Cytosine Methyltransferase Research Articles

Regulation of the DNA‐binding protein CFP1 by ERK1/2

CFP1 (CXXC finger protein 1) was identified in our lab as an ERK2 interactor through a yeast two‐hybrid screen. CFP1 binds to the maintenance cytosine methyltransferase DNMT1 and is required for DNA methylation in an embryonic model. Methylation of DNA in promoter regions is critical for transcriptional silencing throughout development and is often dysregulated in cancer. ERK2 is required for KRAS‐driven aberrant gene silencing via promoter methylation by DNMT1, but ERK2 substrates that may mediate this effect have not been identified. I propose that CFP1 is the major ERK2 substrate that directs DNA methylation by promoting the targeting or activity of DNMT1. ERK2‐bound mononucleosomes are enriched for CFP1. Active ERK2 can phosphorylate CFP1 on multiple sites in vitro. Current work is focused on determining the requirement for CFP1 in KRAS‐driven gene silencing, and dissecting the relationship between promoter methylation and the presence of active ERK2 at DNA methylation sites guided by ERK2 ChIP‐sequencing data from a lung cancer model. If ERK1/2 targeting of CFP1 helps to drive directed DNMT1 activity and tumor suppressor gene silencing, it would cement a clear mechanistic linkage between a common activating mutation in cancer and conserved downstream epigenetic effects that promote development of the disease.This work was supported by NIGMS Training Grant T32 GM008203 and by NIH R37 DK34128.

Characterization of Undermethylated Sites in Vibrio cholerae

The activities of DNA methyltransferases are important for a variety of cellular functions in bacteria. In this study, we developed a modified high-throughput technique called methyl homopolymer tail mediated sequencing (methyl HTM-seq) to identify the undermethylated sites in the Vibrio cholerae genome for the two DNA methyltransferases, Dam, an adenine methyltransferase, and VchM, a cytosine methyltransferase, during growth in rich medium in vitro. Many of the undermethylated sites occurred in intergenic regions, and for most of these sites, we identified the transcription factors responsible for undermethylation. This confirmed the presence of previously hypothesized DNA-protein interactions for these transcription factors and provided insight into the biological state of these cells during growth in vitro. DNA adenine methylation has previously been shown to mediate heritable epigenetic switches in gene regulation. However, none of the undermethylated Dam sites tested showed evidence of regulation by this mechanism. This study is the first to identify undermethylated adenines and cytosines genomewide in a bacterium using second-generation sequencing technology.

Methylating the DNA of the Most Repressed: Special Access Required

A new study (Zemach etal., 2013) suggests that the chromatin remodeling ATPase DDM1 is specifically required for cytosine methylation at linker histone H1-associated heterochromatin, facilitating access by three cytosine methyltransferases, including a previously uncharacterized CHH methylase, CMT2.

Influence of photoperiod on expression of DNA (cytosine-5) methyltransferases in Atlantic cod

Photoperiod manipulation during early juvenile stages can influence growth in Atlantic cod. In the present study, one group of cod juveniles were reared under natural photoperiod conditions for Bodø (67° N, 14° E), whereas their counterparts were kept under continuous illumination. The mean weight of juvenile cod reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod after 120days of light treatment. The molecular basis of this phenotypic plasticity is currently unknown but it is likely that DNA (cytosine-5)-methyltransferases (dnmts) are involved, since these genes play a crucial role in epigenetic regulation of gene expression. Phylogenetic analysis of Atlantic cod dnmt1, dnmt2 and dnmt3a revealed that within each group, the phylogeny follows the taxonomic relationship between the various species and comparative mapping of dnmt paralogues showed that these genes lie within regions of conserved synteny amongst teleosts. Of the three dnmt paralogues, dnmt3a had the highest expression in fast muscle of adult cod. In addition, dnmt1 and dnmt2 were differentially expressed between tissues but with prominent expression in gonads. Dnmt1 and dnmt3a transcript levels showed a significant increase in fast muscle of juvenile cod from the continuous light group at several time points. Remarkably, dnmt1 and dnmt3a transcript levels were 2-fold higher at 120days, by which point photoperiod conditions between the two light groups had become identical. Our data revealed that photoperiod can have an extended effect on expression of dnmt genes, which may be involved in the epigenetic regulation of muscle growth by photoperiod in Atlantic cod.

Distal Structural Elements Coordinate a Conserved Base Flipping Network

One of the most dramatic illustrations of enzymatic promotion of a high-energy intermediate is observed in DNA modification and repair enzymes where an individual base is rotated (flipped) 180° around the deoxyribose-phosphate backbone and into the active site. While the end states have been extensively characterized, experimental techniques have yet to yield a full description of the base flipping process and the role played by the enzyme. The C5 cytosine methyltransferase M.HhaI coordinates an ensemble of reciprocal DNA and enzyme rearrangements to efficiently flip the target cytosine from the DNA helix. We sought to understand the role of individual amino acids during base flipping. Our results demonstrate that M.HhaI initiates base flipping before closure of the catalytic loop and utilizes the conserved serine 85 in the catalytic loop to accelerate flipping and maintain distortion of the DNA backbone. Serine 87, which forms specific contacts within the DNA helix after base flipping, is not involved in the flipping process or in maintaining the catalytically competent complex. At the base of the catalytic loop, glycine 98 acts as a hinge to allow conformational dynamism of the loop and mutation to alanine inhibits stabilization of the closed loop. Our results illustrate how an enzyme utilizes numerous, distal residues in concert to transform substrate recognition into catalysis.

Structural Origins of DNA Target Selection and Nucleobase Extrusion by a DNA Cytosine Methyltransferase

How DNA 5-cytosine methyltransferases (DCMTases) select their substrate nucleobase for extrusion from DNA duplex is poorly understood. The crystal structure of a pre-extrusion M.HaeIII DCMTase-substrate DNA complex is reported here. M.HaeIII selects its substrate cytosine for extrusion by selectively interfering with its stacking and hydrogen bonding interactions within the DNA duplex. This is the first structural elucidation of the target cytosine selection by a DCMTase. Epigenetic methylation of cytosine residues in DNA is an essential element of genome maintenance and function in organisms ranging from bacteria to humans. DNA 5-cytosine methyltransferase enzymes (DCMTases) catalyze cytosine methylation via reaction intermediates in which the DNA is drastically remodeled, with the target cytosine residue extruded from the DNA helix and plunged into the active site pocket of the enzyme. We have determined a crystal structure of M.HaeIII DCMTase in complex with its DNA substrate at a previously unobserved state, prior to extrusion of the target cytosine and frameshifting of the DNA recognition sequence. The structure reveals that M.HaeIII selects the target cytosine and destabilizes its base-pairing through a precise, focused, and coordinated assault on the duplex DNA, which isolates the target cytosine from its nearest neighbors and thereby facilitates its extrusion from DNA.

Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe, mediates tRNA methylation in response to nutrient signaling

The fission yeast Schizosaccharomyces pombe carries a cytosine 5-methyltransferase homolog of the Dnmt2 family (termed pombe methyltransferase 1, Pmt1), but contains no detectable DNA methylation. Here, we found that Pmt1, like other Dnmt2 homologs, has in vitro methylation activity on cytosine 38 of tRNAAsp and, to a lesser extent, of tRNAGlu, despite the fact that it contains a non-consensus residue in catalytic motif IV as compared with its homologs. In vivo tRNA methylation also required Pmt1. Unexpectedly, however, its in vivo activity showed a strong dependence on the nutritional status of the cell because Pmt1-dependent tRNA methylation was induced in cells grown in the presence of peptone or with glutamate as a nitrogen source. Furthermore, this induction required the serine/threonine kinase Sck2, but not the kinases Sck1, Pka1 or Tor1 and was independent of glucose signaling. Taken together, this work reveals a novel connection between nutrient signaling and tRNA methylation that thus may link tRNA methylation to processes downstream of nutrient signaling like ribosome biogenesis and translation initiation.

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA–M.HhaI–AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA.

Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease

Objective & designNonalcoholic fatty liver disease (NAFLD) is a clinical condition that refers to progressive histological changes ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH). We evaluated the status...

Comparative Transcriptomics of H. pylori Strains AM5, SS1 and Their hpyAVIBM Deletion Mutants: Possible Roles of Cytosine Methylation

Helicobacter pylori is an important human pathogen and one of the most successful chronic colonizers of the human body. H. pylori uses diverse mechanisms to modulate its interaction with the host in order to promote chronic infection and overcome host immune response. Restriction-modification genes are a major part of strain-specific genes present in H. pylori. The role of N6 - adenine methylation in bacterial gene regulation and virulence is well established but not much is known about the effect of C5 -cytosine methylation on gene expression in prokaryotes. In this study, it was observed by microarray analysis and RT-PCR, that deletion of an orphan C5 -cytosine methyltransferase, hpyAVIBM in H. pylori strains AM5and SS1 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. AM5ΔhpyAVIBM mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. hpyAVIBM from strain 26695 is able to complement mutant SS1 and AM5 strains. This study highlights a possible significance of cytosine methylation in the physiology of H. pylori.

Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

Differential expression and cellular localization of novel isoforms of the tendon biomarker tenomodulin

Tenomodulin (Tnmd, also called Tendin) is classified as a type II transmembrane glycoprotein and is highly expressed in developing as well as in mature tendons. Along with scleraxis (scx), Tnmd is a candidate marker gene for tenocytes. Its function is unknown, but it has been reported to have anti-angiogenic properties. Results in a knockout mouse model did not substantiate that claim. It has homology to chondromodulin-I. Single nucleotide polymorphisms of TNMD have been associated with obesity, macular degeneration, and Alzheimer's disease in patients. In the present study, three Tnmd isoforms with deduced molecular weights of 20.3 (isoform II), 25.4 (isoform III), and 37.1 (isoform I) kDa were proposed and verified by Western blot from cells with green fluorescent protein-linked, overexpressed constructs, tissue, and by qPCR of isoforms from human tissues and cultured cells. Overexpression of each Tnmd isoform followed by immunofluorescence imaging showed that isoforms I and II had perinuclear localization while isoform III was cytoplasmic. Results of qPCR demonstrated differential expression of each Tnmd isoform in patient's specimens taken from flexor carpi radialis, biceps brachii, and flexor digitorum profundus tendons. Knockdown of Tnmd increased the expression of both scleraxis (scx) and myostatin, indicating a potential negative feedback loop between Tnmd and its regulators. Knockdown of all Tnmd isoforms simultaneously also reduced tenocyte proliferation. I-TASSER protein three-dimensional conformation modeling predictions indicated each Tnmd isoform had different structures and potential functions: isoform 1, modeled as a cytosine methyltransferase; isoform 2, a SUMO-1-like SENP-1 protease; and isoform 3, an α-syntrophin, plextrin homology domain scaffolding protein. Further functional studies with each Tnmd isoform may help us to better understand regulation of tenocyte proliferation, tendon development, response to injury and strain, as well as mechanisms in tendinoses. These results may indicate novel therapeutic targets in specific tenomodulin isoforms as well as treatments for tendon diseases.

Development of rationally designed DNA N6 adenine methyltransferase inhibitors

A series of bisubstrate inhibitors for DNA N6 adenine methyltransferase (Dam) have been synthesized by linking an amine analogue of S-adenosylmethionine to an aryl moiety designed to probe the binding pocket of the DNA adenine base. An initial structure–activity relationship study has identified substituents that increase inhibitor potency to the ∼10μM range and improve selectivity against the human cytosine methyltransferase Dnmt1.

Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

ABSTRACTThe strong restriction barrier present in Staphylococcus aureus and Staphylococcus epidermidis has limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain of S. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiency Escherichia coli cloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates of S. aureus and S. epidermidis. We also show that the loss of restriction (both type I and IV) in an S. aureus USA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.IMPORTANCE Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

Mutations in hpyAVIBM, C5 cytosine DNA methyltransferase from Helicobacter pylori result in relaxed specificity

The genome of Helicobacter pylori is rich in restriction-modification (RM) systems. Approximately 4% of the genome codes for components of RM systems. hpyAVIBM, which codes for a phase-variable C(5) cytosine methyltransferase (MTase) from H. pylori, lacks a cognate restriction enzyme. Over-expression of M.HpyAVIB in Escherichia coli enhances the rate of mutations. However, when the catalytically inactive F9N or C82W mutants of M.HpyAVIB were expressed in E. coli, mutations were not observed. The M.HpyAVIB gene itself was mutated to give rise to different variants of the MTase. M.HpyAVIB variants were purified and differences in kinetic properties and specificity were observed. Intriguingly, purified MTase variants showed relaxed substrate specificity. Homologues of hpyAVIBM homologues amplified and sequenced from different clinical isolates showed similar variations in sequence. Thus, hpyAVIBM presents an interesting example of allelic variations in H. pylori where changes in the nucleotide sequence result in proteins with new properties.

Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli

In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5'CCWGG3'. Although the presence of cytosine DNA methylation was reported over 35years ago, the biological role of 5-methylcytosine in E.coli remains unclear. To gain insight into the role of cytosine DNA methylation in E.coli, we (1) screened the 72 strains of the ECOR collection and 90 recently isolated environmental samples for the presence of the full-length dcm gene using the polymerase chain reaction; (2) examined the same strains for the presence of 5-methylcytosine at 5'CCWGG3' sites using a restriction enzyme isoschizomer digestion assay; and (3) quantified the levels of 5-methyl-2'-deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm-mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5-methylcytosine ranges from 0.86% to 1.30% of the cytosines. We also demonstrate that Dcm reduces the expression of ribosomal protein genes during stationary phase, and this may explain the highly conserved nature of this DNA modification pathway.

The Lytic Phase of Epstein-Barr Virus Requires a Viral Genome with 5-Methylcytosine Residues in CpG Sites

Epstein-Barr virus (EBV) is a human herpesvirus which has been studied intensively for its role in certain human tumors. It also serves as a model of herpesviral latency because it establishes an immediate, latent infection in human B cells. When EBV infects quiescent, primary B cells it induces their continuous proliferation to yield growth-transformed B-cell lines in vitro. The lytic or productive phase of EBV's life cycle is induced by the expression of the viral BZLF1 gene in latently infected cells. The BZLF1 protein is a transactivator, which selectively binds to two classes of distinct DNA sequence motifs. One class is similar to the motifs that are bound by members of the AP-1 transcription factor family to which BZLF1 belongs. The second class, which contains CpG motifs, is predominant in viral promoters of early lytic genes and is BZLF1's preferred or exclusive target sequence when methylated. The BZLF1 gene is transiently expressed in newly infected B cells but fails to induce EBV's lytic cycle, potentially because the virion DNA is unmethylated. Here we report that the lack of 5-methylcytosine residues in CpG sites of virion DNA prevents the expression of essential lytic genes indispensable for viral DNA amplification during productive infection. This finding indicates that BZLF1 transactivates these promoters in a methylation-dependent fashion and explains how progeny virus synthesis is abrogated in newly infected B cells. Our data also reveal that viral lytic DNA synthesis precludes CpG methylation of virion DNA during EBV's lytic, productive cycle, which can be overcome by the ectopic expression of a prokaryotic cytosine methyltransferase to yield CpG-methylated virion DNA. Upon infection of B cells, randomly CpG-methylated virion DNA induces high expression of essential lytic genes in contrast to virion DNA free of 5-methylcytosine residues. Our data suggest that unmethylated virion DNA is part of EBV's strategy to prevent the viral lytic phase in newly infected B cells, allowing it to establish its characteristic latent infection in them.

Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.

Identification of Novel Clostridium perfringens Type E Strains That Carry an Iota Toxin Plasmid with a Functional Enterotoxin Gene

Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ∼65 kb. Complete sequence analysis of the ∼65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ∼65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.

Abstract 2537: Novel 2′-deoxycytidine analogs as DNA demethylation agents

Abstract Epigenetic therapies using cytidine/deoxycytidine analogs are proving to be effective as indicated by the recent approvals of 5-azacytidine (5-azaCyd) and 5-aza-2’-deoxycytidine (5-azadCyd) in myelodysplastic syndromes (MDS) and certain leukemias. Studies suggest that the inhibition of DNA cytosine-5 methylation and the re-expression of silenced tumor suppressors contribute to the beneficial effects of these drugs. However inhibition of DNA synthesis and other toxicities of these compounds represent major drawbacks in the clinic. Recently another analog previously examined in our anticancer discovery program, 4’-thio-2’-deoxycytidine (T-dCyd), had been shown to be an inhibitor of methyl transfer by the M. HhaI DNA cytosine-5 methyltransferase. In the current studies we demonstrate that T-dCyd and its 5-aza analogue 4’-thio-5-aza-2’-deoxycytidine (5-aza-T-dCyd), can deplete human DNMT1 protein in NCI-H23 lung carcinoma and the myeloid leukemia lines THP-1 and KG1a. Consistent with this we also found that T-dCyd and 5-aza-T-dCyd were also effective in decreasing DNMT activities in cancer cells. Methylation-specific PCR (MSP) analysis also demonstrated that both T-dCyd and 5-aza-T-dCyd induced CpG demethylation and re-expression of the tumor suppressor p15 in KG1a cells. We have also found that T-dCyd is inserted into replicating DNA at nanomolar doses as readily if not better than the natural 2’-deoxycytidine (dCyd) and exhibited very little toxicity at these doses. T-dCyd is therefore readily activated to its triphosphate T-dCTP, which is a good substrate for DNA polymerase mediated incorporation, and DNA polymerases also readily extend the chain after incorporation. Furthermore we also observed that T-dCyd and 5-aza-T-dCyd are efficacious in in vivo tumor models. Collectively our data suggest that these compounds or their analogs could be developed as novel DNA methylation inhibitors with better properties for cancer therapy. 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 2537. doi:10.1158/1538-7445.AM2011-2537