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New horizons for diagnostic applications of circulating nucleosomes in blood?

In 1974, Kornberg proposed a model of the organization of human chromatin as a nucleosomel chain formed by repeating histone-DNA sequences (1 ). Subsequent x-ray crystallographic analyses have confirmed his electron microscopy findings as advances in methodology and crystallographic resolution have revealed more and more details of the structure of nucleosomel core particles (2). Today, it is clear that nucleosomes consist of a central protein core of the doubly represented histones H2A-H2B and H3-H4 plus 147 by of double-stranded DNA twisted around this complex (2, 3). Another histone, H1, is located outside the nucleosomes at the so-called linker DNA, which connects the various 206-kDa disk-like nucleosomes. This histone stabilizes the chain in its tertiary structure as chromatin fiber (2, 3).

Beyond the essential role of DNA packaging and stabilization, the arrangement of chromatin into multinucleosomal order has other important functions, particularly in regulating the transcription, replication, and repair of DNA. Transcription factors and polymerases gain access to specific DNA sequences only if they are released from their close association with the histone core (3, 4). The flexible and dynamic structure of the nucleosomel organization is facilitated by ATP-dependent chromatin-remodeling complexes that cause uncoupling of the close DNA-histone connection, the transfer of a histone octamer to another DNA molecule, or the core particle to slide along the DNA (3, 4). As more details of these processes have been elucidated, it has become increasingly evident that their coordination is quite complex and can be easily disturbed by various diseases.

Recent findings have revealed that in addition to genetic mutations, such epigenetic modifications as DNA methylation, histone modifications, and the pro duction of noncoding micro-RNAs are key features in the regulation of gene transcription, replication, and DNA repair (5, 6). Those epigenetic patterns are regarded as heritable markers that ensure accurate transmission of gene expression profiles and chromatin states over many cell generations. Of particular interest is the cross talk between the epigenetic components involved in transcription processes. Although many molecular mechanisms are well known to lead to gene activation and silencing, the hierarchical order and dependencies of these mechanisms, as well as the role of interplay between chromatin components of higher-order structure, have yet to be elucidated (4-7).

During the last decade, extensive efforts have been undertaken to map the human DNA-methylation profile and to identify characteristic associations with various diseases. Although 50%-70% of cytosine bases in CpG dinucleotides are methylated in healthy humans, CpG-rich regions near the promoters of activated genes are hypomethylated. In contrast, the DNA in many cancer cells features a global genomic hypomethylation and specific hypermethylation of so-called CpG islands in promoter regions (5, 6). These changes are often already present at early stages of carcinogenesis and lead to the silencing of relevant tumor suppressor genes that are involved in cell cycle regulation [GDKN2A [3] /[p16.sup.TNx4a]) CDKN2B ([p15.sup.INx4b]) RB1 (Rb)], apoptosis [DAPK1, WIF1, PYCARD (TMS1)], WNT signaling (APC, DKK1, IGFBP3), growth signal autonomy (RASSFIA, SOCS1), hormone response (ESR1, PGR, AR), angiogenesis [THBS1 (thrombospondin)], cell adherence and invasion [CDH1 (E-cadherin), CDH13], detoxification (GSTP1), and DNA repair (MLH1, MGMT) (5, 6). Many examples of CpG hypermethylation in gene promoters have been identified, characterized, and mapped for several cancers (8).

Posttranslational histone modifications include the addition of small acetyl, methyl, or phosphoryl groups and the attachment of larger moieties, such as poly(ADP-ribose) and small proteins [e.g., ubiquitin or small ubiquitin-like modifier (SUMO)]. These modifications occur at amino acid residues, mainly lysine or arginine, of the N-terminal ends of histones (tails) that protrude from the nucleosome core (5, 6, 9). A set of enzymes, such as histone acetylases, histone deacetylases (HDACs), [4] histone methyltransferases, and histone demethylases, regulates these reversible processes (5, 6, 9). The variety and combinatorial nature of histone modifications have led to the development of a unique nomenclature for facilitating our understanding of the comprehensive "histone code" (9).

Although the deciphering of the histone language is still in its infancy, certain characteristic features of transcriptional status have been elucidated. Methylated cytosines of silenced promoters bind to methyl-CpG-binding domain proteins (MBDs) in a complex that also includes HDACs, which remove acetyl groups from histones and transform the heterochromatin into a closed, transcriptionally inactive status (5, 6). Conversely, histone acetylases add acetyl groups to histone tails at nonmethylated promoters. This process opens the chromatin structure, facilitating transcription by allowing promoter binding to transcription factors and coactivator proteins (5, 6). Methylation of histones often leads to gene silencing; however, such findings are not universal. For example, although trimethylation of lysine 9 and lysine 27 on histone 3 (H3K9me3 and H3K27me3) has inactivating properties, trimethylation oflysine 4 on histone 3 (H3K4me3) activates gene expression (5, 6). In human cancer cells, the loss of monoacetylated lysine 16 and trimethylated lysine 20 on histone 4 (H4K16ac and H4K20me3) was found to be a common hallmark of neoplastic disease (5, 10). Dimethylated lysine 4 on histone 3 (H3K4me2) is a prognostic indicator of a favorable outcome for patients with resected early-stage lung cancer, whereas acetylated lysine 9 on histone 3 (H3K9ac) has been associated with poor survival prospects (11 ). As genomewide screening and the mapping of histone modifications progress, the diagnostic and prognostic relevance of panels of histone and DNA markers will become more clear so that patients can be stratified for specific therapies. This approach becomes all the more interesting as HDAC inhibitors and DNA-demethylating agents are developed as new anticancer therapies (5, 6).

For the use of biomarkers in routine clinical practice, particularly in serial measurements for therapymonitoring purposes, the ability to follow such changes in easily available bodily fluids such as blood and urine would be desirable. Nucleosomes are released by apoptotic and necrotic cells or are secreted actively into the circulation; therefore, they are detected in the blood in increased amounts during cancer and in other acute diseases [reviewed in (12)]. Because of the nonspecific nature of nucleosome release, measurements of the nucleosome concentration are minimally helpful in diagnosing cancer, but they have been shown to have some prognostic value. Importantly, nucleosome measurements made during cytotoxic therapies have revealed characteristic patterns and have identified nonresponses with high diagnostic accuracy (12).

Analyses of qualitative alterations in circulating DNA, such as specific mutations, loss of heterozygosity, microsatellite instabilities, and epigenetic modifications, have been performed to improve the tumor specificity of these markers (5, 6, 8). Although tumor DNA is known to be quite rare in blood, the use of sensitive amplification techniques has facilitated the detection of such changes, such as tumor-derived methylated DNA in the sera of affected patients (13). CpG island methylation of certain genes in the blood of patients with breast and colorectal cancers has recently been shown to be correlated with prognosis and has been identified as an independent predictor of survival (13, 14). Moreover, analysis of DNA methylation in serum may also serve to monitor the efficacy of therapy.

In this issue of Clinical Chemistry, Deligezer and colleagues report the detection of histone modifications on circulating nucleosomes (15). These authors immunoprecipitated monomethylated lysine 9 residues on histone 3 (H3K9me1) of nucleosomes from plasma samples of 21 patients with multiple myeloma and amplified sequences of DNA isolated from these DNA-histone complexes. In line with earlier findings that H3K9me1 is enriched in nonspecific repetitive ALU sequences, this modification was detected on ALU115 fragments, which cover all kinds of mono-and oligonucleosomes, in 81 % of the samples, whereas it was present on oligonucleosomal ALU247 fragments in only 48% of the samples. The authors explained this observation as indicating the predominance of mono-nucleosomal fragments in plasma. On the tumor-associated sequence of the CDKN2A promoter, H3K9me1 was detected in 33% of the samples and in lower concentrations than in ALU115 fragments. The authors deduced a sequence specificity for this marker that was independent of the total amount of circulating nucleosomes or DNA (15).

Although the presence of posttranslational histone modifications in circulating nucleosomes was expected, combining chromatin immunoprecipitation and real-time PCR (as standard methods for measuring histone and DNA) to detect such histone markers in the plasma of cancer patients is a promising new approach toward closing the gap between basic research and clinical application. The absence of a correlation between H3K9me1 and the amounts of circulating nucleosomes or DNA is not surprising and may enhance the appeal of circulating nucleic acids as markers of tumor specificity. Unfortunately, comparisons of cancer patients with healthy control individuals and patients with different diagnostically relevant benign diseases were not included in this study. Nevertheless, the study may still serve as a proof of principle that may stimulate future analyses of histone markers as potentially new tools in cancer diagnostics.

Many questions remain unresolved with this "door-opener" publication. For example, which histone sites and what kinds of modifications (e.g., single, double, or triple methylations, acetylation, or phosphorylation) will be the most relevant? Thus, many antibodies remain to be developed and tested. Second, are there characteristic histone markers of cancer at specific gene sequences, or will a more global epigenetic landscape of histone and DNA alterations be the most help in cancer diagnosis and prognosis? The development of sensitive diagnostic methods that facilitate genome-wide histone and DNA screening may be possible (5, 6). Finally, what will be the clinical relevance of histone modifications on plasma nucleosomes? As with the development and validation of other DNA or protein biomarkers, we are a long way from answering this important question in prospective patient studies. In particular, comparisons with existing biomarkers and imaging techniques may reveal histone markers' specific and independent benefit in cancer diagnosis, prognosis, and therapy monitoring.

The early occurrence of histone and DNA modifications during carcinogenesis and the early changes in these epigenetic markers that have been found during cancer treatment with HDAC inhibitors and DNA demethylases (5-7) hint that plasma histone markers maybe powerful diagnostic tools in oncology. Whether "nucleosomics" will become valuable for the management of individual cancer patients remains to be determined.

References

(1.) Komberg R. Chromatin structure: a repeating unit of histones and DNA. Science 1974;184:868-71.

(2.) Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 1997; 389:251-60.

(3.) Khorasanizadeh S. The nucleosome: from genomic organization to genomic regulation. Cell 2004;116:259-72.

(4.) Eberharter A, Ferreira R, Becker P. Dynamic chromatin: concerted nucleosome remodelling and acetylation. Biol Chem 2005;386:745-51.

(5.) Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 2007;8:286-98.

(6.) Gronbaek K, Hother C, Jones PA. Epigenetic changes in cancer. APMIS 2007;115:1039-59.

(7.) Vaissiere T, Sawan C, Herceg Z. Epigenetic interplay between histone modifications and DNA methylation in gene silencing. Mutat Res (Forthcoming zoos>.

(8.) Esteller M. Epigenetics in cancer. N Engl J Med 2008;358:1148-59.

(9.) Turner BM. Reading signals on the nucleosome with a new nomenclature for modified histones. Nat Struct Mol Biol 2005;12:110-2.

(10.) Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 2005;37:391-400.

(11.) Barlesi F, Giaccone G, Gallegos-Ruiz MI, Loundou A, Span SW, Lefesvre P, et al. Global histone modifications predict prognosis of resected non small-cell lung cancer. J Clin Oncol 2007;25:4358-64.

(12.) Holdenrieder S, Nagel D, Schalhom A, Heinemann V, Wilkowski R, v Pawel J, et al. Clinical relevance of circulating nucleosomes in cancer disease. Ann N Y Acad Sci (Forthcoming 2008).

(13.) Wallner M, Herbst A, Behrens A, Crispin A, Stieber P, Gbke B, et al. Methylation of serum DNA is an independent prognostic marker in colorectal cancer. Clin Cancer Res 2006;12:7347-52.

(14.) Muller HM, Widschwendter A, Fiegl H, Ivarsson L, Goebel G, Perkmann E, et al. DNA methylation in serum of breast cancer patients: an independent prognostic marker. Cancer Res 2003;63:7641-5.

(15.) Deligezer U, Akisik EE, Erten N, Dalay N. Sequence-specific histone methylation is detectable on circulating nucleosomes in plasma. Clin Chem 2008;54:1125-31.

[3] Human genes: CDKN2A, cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4); CDKN28, cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4); R81, retinoblastoma 1 (including osteosarcoma); DAPK1, death-associated protein kinase 1; WIF1, WNT inhibitory factor 1; PYCARD, PYD and CARD domain containing; APC, adenomatous polyposis coli; DKK1, dickkopf homolog 1 (Xenopus laevis); IGFRP3, insulin-like growth factor binding protein 3; RASSF1A, Ras association (RaIGDS/AF-6) domain family member 1A; SOC51, suppressor of cytokine signaling 1; ESR1, estrogen receptor 1; PGR, progesterone receptor; AR, androgen receptor; TH851, thrombospondin 1; CDH1, cadherin 1, type 1, E-cadherin (epithelial); CDH13, cadherin 13, H-cadherin (heart); GSTP1, glutathione S-transferase pi; MLH1, mutt homolog 1, colon cancer, nonpolyposis type 2 (E. coli); MGMT, 0-6-methylguanine-DNA methyltransferase.

[4] Nonstandard abbreviations: HDAC, histone deacetylase; H3K9me, trimethylation of lysine 9 on histone 3.

DOI: 10.1373/clinchem.2008.108688

--Stefan Holdenrieder [1] * --Frank T. Kolligs [2] --Petra Stieber [3]

[1] Institute of Clinical Chemistry University Hospital Munich-Grosshadern Munich, Germany

[2] Medical Clinic II University Hospital Munich-Grosshadern Munich, Germany

* Address correspondence to this author at:

Institute of Clinical Chemistry

University Hospital Munich-Grosshadern

Marchioninstrasse 15

D-81366 Munich, Germany

Fax 0049-89-7095-6298

E-mail Stefan.Holdenrieder@med.uni-muenchen.de
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Title Annotation:Editorial
Author:Holdenrieder, Stefan; Kolligs, Frank T.; Stieber, Petra
Publication:Clinical Chemistry
Date:Jul 1, 2008
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