our study, we have analyzed the epigenetic regulation of EPO in human cancer cell lines by DNA methylation assays, chromatin immunoprecipitation, RT-PCR, and promoter analysis under different growth conditions. Moreover, the growth-related effects of ectopic EPO expression were analyzed in a head and neck cancer cell line. We found frequent DNA hypermethylation of the CpG island promoter and enhancer of EPO in different cancer cell lines. Aberrant methylation of EPO promoter was observed in primary lung, head and neck, breast, and liver cancers. Hypermethylation of EPO was associated with a decreased expression of EPO in cancer cells. Treatment of cancer cell lines with 5-aza-2'-deoxycytidine (Aza), an inhibitor of DNA methylation, reactivated EPO expression under hypoxia. In contrast, in the liver cancer cell line HepB3, the EPO promoter was unmethylated, and a high EPO expression was observed independently of Aza treatment. Moreover, in vitro hypermethylation of the EPO promoter and enhancer reduced expression of a reporter gene under normoxia and hypoxia. Induction of EPO under hypoxia was accompanied by increased histone H3 acetylation and reduced histone H3 lysine 9 trimethylation. In a head and neck cancer cell line, which exhibited low EPO levels, ectopic expression of EPO significantly enhanced proliferation under normoxia and hypoxia. In summary, we show that hypermethylation of regulatory sequences of EPO is frequently observed in tumors and that this aberrant methylation induces epigenetic silencing of EPO in cancer cells.

712: The effects of exposure to BPA and BBP on the DNA methylation profile of the IGF2/H19 imprinting control region in HTR-8 cells

Bisphenol-A (BPA) and Butyl Benzyl Phthalate (BBP) affect a range of endocrine, metabolic and developmental outcomes. While not mutational, these plasticizers may disrupt the placental and fetal epigenetic profile of imprinted genes. Disruption of two of these imprinted genes, IGF2 and H19, may lead to alterations in fetal development and programming of adult disease. Our objective was to measure the effects of exposure to BPA and BBP in first trimester extravillous HTR8 trophoblast cells by: 1.Profiling the DNA methylation of the imprinting control region (ICR) of IGF2 and H19 2.Measuring IGF2 and H19 gene expression HTR8 cells were treated with two sublethal concentrations of BPA and BBP. 5-Azacitidine (AZA), a demethylating agent, was used as a positive control. Cells were harvested on post-treatment days 1 and 4. Extracted DNA was bisulfite-treated and purified. The methylation profile of six CpG dinucleotides, part of the CTCF6 binding site of the IGF2/H19 ICR, was determined by pyrosequencing. Extracted mRNA was quantified by real-time PCR for changes in IGF2 and H19 gene expression. There were no significant differences in the methylation profiles of the groups at the various time points. The CTCF6 binding site was shown to be insensitive to treatment with AZA in HTR8 cells. The commercially-available AZA-treated “control-unmethylated DNA” from Jurkat cells was shown to be 50% methylated. Finally, no differences were found in IGF2 and H19 gene expression. This data demonstrates that the epigenetic effects of BPA and BBP are not due to changes in methylation of the CTCF6 binding site of the IGF2/H19 ICR. Additionally, BPA and BBP do not cause dysregulation of IGF2 nor H19 gene expression in HTR8 cells. Interestingly, the CTCF6 binding site appears to be stably methylated and resistant to AZA treatment in the control Jurkat cells. This suggests that a different mechanism exists to control the epigenetic profile of this ICR. This finding is now being explored in the villous 3A first trimester placental cells and freshly prepared dendritic cells.

Tissue-specific expression of IL-15RA alternative splicing transcripts and its regulation by DNA methylation

The human IL-15RA gene encoding the alpha chain of the IL-15 receptor is expressed in a variety of immune and non-immune cell types from different tissues, and generates multiple splicing events of functional importance. We aimed to evaluate expression of IL-15RA transcripts generated by alternative usage of transcription start site (Var1 and Var2) and by deletion of exon 3 (Del3), exon 2 (Del2), or both (Del2,3) in different human tissues. Since a CpG island was found near to the IL-15RA gene transcription start site, we also investigated the role of DNA methylation on the expression of IL-15RA full-length and alternative transcripts fragments in peripheral blood mononuclear cells (PBMC). IL-15RA transcription of functional (full-length and del 3) and non-functional (del 2 and del 2,3) variants was detected in many tissues, however, the number of different IL-15RA transcripts variants detected in each tissue did not correlate with the level of gene expression. IL-15RA transcript variants Var1 and Var2 presented similar expression levels in different human tissues. However, we found a distinct expression profile of functional and non-functional IL-15RA transcripts fragments. A preferential expression of transcripts that bind IL-15 compared to IL-15 non-binding transcripts was seen in the tissues investigated. When PBMC cultures were treated with 5-azacitidine (AZA), a DNA methyltransferase inhibitor, we detected a significant increase in IL-15RA copy number. Only alternative exon skipping events of Var1 (Del 2, Del 3 and Del 2, 3) were altered by AZA treatment, which is consistent with the CpG island localization in the regulatory region 5' upstream of the transcription start site of Var1 and not of Var2. Therefore, this work shows a broad expression pattern of functional IL-15RA splicing forms and suggests a regulatory role of DNA methylation in IL-15RA transcript Var1 expression in mononuclear cells.

The Hypomethylating Agent 5-Azacytidine Can Target Leukaemic Stem and Progenitor Cells In Acute Myeloid Leukemia (AML) by Inducing DNA Damage and Stress Response Pathways

AbstractAbstract 292Acute Myeloid Leukemia (AML) is a heterogeneous disease that arises from malignant leukaemic stem and progenitor cells (LSPC). LSPC are relatively resistant to current chemotherapy and considered to contribute to disease progression and relapse in patients due to their ability to re-initiate long-term leukaemic cell growth following initial treatment. We have previously shown that CD34+CD38−CD123+ LSPC are less sensitive to cytarabine and to the FLT3 inhibitor AG1296 whilst exhibiting differential sensitivity to Mylotarg (Gemtuzumab Ozogamicin) when grown under niche support, compared to LSPC with no niche support. As the hypomethylating agent and nucleoside analog 5-Azacytidine (Aza) is showing promise clinically in AML and MDS patients with effects, including induction of ATM, seen as early as 5 days post chemotherapy, we assessed this agent in our model. LSPC from AML samples treated for 72hrs with Aza (1microM) were found to be relatively cytotoxic, resulting in 33% cell kill (n=15) compared to 15% AML bulk cell kill (n=19; p=0.031). We used a protein profiler assay to depict the expression of 46 known human phosphokinase proteins when treated with Aza using the CD34+CD38− TF1A cell line. Two phosphokinase proteins were highly activated upon Aza treatment and these were the cell cycle checkpoint regulator and putative tumor suppressor Chk2 and the stress-induced p38 mitogen-activated protein kinase (p38MAPK). Drug-induced DNA damaged cells are known to stall the cell cycle to allow DNA repair by activating the traditional ATM/Chk2/ATR/Chk1 pathway and the more recently described ATM/ATR/p38MAPK/MK2 checkpoint pathway, known to be essential in p53 deficient cells. Thus, we next aimed to examine the DNA damage and stress pathways activated by Aza treatment. Using several AML cell lines, including the CD34+CD38− KG1A and TF1A cells, we demonstrate that Aza treatment eliminates S-phase cells while inducing massive DNA damage response (increase in gH2AX), leading to the activation of the ATM/Chk2 and Chk1 and thus triggering G2/M cell cycle arrest. To date 3 primary AML samples have been treated in vitro with Aza and downstream effects analysed: only 1/3 was sensitive to Aza and this was the only sample that showed an increase in ATM and Chk2 in response to the Aza treatment. As we wanted to investigate whether activation of the stress response pathway encourages any additional cell survival, we pre-treated the same cell lines with the p38MAPK inhibitor SB203580 for 30minutes prior to Aza treatment. Our data shows that there is enhanced cell kill if the p38MAPK pathway is inhibited first and then Aza treated later. We are currently in the process of assessing induction of downstream effector proteins involved in the p38MAPK pathways after Aza treatment. In conclusion, the success of Aza treatment in AML is maybe due to the specific targeting of the relapse-causing LSPCs that is achieved by the activation of DNA damage and stress response pathways leading to G2/M cell cycle arrest. We are currently assessing the efficacy of Aza in in vivo animal models and investigating any dysregulated methylation patterns in Chk2 and p38MAPK in AML cells. Disclosures:No relevant conflicts of interest to declare.