DNA hypomethylation as Achilles’ heel of tumorigenesis: A working hypothesis

Cell Biology International 33 (2009) 904e910 www.elsevier.com/locate/cellbi

DNA hypomethylation as Achilles’ heel of tumorigenesis: A working hypothesis L.P. Shvachko* Institute of Molecular Biology and Genetics of NAS of Ukraine, Department of Molecular Genetics, 150, Zabolotny Street, Kyiv 03143, Ukraine Received 18 November 2008; accepted 20 February 2009

Abstract There are at least two findings that show DNA hypomethylation plays a key role in carcinogenesis. The first major evidence is that DNA hypomethylation induces target chromosomal and genomic instability with cancer manifestations. The second reason that cancer progression is associated with deepening DNA hypomethylation. Nevertheless, the evolution of this crucial epigenomic alteration in the somatic cellular malignant transformation remains unclear. From some of the experimental data to be present, a key role of DNA hypomethylation in early development of epigenetic somatic cancer biology is proposed. We have observed the significant increasing of genome ploidy at the level of peripheral blood lymphocytes taken from the patients with different solid carcinomas. Similarly, 5-azacytidine demethylating DNA treatment of cultured healthy lymphocytes induces increased nuclear DNA content. We argue that somatic lymphocyte ploidy induced by genomic DNA hypomethylation during carcinogenesis is related to global demethylation and decondensation of mitotic constitutive pericentromeric heterochromatin. This results in disturbances of pericentromeric heterochromatin that are expressed in nuclear heterochromatinization on the basis of extrachromosomal chromomerization. On the basis of literature searches and experimental findings, it is proposed that DNA hypomethylation plays the role of an initiator in epigenetic somatic cancer biology. Ó 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: DNA hypomethylation; 5-Azacytidine; Pericentromeric heterochromatin; Latent polytene heterochromatinization; Somatic cancer cell biology

1. Introduction Cancer therapy could be improved if we understood the epigenetic causes of carcinogenesis. It is well known that cancer is a genetic disease, caused mainly by the acquisition of mutations in somatic cells (Sonnenschein and Soto, 2001; Soto and Sonnenschein, 2004). The authors have argued that cancer is a disorder of cell ‘‘societies’’, and does not necessarily involve genetic changes/mutations at the individual cell level (Sonnenschein and Soto, 1999). In this regard, there is little understanding of the uniqueness of somatic cellular malignant transformation. The causes and detection of somatic cancer mutations using highly sensitive DNA microarray technologies

* Tel.: þ38 044 526 0729; fax: þ38 044 526 0759. E-mail address: [email protected]

is not sufficient to establish the nature of the cell neoplastic transformation on the basis of genomic DNA sequences (Somasundaram et al., 2002). There is increasing evidence that the pathogenesis of cancer follows a general mechanism regardless of the origin of transformed cells (Ponder, 2001). Epigenetic evidence seems to indicate that progression of any type of cancer follows a unique course, as recently attested by a growing number of groups (Feinberg, 2004; Feinberg et al., 2006; Jones and Baylin, 2002; Laird, 2005; Verma and Srivastava, 2002; Herman, 2005; Herranz and Esteller, 2006; Haslberger et al., 2006). There is increasing evidence that abnormalities of DNA methylation are involved in early carcinogenesis (Baylin et al., 1998; Wajed et al., 2001; Baylin and Bestor, 2002; Worm and Guldberg, 2002; Kourmouli et al., 2005; Szyf, 2006; Shames et al., 2007), mainly as global genomic DNA hypomethylation (Feinberg and Vogelstein, 1983; Gaudet et al., 2003; Hoffmann and Schulz, 2005; Kisseljova and Kisseljov,

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L.P. Shvachko / Cell Biology International 33 (2009) 904e910

2005) and the regional genomic CpG-island promoter DNA hypermethylation of the major tumor suppressor genes (Baylin and Herman, 2000; Herman and Baylin, 2000; Garinis et al., 2002; Clark and Melki, 2002; Paz et al., 2003; Sherr, 2004). If DNA hypermethylation is associated with transcriptional silencing of tumor suppressor genes, the biological significance of DNA hypomethylation is difficult to explain. The pattern of genomic DNA hypomethylation appears to be a critical determinant in carcinogenesis because the effect of DNA hypomethylation on chromosome instability (CIN) can manifest itself in cancer (Chen et al., 1998; Xu et al., 1999; Ehrlich, 2002a,b; Eden et al., 2003; Karpf and Matsui, 2005; Rodriguez et al., 2006; Chen et al., 2007). To gain a better understanding of such the mechanisms, it is essential to know how DNA hypomethylation causes genomic instability. Since both genomic instability and genome-wide hypomethylation are observed in the early stages of carcinogenesis, this has led to some speculation on how genomic DNA hypomethylation facilitates the appearance of incipient cancer cells by destabilization of genome, along with the acquisition of more somatic mutations (Hoffmann and Schulz, 2005). However, knowledge of generalized epimutations of carcinogenesis leads to an avalanche of cancer molecular heterogeneity, which is sometimes contradictory. An example is the abnormal DNA hypomethylation and hypermethylation paradox in cancer progression (Counts and Goodman, 1995; Jones, 1998; Rhee et al., 2000; Ehrlich, 2002a,b; Szyf, 2005). But the changes in DNA methylation during carcinogenesis are not isolated events, and have to occur in the context of more complicated epigenomic deregulation. The aim of the present analysis is to address the biological significance of aberrant epigenetic DNA hypomethylation in somatic carcinogenesis. 2. Relevant experimental data Some of the experimental data which underpins our hypothesis were obtained in the following way. Peripheral blood lymphocytes had been obtained from the patients with solid tumors (thyroid cancer, colorectal cancer, breast cancer, neuroblastoma, Wilms’ tumor, total n ¼ 75). Healthy donors acted as controls (n ¼ 20). Genomic DNA samples (5e10 mg) were prepared by conventional phenol-chloroform extraction followed by ethanol precipitation (Klaus, 1987). DNA hypomethylation profiles of somatic genome lymphocytes were determined using unmethyl-CpG selected HpaII restriction of genomic DNAs under the following conditions: SL buffer e 10 mM TriseHCl, pH 7.5; 10 mM MgCl2, 1 mM dithioerythritol (DTE), at 37  C within 16 h. l DNA was used as positive control of HpaII restriction activity. The reaction of restriction was restrained by enzyme HpaII inhibition using 0.5 M EDTA. Blot-hybridization of satellite DIG-pUC (Alu) DNA repeats with cancer-associated genomic DNA/HpaII hypomethylation profile was performed according to (Shvachko, 2008). Mitogen-stimulated cultures of peripheral blood lymphocytes were grown in conventional RPM1640 medium with 10% fetal calf serum, 1% penicillin, and streptomycin.

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Healthy control lymphocyte cultures were given 5-azacytidine (105 M), as a model of genomic DNA demethylation, for 72 h. Cytomorphological investigation of their metaphase chromosome plates (n ¼ 500; p < 0.001) and interphase nuclei (n ¼ 500; p < 0.001) was done by light and fluorescent microscopy (Axiotar FL, Zeiss, Germany) with Giemsa, DAPI, and Hoechst 33258 dyes. Quantitative analysis of these agents with in situ hybridization of interphase nuclei (Scion Image Zeiss, Germany) was also carried out. 3. Relevant findings and discussion Recently the causal role of DNA hypomethylation in carcinogenesis has been suggested (Gaudet et al., 2003; Eden et al., 2003; Fraga et al., 2004; Hoffmann and Schulz, 2005; Kisseljova and Kisseljov, 2005). Regardless of global genomic DNA hypomethylation in many human tumors and its significance as a hallmark of carcinogenesis, understanding about this epigenetic alteration during somatic cell malignancy is limited. Previously, we found a striking association between the aberrant pattern genomic DNA hypomethylation profiles of peripheral blood lymphocytes of those patients with different types of solid cancer, and an increase in genomic DNA ploidy during cancer progression, as well under 5-azacytidine DNA demethylating treatment of healthy lymphocytes (Shvachko, 2008). The targeted increase of the heterochromatic DNAcontents shown by DAPI and Hoechst 33258 fluorescence took place under a generalized genomic DNA hypomethylation/demethylation background (Fig. 1). The DNA hyperploidy of cancer cells is connected only with increased nuclear DNA content (Pinto et al., 1997, 2002; Ganina et al., 1998). However, data concerning the correlation between cancer-associated micronuclear sizes and an increase in nuclear DNA content deserves closer attention (Nusse et al., 1996). In this connection, there is insufficient information about the contribution epigenetic DNA demethylation in this phenomenon. The strong link between malignant stage of the cancer progression and acute loss of DNA methylation is well referenced (Habets et al., 1990; Cravo, 1999; Qu et al., 1999; Lin et al., 2001; Widschwendter et al., 2004; Pogribny et al., 2006). Moreover, an aberrant pattern of DNA hypomethylation often takes place even in premalignant stages of tumor development (Pogribny et al., 2004; Herman, 2005). In contrast, a DNA methylation pattern similar to that in normal tissue is associated with a better prognosis in human cancer (Muller et al., 2004). These results clearly suggest that DNA hypomethylation is a key event in the initiation of carcinogenesis and may contribute to earlier formation of a somatic cancer phenotype. Recently the important role of epigenetic chromatin reprogramming associating with DNA hypomethylation has become increasingly evident in somatic neoplastic transformation (Lewis and Bird, 1991; D’Alessio and Szyf, 2002; Esteller and Herman, 2002; Robertson, 2002; Ballestar and Esteller, 2005; Brock et al., 2007; Jones and Baylin, 2007). We have focused attention on the alterations of

L.P. Shvachko / Cell Biology International 33 (2009) 904e910

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Fig. 1. The increasing of nuclear heterochromatic DNA content of the interphase peripheral blood lymphocytes from the patients with different solid cancers and healthy interphase lymphocytes (control) after 5-azacytidine DNA demethylation treatment using DAPI and Hoechst 33258 quantitative cytofluorescence.

the constitutive heterochromatin in particular pericentromeric heterochromatin in cancer patients. It is known that DNA methylation acts as the guardian of condensed constitutive heterochromatin during cell division (Rountree et al., 2001; Kourmouli et al., 2005; Muegge, 2005). Our observations are in accord with (Ehrlich, 2002a; Ehrlich et al., 2003; Grunau et al., 2005) that cancer-binding genome-wide DNA hypomethylation reflects the global pericentromeric demethylation of DNA repeats, resulting in crucial decondensation of pericentromeric heterochromatin in mitosis (Shvachko et al., 2006). This decondensation of pericentromeric/centromeric heterochromatin observed in the somatic blood lymphocytes from the patients with different solid carcinomas appears typically as in Fig. 2. Such significant epigenetic deregulation of mitotic constitutive heterochromatin may clarify the consequently role of epigenetic somatic tumor phenotype. In part, the consequent morphological alterations of decondensed pericentromeric heterochromatin regions were detected, developing extrachromosomal organization similar to latent polyteny. This phenomenon takes place in metaphase

chromosomes of the lymphocyte (Figs. 3A,B), and as heteropycnosis in the interphase nucleus (Figs. 4A,B) in patients with a poor cancer prognosis, as shown by Giemsa and DAPI staining before cytomorphological assay (n ¼ 75; p < 0.001). Moreover, the latent polyteny feature of pericentromeric heterochromatin regions associated with cancer is indicated by their tendency towards bilateral ectopic conjugation between metaphase sister chromatids (Fig. 3B). In addition, data concerning the induction by 5-azacytidine DNA demethylating reagent actual metaphase chromosome decondensation and the target chromocenters organization of heterochromatin regions of pig’s lymphocytes have been reported by Stefanova et al. (1987). This could confirm our observations showing that cancer-associated genome-wide DNA hypomethylation evidently causes the development of epigenetic alterations of the constitutionally silenced heterochromatin accompanying extrachromosomal latent polyteny development. In addition, and crucially, nuclear heteropycnosis was expressed during the 5-azacytidine DNA demethylating

Fig. 2. The expressed decondensation of pericentromeric/centromeric heterochromatin of the blood lymphocyte metaphase chromosomes from the patients with solid cancer type progression (the thyroid cancer and neuroblastoma).

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Fig. 3. (A) The latent polytene chromomerization feature of pericentromeric heterochromatin of metaphase chromosomes of the peripheral blood lymphocytes from the patient with poor thyroid gland cancer progression. (B) The bilateral ectopic conjugation event of the chromomeric heterochromatin regions between the sister chromatids.

treatment of the healthy lymphocyte culture after 72 h period (Fig. 5). A similar somatic heterochromatinization observed during malignant transformation against a pattern genomic DNA hypomethylation developed abundant heterochromatic chromocenters granules, which consist of dense a-heterochromatin with friable looping b-heterochromatin (Fig. 4B). The rather dramatic appearance of b-heterochromatin may result in chromosomal and genomic imbalances generally in gene expression and DNA replication. b-heterochromatin loops (Fig. 6) are highly polytenized and characterized by very active gene expression and DNA amplification, mainly by satellite and mobile genetic DNA sequences (Zhimulev, 1994), resulting in global mitotic genomic disturbances in carcinogenesis. In addition, Chen et al. (2007) state that global DNA hypomethylation in carcinogenesis might contribute to ‘‘mitotic catastrophe’’.

We have observed that 5-azacytidine induces irreversible nuclear heteropycnosis phenotype by forming massive chromocenters in the healthy interphase lymphocytes cultured in 105 M 5-azacytidine presence for 72 h (Fig. 5). Our observations may indirectly corroborate previous reports (Habets et al., 1990; Szyf et al., 2004), showing that increase in metastatic potential of tumor cell lines treated with 5-azacytidine occurs. We have proposed that genome-wide DNA hypomethylation underlies the somatic nuclear hyperploidization taking place in the oncological process, which evidently results in the potential progression of latent polyteny of pericentromeric heterochromatin. Altogether, we can argue that the initial role of genomewide DNA hypomethylation in carcinogenesis creates links with the triggering reprogramming of constitutive silence heterochromatin on the extrachromosomal pathway as we saw

Fig. 4. (A) The cancer-associated nuclear heteropycnosis of constitutive heterochromatin in the interphase lymphocytes using DAPI-in situ hybridization fluorescence. (B) The abundant interphase nuclear constitutive heterochromatic chromocenters formation with density a- and looping reticular b-heterochromatin formation using Giemsa detection.

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Fig. 5. The formation of the massive chromocenters of interphase constitutive heterochromatin induced by 5-azacytidine DNA demethylating treatment of the healthy lymphocyte culture for 72 h.

throughout its latent polyteny chromocenters formation during cancer progression. These observations could be important in the prevention of anti-DNA methylation in epigenetic therapy of various somatic malignancies (Szyf et al., 2004; Szif, 2005; Momparler, 2005; Herranz and Esteller, 2006). 4. Summary We have made an attempt to characterize the status of epigenetic morphological alteration of somatic genome in the course of tumorigenesis, which is causally linked to an aberrant pattern of genomic DNA hypomethylation. The crucial decondensation of pericentromeric/centromeric heterochromatin and its consequent latent polyteny features leading to the increase in somatic nuclear DNA ploidy There are three distinct latent polyteny features of mitotic pericentromeric heterochromatin in cancer progression: (i) targeted metaphase and interphase nuclear abounding chromocenters formation; (ii) the appearance of ectopic conjugation between sister chromatids in chromomerized pericentromeric heterochromatin regions; and (iii) commonly nuclear heterochromatinization leading to

heteropycnosis. The accumulated evidence clearly indicates the role of abnormal epigenetic DNA demethylation as key factor in the initiation of latent polyteny development of pericentromeric heterochromatin during the somatic malignant transformation. Thus, DNA hypomethylation may be key in unravelling the mechanism of carcinogenesis. These results may shed some light on epigenetic tumor biology and provide improved strategies for early diagnosis, prognosis, and epigenetic cancer therapy.

Acknowledgements The author is grateful to Drs AP Stepanenko and MV Gulchiy from Kyiv Thyroid Gland Centre; Dr GI Klimnjuk, Head of Department of Children’s Oncology, Prof VS Procyk, Head of Department of Head and Neck Oncology, Prof VA Kikot’, Head of Department of Colorectal Oncology from the Institute of Oncology, AMS of Ukraine, for the peripheral blood samples kindly made available for our investigations. This work was supported by the National Ukrainian Scientific Program ‘‘Genomics organization and regulation’’ (No. 0107U00337).

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Fig. 6. The schematic organization of pericentromeric heterochromatin in mitotic (a); and polytene chromosomes (b); black regions e a-heterochromatin, white regions e b-heterochromatin (Zhimulev, 1994).

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