80 EFFECT OF DNA METHYLATION ON SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT IN RHESUS MONKEY

2006 ◽  
Vol 18 (2) ◽  
pp. 148
Author(s):  
J. F. Yang ◽  
S. H. Yang ◽  
Y. Y. Niu ◽  
Q. Zhou ◽  
W. Z. Ji
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Methylation Pattern ◽  
Serum Starvation ◽  
Global Dna Methylation ◽  
Blastocyst Development ◽  
Donor Cells ◽  
Morula Stage ◽  
Asymmetric Methylation

Up to now, no primate animals have been successfully cloned with somatic cell nuclear transfer (SCNT) and little is known about molecular events occurring in SCNT embryos. DNA methylation reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Epigenetic characteristics of donor cell nuclei and their epigenetic reprogramming in oocyte cytoplasm have been supposed as major factors influencing the development of SCNT embryos. In Experiment 1, on donor cells used in a previous SCNT at our laboratory, global DNA methylation and histone 3 lysine 9 acetylation (H3K9ac) of three cell lines (S11, S1-04, and S1-03) derived from ear skin were examined after serum starvation by immunofluorescence with monoclonal antibody to 5-methyl cytosine (Oncogene, Science, Inc., Cambridge, MA, USA) and anti-acetyl-Histone H3 (Lys 9) (Upstate Jingmei Biotech, Ltd., Shenzhen, China). In the results, two cells lines, S11 and S1-04, supporting higher blastocyst development (about 20%) than that (7.8%) of S1-03, showed a higher level of H3K9ac than the S1-03 cell line. Global DNA methylation levels in the three cell lines were decreased after serum starvation, but no obvious correlation between the level and SCNT embryo developmental potential was found among the three cell lines. In Experiment 2, on SCNT and IVF embryos, global DNA methylation reprogramming during pre-implantation development was investigated with immunofluorescence and laser scanning microscopy techniques. In IVF embryos, active demethylation of paternal genome occurred soon after fertilization; subsequently, passive demethylation resulted in remarkably reduced global methylation level at the 8-cell stage and the morula stage. Thereafter, genomewide remethylation started at the late morula stage and an asymmetric methylation pattern was formed in blastocysts, with higher methylated trophectoderm than inner cell mass (ICM). Compared with IVF embryos, most SCNT 2-cell embryos and ICM in blastocysts showed higher methylation levels, and the asymmetric methylation pattern was not as evident as that in IVF blastocysts. Some SCNT 8-cell embryos showed higher methylation, but others were slightly stained, even lower than IVF embryos. In conclusion, the higher global H3K9 acetylation level of donor cells may benefit chromatin remolding and development of SCNT embryos. Abnormal methylation reprogramming in most SCNT embryos, especially in ICM of blastocysts, may be one main obstacle for primate cloning, although relatively high blastocyst development rate was obtained. DNA methylation reprogramming in rhesus monkey pre-implantation embryos, on the whole, was as conservative as that reported in other mammals.

Cellular reprogramming of adult goat fibroblast: Toward pluripotency

2016 ◽  
Author(s):  
Dharmendra Kumar ◽  
Rakesh Ranjan ◽  
Ajit P. Singh ◽  
Bikash C Sarkhel
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Cellular Reprogramming ◽  
Serum Starvation ◽  
Methyl Transferase ◽  
Donor Cells ◽  
Key Factor ◽  
Gene Transcripts

Cellular reprogramming erases the epigenetic constraints of somatic cells genome and thus considered as key factor for success of somatic cell nuclear transfer technology. To achieve the reprogramming, different strategies are used which are mostly based on arresting the cell cycle at G0 or G1 stage. The present study was based on molecular investigation of reprogrammed cells for expression of pluripotent genes that are crucial for development of cloned embryos. The fibroblast cell lines were treated by four methods to induce cellular reprogramming viz., serum starvation, Roscovitin, aphidicolin and overconfluent. These treated cell lines were used for quantification of pluripotent gene transcripts by using real time PCR machine. The results showed that the relative expression of different pluripotent genes as Oct-4 and Nanog along with DNA methyl transferase gene (Dnmt-1) was observed in four treated cells. In case of normal cells, only Dnmt-1 gene was expressed, but pluripotent genes were not expressed at detection level. The expression of pluripotent genes in the donor cells prior to nuclear transfer have significant impact on cloning as because it facilitates the expression of that gene in the resulting embryo after nuclear transfer. The finding of this study may be extended for stem cell generation as it showed that pluripotent genes could be induced in the somatic cells without any transgenic incorporation.

scholarly journals 33DEVELOPMENT OF BOVINE EMBRYOS CLONED WITH FETAL FIBROBLASTS ARRESTED AT G0/G1 PHASE BY DIFFERENT TREATMENTS

2004 ◽  
Vol 16 (2) ◽  
pp. 139
Author(s):  
S.R. Cho ◽  
W.J. Son ◽  
C.S. Park ◽  
S.Y. Choe ◽  
G.J. Rho
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Serum Starvation ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Group 2 ◽  
Group 1

Numerous factors have an effect on the development of cloned embryos, and one of the most important might be the synchronization between donor nuclei and recipient ooplasts. The objective of this study was to examine the effect of donor cell treatments for G0/G1 synchronization and the donor cell type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on Day 50 of gestation and adult ear skin biopsies. The cells were used for assessements of cell cycle and apoptosis, and for nuclear transfer. Cells were randomly allocated into 3 experimental treatment groups after 6–8 passages: Group 1 (confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent; Group 2 (serum-starvation), cells were cultured in DMEM supplemented with 0.5% FBS for 5 days; Group 3 (Roscovitine), cells were cultured in DMEM supplemented with 10% FBS and 30μM Roscovitine for 12h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1, respectively. At 19h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6kV/cm, 60μs) delivered by a BTX 200. After activation with the combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1, 5h), the eggs were cultured in CR1aa medium for 3 days and additionally cultured in CR1aa medium supplemented with 30mgmL−1 BSA for 5 days at 39°C in a humidified atmosphere of 5% CO2 in air. Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. There were no significantly differences in the incidence of cells arrested at G0/G1 for fetal fibroblasts cultured in the three treatment groups (87%, 83% and 80%; confluent, serum starvation and Roscovitine, respectively). More cells were apoptotic in Group 2 compared to the cells in Groups 1 and 3 (12% v. 6 and 6%, respectively) (P<0.05). Blastocyst development of cloned embryos was significantly (P<0.05) higher when fetal fibroblasts from Group 1 were used, compared to Groups 2 and 3 (35.1%, v. 31 and 29.7%, respectively). Similar results were observed in the use of ear skin fibroblasts as nuclear transfer donor cells (32.7%, v. 24 and 24%, respectively). These results suggest that fetal fibroblasts can be effectively synchronized at G0/G1 by three different treatments, including growth to confluence, serum-starvation and Roscovitine treatment. However, based on blastocyst development and levels of apoptosis, the use of confluent fetal fibroblasts as donor cells is more effective than using cells synchronized by serum-starvation or Roscovitine treatment in the production of cloned bovine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 30012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

Methylation characteristics and developmental potential of Guangxi Bama minipig (Sus scrofa domestica) cloned embryos from donor cells treated with trichostatin A and 5-aza-2′-deoxycytidine

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 178-186 ◽  
Author(s):  
Shu-Fang Ning ◽  
Qing-Yang Li ◽  
Ming-Ming Liang ◽  
Xiao-Gan Yang ◽  
Hui-Yan Xu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Formation Rate ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Donor Cells ◽  
Aberrant Dna Methylation ◽  
Methylation Patterns ◽  
Sus Scrofa Domestica

SummaryReprogramming of DNA methylation in somatic cell nuclear transfer (SCNT) embryos is incomplete, and aberrant DNA methylation patterns are related to the inefficiency of SCNT. To facilitate nuclear reprogramming, this study investigated the effect of treating Guangxi Bama minipig donor cells with trichostatin A (TSA), 5-aza-2′-deoxycytine (5-aza-dC), or combination of TSA and 5-aza-dC prior to nuclear transfer. Analyses showed that there were no major changes in cell-cycle status among all groups. We monitored the transcription of DNMT1, DNMT3a, HDAC1 and IGF2 genes in donor cells. Transcription levels of HDAC1 were decreased significantly after treatment with a combination of TSA and 5-aza-dC, along with a significantly increased level of IGF2 (P < 0.05). Although treatment of donor cells with either TSA or 5-aza-dC alone resulted in non-significant effects in blastocyst formation rate and DNA methylation levels, a combination of TSA and 5-aza-dC significantly improved the development rates of minipig SCNT embryos to blastocyst (25.6% vs. 16.0%, P < 0.05). This change was accompanied by decreased levels of DNA methylation in somatic cells and blastocyst (P < 0.05). Thus in combination with TSA, lower concentrations of 5-aza-dC may produce a potent demethylating activity, and lead to the significantly enhanced blastocyst development percentage of Bama minipig SCNT embryos.

40 TREATMENT OF DONOR CELLS AND ITS EFFECT ON INTERSPECIES NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 138
Author(s):  
M. A. Hashem ◽  
D. P. Bhandari ◽  
S. K. Kang ◽  
B. C. Lee
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Development Rate ◽  
Serum Starvation ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Significant Difference ◽  
Morula Stage

The present study was undertaken to examine the effect of donor cells, under a variety of treatment effects, on the development of goral porcine reconstructed embryos. Three experiments were performed, each with a one-way completely randomized design involving 3 to 4 replicates of all. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved (SS), and fully confluent stages of goral cells when reconstructed with porcine enucleated oocytes. In Experiment II, the effects of 2 antioxidants, β-mercaptoethanol (β-ME, 10 �M) and cysteine (2 mM), were examined after cells were fully confluent without serum starvation for 4 h. In Experiment III, the effect of different levels of dimethylsulfoxide (DMSO) at 0%, 0.5%, and 1.0% were tested, after 4 h of treatment, on the development rate after reconstruction with enucleated porcine oocytes. From the results, it appears that there were no significant (P &gt; 0.05) differences from cleavage to morula among cyclic, SS, and fully confluent stages of the cell cycle. None of the treated group reached the blastocyst stage. There were no significant differences at the fused, 2- to 4-cell, and morula stages of embryo development after treatment of the donor cells with β-ME and cysteine before nuclear transfer. However, in the case of 8- to 16-cell stages, there were significant differences between β-ME and cysteine; the donor cells treated with β-ME had a better development rate than those treated with cysteine. No significant differences were observed in fusion, 2- to 4-cell, 8- to 16-cell, blastocyst, and hatching blastocyst stages at the 0.0, 0.5, and 1.0% levels of DMSO. However, there were statistically significant (P &lt; 0.05) differences observed at the morula stage of embryo development. When donor cells were treated for 4 h with 0.5 and 1.0% levels of DMSO, goral-porcine reconstructed embryos reached the morula stage. From the results it can be concluded that goral somatic cells can be de-differentiated in porcine oocytes after treated with antioxidants and DMSO.

62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 139 ◽  
Author(s):  
S. Mitalipov ◽  
Q. Zhou ◽  
J. Byrne ◽  
W.-Z. Ji ◽  
D. Wolf
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Developmental Competence ◽  
Lamin A ◽  
Cell Nuclei ◽  
Blastocyst Development ◽  
Morula Stage

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

28 GENERATION OF REACTIVE OXYGEN SPECIES IN BOVINE CULTURED SOMATIC CELLS AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DURING MICROMANIPULATION PROCEDURES AND EARLY IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 120 ◽  
Author(s):  
H. K. Bae ◽  
J. Y. Kim ◽  
I. S. Hwang ◽  
C. K. Park ◽  
B. K. Yang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Serum Starvation ◽  
Ros Generation ◽  
Oh Radical ◽  
Oxygen Species ◽  
Donor Cells

The present study was conducted to examine the reactive oxygen species (ROS) generation levels in the donor cells, recipient oocytes, and somatic cell nuclear transfer (SCNT) embryos during nuclear transfer procedures. Bovine ear skin cells were classified by serum starvation, confluence, and cycling cells. Bovine metaphase II (MII) oocytes matured in vitro for 22 h and denuded by vortexing were enucleated and electrofused with serum-starved donor cells, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture for 4 h. In vitro fertilization (IVF) was performed for controls. SCNT and IVF embryos were cultured in CR1aa supplemented with 3 mg mL–1 BSA for ∼36 h. Donor cells, recipient oocytes, and SCNT embryos were stained in 10 μM dichlorohydrofluorescein diacetate (DCHFDA) or 10 μM HPF dye each for 30 min at 39°C to measure the H2O2 or ·OH radical levels after various micromanipulation steps. SCNT and IVF embryos were also stained at the 1-, 2-, and 4-cell stages after 8, 24, and 42 h of fusion or insemination, respectively. The fluorescent emissions from the samples were recorded as JPEG file using a digital camera (F5.0, 4 s) attached to a fluorescent microscope with filters at 450 to 480 nm for excitation and at 515 nm for emission. The images were analysed using ImageJ software 1.37 (NIH) by the intensity of fluorescence (pixels) in each cell (total 70 to 75 cells in each group), oocyte and embryo (total 50 to 60 eggs or embryos in each group). 4 to 7 replicates were performed for each experiment, and data were analysed by Duncan′s multiple-range tests. H2O2 and ·OH radical levels of cultured somatic cells were high in confluence group and significantly low in serum starvation group (P < 0.05). During micromanipulation, H2O2 levels in recipient oocytes and SCNT embryos were increased by enucleation (37.2 pixels), electrofusion (49.7 pixels), and activation (40.6 pixels) treatments (P < 0.05) compared to that in MII oocytes (33.1 pixels), and the level of H2O2 was extremely increased immediately after electrofusion. ·OH radical levels were significantly higher during manipulation procedures (51.6 to 55.7 pixels; P < 0.05) compared to MII oocytes. During in vitro culture, the H2O2 and ·OH radical levels of SCNT embryos were significantly higher (P < 0.05) compared to IVF embryos at 1- (32.4 v. 17.3 and 52.0 v. 29.6 pixels, respectively), 2- (27.2 v. 22.0 and 33.4 v. 26.0 pixels, respectively), and 4-cell (25.1 v. 16.5 and 26.9 v. 20.7 pixels, respectively) stages. These results suggest that the culture type of donor cells can affect the ROS generation level and the cellular stress during micromanipulation procedures also can generate the ROS in bovine SCNT embryos, which may lead the cellular damages in bovine SCNT embryos. This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008–313-F00067).

38 EFFECT OF OOCYTE MATURATION DURATION ON BLASTOCYST RATES AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 112 ◽  
Author(s):  
Y. H. Choi ◽  
I. C. Velez ◽  
B. Macías-García ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Direct Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Donor Cells

In equine cloning, the scarcity of equine oocytes places emphasis on development of the most efficient nuclear transfer (NT) methods possible. In other species, using oocytes matured for the shortest duration needed to reach metaphase II has increased NT efficiency. In the present study, we examined the effect of duration of oocyte maturation at the time of enucleation on equine cloned blastocyst production. Oocytes were collected from live mares by transvaginal ultrasound-guided aspiration of all visible follicles ≥5 mm in diameter. The oocytes were held overnight (16–22 h) at room temperature, matured in vitro, and reconstructed with donor cells as described in our previous study (Choi et al. 2013 Theriogenology 79, 791–796). In Experiment 1, oocytes were divided into 2 groups and matured for 20 or 24 h. After enucleation, oocytes were reconstructed by direct injection of donor cells. Reconstructed oocytes were held for 5 h and then activated by treatment with 5 μM ionomycin for 4 min, then injection with sperm extract, followed by incubation in 2 mM 6-DMAP for 4 h. The activated reconstructed oocytes were cultured in global human embryo culture medium under 5% CO2, 6% O2, and 89% N2 at 38.2°C for 7 to 11 days (20 mM glucose was added at Day 5) and blastocyst rate was recorded. Because a low maturation rate was found at 20 h in Experiment 1, in Experiment 2 oocytes were denuded at 20 h and those that were mature were enucleated and used for NT; those that had not cast out a polar body at 20 h were cultured for an additional 3 h (20 + 3h) and then evaluated for polar body formation and used for NT, which was conducted as in Experiment 1. Data were analysed by Fisher's exact test. In Experiment 1, 203 oocytes were collected in 46 aspiration sessions. The rate of oocyte maturation to metaphase II was significantly lower for oocytes cultured for 20 h (35/116, 30%), than for those cultured for 24 h (47/80, 59%). However, the rate of blastocyst development was significantly higher for oocytes cultured for 20 h (11/27, 41%) than for 24 h (2/38, 5%). In Experiment 2, 89 oocytes were collected in 18 aspiration sessions. After 20 h of maturation culture, 22 oocytes were mature (25%). After an additional 3 h of culture, 21 additional oocytes had matured. There were no significant differences between the two treatments (20 and 20 + 3h) in reconstruction rates (77%, 17/22, and 90%, 19/21, respectively) or blastocyst rates (24%, 4/17, and 32%, 6/19, respectively). These results indicate that duration of in vitro maturation, or the duration of presence of cumulus cells, influences blastocyst development after somatic cell NT in the horse. This appears to be due to a benefit of using oocytes immediately after they reach metaphase II; if this is ensured as in Experiment 2, the duration of maturation itself had no effect.This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

Sensitivity of Diffuse Large B-Cell Lymphomas to DNA Methyltransferase Inhibitors Is Associated with a Specific Epigenetic Signature.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 831-831
Author(s):  
Megan Ryan ◽  
Leandro Cerchietti ◽  
Maria E. Figueroa ◽  
John Greally ◽  
Ari Melnick
Keyword(s):  
Dna Methylation ◽  
B Cell ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Serum Starvation ◽  
Gene Promoters ◽  
B Cell Lymphomas ◽  
Response Curves ◽  
Differentially Methylated Genes ◽  
Large B Cell

Abstract DNA methyltransferase inhibitor drugs (MTIs) such as decitabine can overcome gene silencing due to aberrant hypermethylation of gene promoters. Presumably, this effect is responsible for the therapeutic activity of MTIs as clinically demonstrated in myelodysplasias (MDS) and leukemias. Other tumors such as diffuse large B-cell lymphomas (DLBCLs) can also present with aberrant promoter hypermethylation. However, it is currently difficult to prospectively identify patients likely to respond to MTIs, since specific methylation markers or signatures have not yet been identified. We predicted that decitabine would have anti-lymphoma activity in a subset of DLBCLs, and that these cases would exhibit specific methylation signatures predictive of response to these drugs. To determine whether this is the case we first exposed a panel of 7 DLBCL cell lines (Ly1, Ly7, Ly10, SU-DHL6, Farage, Pfeiffer and Toledo) to increasing concentrations of decitabine (0.5, 1, 2.5, 5, 10, 50 and 100 μM) administered after synchronization by 12 hr serum starvation. Viability was assessed after 48 hr of culture by MTS-based assay and Trypan blue exclusion. The IC25 and IC50 were calculated for all cell lines by constructing dose-response curves. The IC25 was used to discriminate sensitive (6.3 ± 1.2 μM) vs. resistant (49.4 ± 5 μM, p &lt; 0.01) cell lines. Interestingly, there was no correlation between MTI sensitivity and DLBCL subtype as defined by recent gene expression profiling classification efforts (i.e. GCB vs. ABC, or BCR vs. OxPhos). To identify the methylation signatures of these DLBCL cells we used a method that we developed for genome-wide DNA methylation quantification called HELP (HpaII tiny fragment Enrichment by LM-PCR). HELP is based on comparative Msp1 and HpaII digestion of genomic DNA, followed by size specific amplification and co-hybridization to custom high-density oligonucleotide arrays designed to provide uniform data collection over 25,000 promoters. HELP compares favorably to other high throughput methods in that it is highly reproducible (R &gt; 0.98) and has an extremely robust signal-to-noise ratio. DNA was collected from the DLBCL cells for HELP prior to drug treatment. Most significantly we found that unsupervised (i.e. unbiased) clustering of DNA methylation profiles could readily segregate decitabine resistant vs. sensitive DLBCL cell lines. Correspondence analysis clearly identified a methylation signature consisting of 133 differentially methylated genes that distinguishes between decitabine sensitive and resistant cells. Most of these appeared to be functionally relevant including such genes as Caspase-9, RARB, JUNB, and ELK1. Biological assays to determine the contribution of these genes to the phenotype are underway. Taken together, our data suggest that MTIs might be effective in a cohort of DLBCL cases that exhibit the specific methylation signature that we have identified. Prospective evaluation of the predictive value of this signature may allow optimal selection of patients for clinical trials with these agents.

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