Senescence and epigenetic dysregulation in cancer

The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

Molecules in focus

Senescence and epigenetic dysregulation in cancer Peter Neumeister a , Chris Albanese a , Beate Balent c , John Greally b , Richard G. Pestell a,∗ a Department of Development and Molecular Biology, Division of Hormone-Responsive Tumors, Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA b Department of Hematology, Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA c Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

Received 7 December 2001; received in revised form 1 May 2002; accepted 21 May 2002

Abstract Mammalian cells have a finite proliferative lifespan, at the end of which they are unable to enter S phase in response to mitogenic stimuli. They undergo morphological changes and synthesize an altered repertoire of cell type-specific proteins. This non-proliferative state is termed replicative senescence and is regarded as a major tumor suppressor mechanism. The ability to overcome senescence and obtain a limitless replicative potential is called immortalization, and considered to be one of the prerequisites of cancer formation. While senescence mainly represents a genetically governed process, epigenetic changes in cancer have received increasing attention as an alternative mechanism for mediating gene expression changes in transformed cells. DNA methylation of promoter-containing CpG islands has emerged as an epigenetic mechanism of silencing tumor suppressor genes. New insights are being gained into the mechanisms causing aberrant methylation in cancer and evidence suggests that aging is accompanied by accumulation of cells with aberrant CpG island methylation. Aberrant methylation may contribute to many of the physiological and pathological changes associated with aging including tumor development. Finally, we describe how genes involved in promoting longevity might inhibit pathways promoting tumorigenesis. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Senescence; Methylation; Acetylation; Cancer; Aging

1. Introduction Abbreviaions: ␤-gal, ␤-galactosidase; CDK, cyclin-dependent kinase; DNMT, DNA methyltransferase; ER␣, estrogen receptor ␣; ERC, extrachromosomal rDNA circle; hTERT, human telomerase reverse transcriptase; HAT, histone acetyl transferase; HDAC, histone deacetylase; HDF, human diploid fibroblast; MBD, methyl-CpG binding domain protein; PML, promyelocytic leukemia; POD, promyelocytic oncogenic domain; pRb, retinoblastoma; SIR, silent information regulator ∗ Corresponding author. Tel.: +1-718-430-8662; fax: +1-718-430-8647. E-mail address: [email protected] (R.G. Pestell).

Mammalian cells have a finite replicative potential in vitro and in vivo referred to as the Hayflick limit [1–3]. Upon reaching this limit, cells cease to divide and undergo genetic, biochemical and morphological changes indicative of aging, a state referred to as cellular senescence. As these cells do not proliferate, it has been suggested that cellular senescence is a major barrier against cancerous transformation. The ability to overcome senescence and obtain a limitless

1357-2725/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 0 7 9 - 1

1476

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

replicative potential (termed immortalization), is one of the prerequisites for tumor formation [4,5]. The purpose of this review is to outline recent findings on the molecular events contributing to tumorigenesis through cellular senescence. Emphasis is placed on the genes involved in cellular senescence and immortalization, and on the possible role of epigenetic dysregulation in the process. We highlight recent evidence emphasizing the parallels between these molecular events and the aging process. Ultimately, specific genes regulate many of these changes. However, the majority of mammalian DNA does not encode expressed sequences but is nevertheless conserved. This finding highlights the presence in the genome of a second type of information, the function of which appears to be to regulate the expression of the information encoded by genes. These DNA sequences, and the chromatin constituents that are organized by them, exert epigenetic (epi— upon, in the sense of higher than) regulation of the genome. Epigenetics is defined as the study of heritable changes in gene expression that occur without a change in DNA sequence [6]. Herein we focus on two of the better-characterized epigenetic regulatory mechanisms in mammalian cells, methylation of cytosine residues and modifications of the tails of the core histones. Epigenetic processes regulate gene expression patterns during cell differentiation, and can also be extremely stable, as demonstrated by the example of genomic imprinting, in which gametic epigenetic patterns are maintained in somatic cells through multiple lineages of cell growth and differentiation. With the possibility that the normal stability of epigenetic regulation can be stressed by time and cell divisions, we describe studies linking epigenetic regulation with immortalization, cancerous transformation and aging.

2. Senescence Replicative senescence represents an irreversible arrest of cell proliferation and altered cell function. It is a process dependent on the number of cell divisions but not time. Senescent cells are stably arrested with a G1 DNA content and are unable to enter S phase in response to mitogenic stimuli. The cells remain metabolically active and resemble terminally

differentiated cells. Furthermore, they acquire resistance to the induction of programmed cell death [7,8]. Senescence is associated with altered cellular morphology and size [9], altered expression of genes involved in extracellular matrix remodeling, reduced serum-induced expression of both, immediate-early genes and cyclins, and increased expression of neutral senescence ␤-gal activity [10]. While senescence is consistently observed in vitro, the existence of a similar state in vivo has been difficult to prove. The most convincing argument for the latter is based on the correlation between the in vitro lifespan of cells obtained from younger donors versus older, in which the onset of senescence is markedly accelerated [11]. The properties contributing to cellular senescence and subsequent immortalization differ between human and murine cells (Fig. 1). Cellular senescence in human fibroblasts occurs in two phases, mortality stages 1 and 2 (M1 and M2 ). In normal replicative senescence, M1 likely occurs when telomeres are sufficiently short that they are recognized as requiring double strand break repair. DNA damage checkpoints control proteins are thereby activated and are capable of blocking cell division. Inactivation of these DNA damage checkpoints control proteins by expression of transforming oncogenes or mutation of the checkpoint control proteins (p53 and pRb), results in ongoing cellular division until M2 (crisis). This state is characterized by massive cell death and gross karyotypic changes associated with end to end fusion of chromosomes. Occasionally, a cell with the ability to multiply without limits emerges [12], a trait termed immortalization. Most tumor types in culture appear to be immortalized, suggesting that limitless replicative potential is a prerequisite of malignant growth [3]. In tissue culture, various human cell types seem to have the ability of 60–70 population doublings [5]. This number vastly exceeds the number of cells in the human body, thus casting some doubt on the physiological role of replicative senescence as a mechanism of tumor suppression. This apparent paradox can be resolved by considering the apoptotic rates in certain human cancers [13] and transgenic mouse systems [14,15], in which there is considerable evidence of widespread apoptosis and cell loss during concomitant cell accumulation. Therefore, the absolute number of cells in a tumor is a great under-representation of

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

1477

Fig. 1. There is a fundamental difference in telomere biology of human and mouse telomeres. Murine somatic cells express telomerase activity and have much longer telomeres than their normal human counterparts, which lack telomerase activity. Primary rodent cells are efficiently transformed by co-expressing cooperative oncogenes, and immortalization is achieved solely by disruption of the p53 pathway. Conversely, telomerase maintenance mechanism together with disruption of the p53 and the Rb pathway are necessary to immortalize human cells.

the generations required to produce them. Thus, the generational limit of a normal cell may still represent a major barrier to cancer formation [5]. It is important to note that human and murine cells differ in their mechanism regulating cellular senescence and immortalization (Fig. 1). p53, but not Rb is required for replicative arrest of mouse embryonic fibroblasts (MEFs). Murine cells immortalize with much higher frequencies than human cells, and inactivation of the ARF/p53 pathways is sufficient for immortalization. It has been proposed that the cell cycle arrest observed in cultured murine cells that resembles senescence of human cells is a distinct “stress” response [16,17]. This conclusion is based on the ease with which cellular stressors (genotoxic drugs, DNA damage, oxidative damage) overexpression of oncogenes (ras) and loss of checkpoint proteins (p16INK4a ) induce features of senescence, and the escape of cells from senescence if deleted of genes encoding stress sensors (i.e. Ku80 and Brca2).

3. The telomere hypothesis A cell division cycle counting machinery or ‘event counter’ has been hypothesized to exist in normal somatic cells to monitor the finite cumulative cell divisions implicit in Hayflick’s senescence model. Early experiments by Hayflick and others suggested the ‘replicometer‘ was nuclear in location. According to the telomere hypothesis of senescence, the event counter measures the progressive telomere shortening that occurs with each cell division [18–20]. The ends of human chromosomes are nucleoprotein structures where non-coding DNA sequences are arranged in tandemly repeated units of TTAGGG hexanucleotides, which may contain up to 25,000 bp [21,22]. These terminal structures are called telomeres and protect chromosomes against degradation, rearrangement and fusion to other chromosomal ends [23]. Due to the nature of lagging strand DNA synthesis, conventional DNA polymerases are incapable of completing

1478

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

replication to the very end of linear DNA, thus leading to the loss of 50–200 bp of telomeric DNA per round of replication [20,24,25]. It is hypothesized that telomere erosion ultimately leads to a shortening of ends that are no longer protected, triggering the cell senescence phenotype. This model predicts that residual telomere length predicts the remaining lifespan of a cell [20]. The estimated loss of 50–200 bp of DNA is consistent with the relative telomere length of young and senescent human fibroblasts (20–25 and 8–10 kb, respectively) [22,26]. The demonstration of a tight correlation between telomeric length and age in multiple human tissues [27] as well as the continuously decreasing telomere length with increasing age of human fibroblasts in culture [25] suggests a causal relationship between telomere erosion and proliferative arrest in senescent human diploid cells.

4. Telomerase and immortalization One prediction of the telomere model is that cellular senescence would be circumvented if telomere shortening were prevented [20]. There is experimental evidence to support a role for telomerase in both M1 and M2 in human cells. The cloning of hTERT [28], the catalytic subunit of the telomerase holoenzyme, an enzyme responsible for telomere length maintenance made it possible to ectopically express hTERT in telomerase negative pre-senescent HDFs and endothelial cells. The ectopic expression of hTERT resulted in telomere maintenance, which circumvented senescence and contributed to unrestricted growth and immortalization in some, but not all cell types [20,29,30]. Telomere maintenance is evident in virtually all types of malignant tumors [31]. Telomerase activity is upregulated in 85–90% of tumors. In the remaining 10–15% of tumors a mechanism called alternate lengthening of telomeres (ALT), which is a recombination based interchromosomal exchange of sequence information, maintains telomere length [5,32]. In all human tumors, telomeres are maintained by one or the other mechanism and both mechanisms seem to be strongly suppressed in most normal human cells in order to prevent unrestricted replicative potential [5]. However, recent data suggest that senescence is induced by an altered telomere state, rather than a complete loss of telomeric DNA. TRF2, a telomeric

DNA binding protein, increased the rate of telomere shortening in primary cells without accelerating senescence. TRF2 protected critically short telomeres from fusion and repressed chromosome-end fusions in pre-senescent cultures [33]. Introduction of hTERT is sufficient to immortalize fibroblasts and endothelial cells, but is insufficient to bypass senescence in human keratinocytes and mammary epithelial cells. The cellular transformation of mammary epithelium required not only the ectopic expression of hTERT but also the expression of both, SV40 large T antigen which inactivates the Rb and p53 pathway, and SV40 small t antigen, a potent inducer of cyclin D1 [34,35]. In contrast with this model, however, several studies have shown that cells undergo replicative senescence despite the continuous presence of telomerase [32,36]. Furthermore, telomerase negative (mTR−/− ) MEFs stop dividing after 10–15 doublings, before telomere erosion occurs. Ectopic expression of hTERT was also insufficient to protect human cells from Ras-induced senescence [37]. Therefore, in some but not all cell types, telomere length regulation is a key event in the induction of senescence [20]. How the shortened telomeres signal their impaired state to the cell is uncertain. It has been mentioned above that the critically shortened telomeres may be recognized by the DNA damage checkpoint control proteins, which could be sufficient to cause replicative arrest. An alternative hypothesis is based on the properties of telomeres in model eukaryotic organisms. In organisms such as Saccharomyces cerevisiae and Drosophila melanogaster, juxtaposition of a normally euchromatic gene to the telomeric heterochromatin causes its decreased expression, a phenomenon referred to as a position-effect [38]. Recently, a similar process was demonstrated in mammalian cells, prompting the authors to propose that telomere degradation may alter the expression of subtelomeric genes, possibly contributing to the senescence process without inducing the DNA damage checkpoint control process [39].

5. Other genes involved in replicative senescence During replicative senescence altered expression of genes that are involved in cell cycle control and

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

oncogenesis is well documented. Forced expression of these gene products can also induce a senescence-like state. During replicative senescence HDFs undergo a series of changes in the abundance and activity of the cell cycle proteins. In early studies, senescent cells were characterized by reduced mitogen-induced expression of D-type cyclins [40]. Expression of the cellular CDK inhibitor p21CIP1 protein increases during the final two–three passages of M1 when the majority of cells lose their growth potential, then levels sequentially decline as cells enter senescence. p21CIP1 binds to, and inactivates cyclin E–CDK2 complexes in senescent HDFs, whereas in “young” cells only p21CIP1 free CDK2 complexes are active. Since p21CIP1 accumulates prior to p16INK4a , p21CIP1 might be key for the induction of the senescent state in HDFs. The cyclin D1–CDK4 and cyclin D1–CDK6 complexes in senescent cells are associated with increased p21CIP1 suggesting p21CIP1 inhibition of cyclin D-associated kinase activity contributes to senescence. p21CIP1 and cyclin D1 bind PCNA in senescent cells. As PCNA is essential for replication and repair, the increased binding and inactivation of PCNA may contribute to the cell cycle arrest of senescence [41]. p16INK4a mRNA and protein increases up to 40-fold in senescent HDFs compared to early passage cells. p16INK4a complexes with CDK4 and CDK6 in senescent cells. Loss of p16INK4a accompanies spontaneous immortalization of Li-Fraumeni fibroblasts, and INK4a(Ex2/3)−/− MEFs fail to senesce, supporting an important role for the pRb/p53 pathway in cellular senescence [42]. p16INK4a accumulates after the induction of p21CIP1 in parallel with senescence associated ␤-gal activity and induction of the senescent phenotype, whereas p21CIP1 decreases after achievement of senescence. It has been proposed, therefore, that p16INK4a upregulation may be essential for the maintenance of the senescence associated cell cycle arrest [43]. Based on these observations it was proposed that p16INK4a upregulation is the key event of the terminal stages of growth arrest in senescence, perhaps explaining why p16INK4a but not p21CIP1 is commonly mutated in human tumors [42]. The Ras/Raf/MEK pathway induces cellular senescence in several cell types. Expression of oncogenic Ras in primary human or rodent cells is able to induce a permanent G1 arrest. This arrest is accompanied by accumulation of p53 and p16INK4a and is

1479

phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16INK4a prevented the G1 arrest induced by Ras in rodent cells and led to a tumorigenic state [42]. This work further supports a model in which the cell cycle arrest of cellular senescence functions as a tumor suppression mechanism. The Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins and constitutively active forms of Raf also elicit a cell cycle arrest and the premature onset of senescence in non-immortalized human lung fibroblasts [44]. Concomitant with the activation of Raf-ERK, induction of the CDK inhibitors p21CIP1 and p16INK4a has been observed. Pharmacological inhibition of the Raf/MEK/MAP kinase cascade prevented Raf from inducing p16INK4a and also from Raf-induced senescence. Constitutive activation of MEK, an activator of the MAPK cascade, induced both p53 and p16INK4a and MEK is required for Ras-induced senescence. Primary human fibroblasts are arrested in G1 by constitutively active MEK, but in cells lacking either p53 or p16INK4a induced uncontrolled mitogenesis and transformation [45]. Gene expression profiling of Ras-arrested cells and quiescent human IMR90 fibroblasts revealed induction of the PML protein, a potential tumor suppressor that encodes a component of the nuclear structure referred to as PODs. Forced PML expression was sufficient to promote premature senescence. Like oncogenic Ras, PML increased the levels of p16INK4a , hypophosphorylated pRb and p53 and expression of p53 transcriptional target genes. The fraction of pRb and p53 co-localizing with PML markedly increased upon expression of Ras [46]. Both Ras and PML can induce premature senescence in a p53-dependent manner in primary human fibroblasts [46,47]. Opposing effects of Ets and Id proteins on p16INK4a expression in HDFs during senescence have also been described. Unless provoked by oncogenic Ras early passage HDFs express low levels of p16INK4a and signaling through the predominant Ets protein Ets2 is counterbalanced by Id1. In senescent cells, however, upregulation of p16INK4a depends on the increased expression of Ets1 in the absence of any interference by Id1 [48]. pRb plays a key role in regulating senescence in human cells. Reintroduction of pRb into pRb−/− tumor cell lines induces senescence [49]. The

1480

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

induction of senescence by pRb correlates with the accumulation of the CDK inhibitor p27KIP1 , an increase of p27KIP1 –cyclin E complexes and a decrease in cyclin E-associated kinase activity. Therefore, the pRb-induced cell cycle arrest seems to be mediated by specific inhibition of CDK2 activity by p27KIP1 [50]. Together studies suggest, a senescence-like arrest of HDFs can be induced by ectopic expression of p15INK4b , p16INK4a , p21CIP1 or p27KIP1 or by DNA damaging agents that activate p53 or p27KIP1 . These findings are consistent with a model in which the concurrent induction of a mitogenic stimulus with a forced G1 arrest may be sufficient to induce a senescent phenotype. Senescence, much like apoptosis, represents a major barrier to tumorigenesis and it can be achieved by shortened telomeres or conflicting growth signals that forces aberrant cells into a G0 like state [5]. Immortalization is not sufficient for tumorigenesis, but may provide the necessary prerequisite for accumulating the total number of genetic events required for malignancy [19]. The genetic changes associated with immortalization, namely loss of p53 and p16INK4a /pRb function and activation of telomerase, are among the most common known cancer-related changes and the major regulators of cellular senescence. A deeper understanding of the molecular mechanisms by which these multiple distinct pathways lead to immortalization may result in new powerful tools for diagnosis and therapy of cancer.

6. Epigenetic changes involved in tumorigenesis: methylation and histone tail modification Abnormal methylation of the genome has been described in different cancers. Methylation is generally targeted at cytosine residues at so-called CpG dinucleotides (5 -CG-3 ). As 5-methylcytosine can be deaminated to produce a thymine nucleotide which is not recognized by the cell as the result of mutation [51], 5-methylcytosines are under a constant mutational pressure that exceeds all other sequences, explaining why CpGs are extremely under-represented in the mammalian genome [52]. Methylation governs several distinct processes including genomic stability, retroelement suppression and gene promoter regulation. CpGs in the mammalian genome can be described as associated with large (>500 bp) islands,

frequently associated with promoters, associated with small CpG islands (200–500 bp), more characteristically associated with transposons (usually SINEs), and non-island CpGs [53]. In neoplasia, two seemingly incompatible events occur. Demethylation of the genome as a whole occurs, while increased methylation of promoter-associated CpG islands is concurrently observed, leading to silencing of some genes with normal tumor-suppressive activity [54]. In vitro evidence suggests that methylated genes are initially transcriptionally active, but after a few hours are packaged into a chromatin formation less accessible for transcription [55]. These results indicate that DNA methylation itself does not interfere with transcription in most cases, but rather marks DNA for establishment of a transcriptionally incompetent state. However, whether DNA methylation initiates the repressive state or methylation is a secondary event following formation of incompetent chromatin is still disputed. The demethylation process appears to occur largely at transposable elements, which increase in expression as a consequence [56]. This increased expression has the potential to cause mutational events through transposition [57,58]. The pattern observed is, therefore, a redistribution of methylation, involving both demethylation and de novo methylation. It is critical to understand how methylation and demethylation processes are mediated normally to understand how they might be dysregulated in neoplasia. The signal to methylate a sequence in the genome is principally provided by a pre-existing methylation of that sequence, so that replication of the sequence to daughter chromatids only temporarily results in hemi-methylated DNA. DNMTs recognize these sequences and mediate the restoration of their symmetrical methylation on both DNA strands [59]. It is, therefore, not surprising that DNMTs are found to be part of the replication complex in mammalian cells [60]. What is surprising is that DNMT1 is associated with the replication complex only for part of S phase, after which DNMT3a and DNMT3b replace it in the complex to perform the methyltransferase activity on late-replicating DNA [61]. The other major signal for methylation identified in mammalian cells is the presence of SINE elements in cis [61–63]. B2 SINEs at the rat Afp [62] and the mouse Aprt [60] loci induce methylation of CpGs located on the same reporter construct. A similar

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

phenomenon has been observed with plant SINEs [64]. It appears that the targeting of methylation to transposons is not entirely clean, as innocent bystanders can also become methylated. In Neurospora crassa, cytosine methylation is also a downstream effect of a primary signal, in this case the methylation of the tails of core histones [65]. The Dim-5 protein identified by these authors is related to Clr4 in S. pombe and Suv39h in Drosophila and mammals. The last-mentioned has recently been reported to control histone H3 methylation at the lysine 9-position, although its role in inducing cytosine methylation in mammals has not yet been described. Interestingly, these mice are prone to lymphoma [66]. When CpGs become methylated, what happens? Cytosine methylation has been found to occur to a greater degree at heterochromatic parts of the genome, at loci inactive due to imprinting or X inactivation, and at experimentally-created loci, usually associated with their silencing [67]. The several members of the DNMT and MBD gene families have been described [68]. A member of the latter family, the MeCP2 protein, encodes a transcription repression domain that may have direct effects to cause gene silencing, although the primary mechanism of this domain and the other MBDs to effect transcriptional repression is in part mediated by recruitment of the HDAC1 and HDAC2 as part of a multiprotein complex that also includes mSIN3 [69]. The deacetylation of the tails of the core histones H3 and H4 is believed to cause chromatin compaction by reducing their charge, in turn rendering the DNA less accessible to transcription factors and silencing transcription. As noted above, methylation serves several distinct functions. Methylation may serve as a cellular defense mechanism directed at suppressing the activity of the transposable elements in the genome. It has been proposed on the basis of the analysis of 660 kb of genomic sequence that as the proportion of HpaII sites in retroelements in that sample (57%) is similar to the proportion of HpaII sites that are methylated in differentiated mammalian cells (60%), most if not all methylation occurs at retroelements [70]. Now that more sequence data are available as well as techniques for identifying methylation at CpGs not located at restriction sites [71], this should be more rigorously testable. The other strand of evidence for methylation being directed at transposons comes from the original

1481

methodology for CpG island identification: so-called HpaII tiny fragments (HTFs) isolated from genomic DNA digestion were derived from closely-adjacent and unmethylated HpaII sites [72]. These identified clusters of CpG-rich regions, subsequently found to be CpG islands, by definition unmethylated, and not significantly represented by repetitive DNA (of which transposable elements would be a major element). The relatively non-specific function of methylation to reduce chromatin accessibility is complemented by a more specific process. Transcription factor binding sites may include CpG dinucleotides, the methylation of which may influence the ability of the transcription factor to bind. The MLH1 gene was studied using transient transfection assays, finding that methylation of the construct was associated with decreased promoter activity and impairment of binding of the CBF transcription factor [73]. This indicates a potential role for methylation in the silencing of this mismatch repair gene in colorectal carcinoma. The CTCF transcription factor binds to a site downstream from the Igf2 gene and prevents access to enhancer sequences further downstream. Methylation of this insulator sequence prevents CTCF binding and allows Igf2 expression, a consistent event in Wilm’s tumour and colorectal carcinoma [74,75]. Methylation can thus have specific and relatively non-specific effects on gene regulation. Hypomethylation may also contribute to genomic instability. Embryonic stem cells lacking DNMT1, for example, display both, a globally hypomethylated genome and a higher degree of genetic instability [57,76]. In addition, pericentromeric areas which are normally heavily methylated exhibit increased levels of chromosomal translocations when hypomethylated by either a demethylating substance (5-aza-dC) or in genetic disorders with decreased methyltransferases [57,77,78]. In ICF syndrome (immunodeficiencycentromeric instability-facial anomalies) both alleles of the gene that encodes DNMT3b have mutations [78]. Although many reports show only moderate or nonspecific overexpression in cancer [79,80], others demonstrate an upregulation of DNMT1 in multiple tumor types [59,81]. Like DNMT1, DNMT3a and DNMT3b also appear to be moderately overexpressed in cancer [82]. Forced overexpression of the murine DNMT1 gene in NIH3T3 cells leads to cellular transformation and causes increased CpG island

1482

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

methylation in human cells [83,84]. Antisense inhibition of DNMT1 reverted the transformed cellular phenotype [85]. The effect of methylation on tumorigenesis is in turn dependent upon the genetic makeup of the individual. Mice carrying a mutant Adenoma Polyposis Coli APC allele (the Min mouse model) are predisposed to the development of colonic tumors. In Min mice mated to the DNMT1 knockout animals, despite global hypomethylation in DNMT1 heterozygous animals there was a 50% decrease in colonic tumor formation when compared to their wild-type controls [86,87]. Thus, in certain circumstances, reduction of DNMT1 levels may protect against oncogenesis [57,87]. Further evidence that epigenetic silencing of tumor suppressor genes is essential for neoplastic growth is provided by a study disrupting DNMT1 and DNMT3b in a colorectal cancer cell line [88]. This reduced DNA methylation by greater than 95%. These changes led to the demethylation of repeated sequences, loss of insulin like growth factor imprinting, re-expression of the p16I NK4a gene, and growth suppression. A recent study demonstrated that the leukemia promoting PML–RAR␣ fusion protein induces gene hypermethylation and silencing by recruiting DNMT1 and DNMT3a to target promoters and most importantly that it contributes to its leukemic potential. Retinoic acid treatment induced promoter demethylation, gene re-expression, and reversion of the transformed phenotype [89]. DNA methylation patterns in aging cells reflect the tendencies to aberrant methylation seen in transformed cells. Aging is the most important risk factor for the development of most malignancies of adulthood. A large number of genetic and epigenetic events occur during aging. Some of them directly regulate tumor propensity. Promoter hypermethylation at several genetic loci also increases during aging [86]. Promoter methylation might provide a link between aging and tumorigenesis. Age-related hypermethylation of the ER␣ promoter CpG island was first described in human colorectal tissue [90]. In colon cancer, ER␣ promoter methylation was observed in all cell lines, adenomas and carcinomas analyzed. Even the adjacent tissue showed a significant amount of methylation. Since methylation was virtually undetectable in young individuals it was considered to be age related [86]. Methylation of the locus in breast cancer cell lines also silences the ERα gene in a significant

proportion of primary ERα-negative breast cancers [91,92]. Normal HDFs have also been shown to acquire ER␣ and IGF2 CpG island methylation during several passages in culture [93]. Fibroblasts with extended lifespan achieve higher levels of methylation at senescence than their normal counterparts. Therefore, CpG island methylation parallels the in vitro model of cellular senescence and resembles the telomere shortening mechanism present in these cells [86].

7. DNA methylation and the cell cycle Since DNA methylation controls gene expression in vivo [94], in a cell type specific manner [95] during DNA replication, faithful inheritance of the DNA methylation pattern must be ensured [96]. Replication of the mammalian genome involves two independent tasks: replication of the genome itself and replication of the methylation pattern. Errors in the replication of DNMT1 methylation patterns, as a function of its de novo methylation capacity, do occur. Mechanisms responsible for monitoring and correcting these errors, therefore, exist. Although not confirmed by other groups [97,98], it has recently been shown that MBD2 exhibits a demethylase activity [99,100]. Similar to the de novo methylases DNMT3a and DNMT3b, which are essential for de novo methylation during development and have been implicated in replenishing aberrantly lost methylation sites during replication, the demethylase activity may be used to ensure that ectopic methylation events are corrected. Because neither de novo DNMTs nor the newly cloned demethylases possess a distinct sequence specificity, it was difficult to understand how both enzymes are able to differentiate aberrant from a correct methylation pattern. Since it has been shown that histone acetylation can direct the demethylases to acetylated nucleosomes in order to demethylate a particular sequence, a model has been proposed in which the acetylation status of the nucleosome provides the information for determining the correct methylation status. The replication of DNA methylation patterns during cell division also requires the “repair” functions of demethylases and de novo DNMTs that protect the methylation pattern from drifting. The repair methylation enzymes are guided by the state of acetylation, which is also inherited during replication [96].

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

It is imperative to ensure that cell division does not occur in the absence of DNA methylation [101]. As a consequence of the close link between DNA synthesis and DNA methylation, the regulation of the DNA methylation enzymes are tightly coordinated within the cell cycle and in turn DNMTs regulate cell cycle progression. DNMT1 levels are regulated by mitogenic signals and the dnmt1 gene is induced by the Ras–Jun signaling pathway [102,103]. The abundance of dnmt1 mRNA is post-transcriptionally reduced to non-detectable levels at G0 and induced upon entrance into S phase [101]. The targeting of DNMT1 to the replication fork in S phase through a special fork targeting domain in its N-terminus as well as its association with PCNA at the replication fork, further ensures the concurrent activity of replication and DNA methylation [104]. Expression of dnmt3b is also regulated during the cell cycle peaking earlier than dnmt1 during S phase. dnmt3a is expressed constitutively at low levels throughout the cell cycle [105]. Several lines of evidence support a role for DNMT1 in cell growth. Inhibition of DNMT1 by either antisense oligonucleotides or 5-aza-dC inhibits cell growth [85,106,107] and inhibition of DNMT1 by hairpin directed inhibitors or antisense expression

1483

plasmids inhibits the induction of origins of replication of DNA, suggesting a more direct involvement of DNMT1 in DNA replication [108]. DNMT1 may also regulate growth through methylation-dependent and -independent regulation of tumor suppressor genes. Thus, overexpression of DNMT1 in the bladder carcinoma cell line T24 causes promoter hypermethylation of the p16I NK4a gene and consequently, treatment with 5-aza-CdC results in demethylation and reactivation of this tumor suppressor and inhibition of cell cycle progression [107]. In addition DNMT1 controls expression of critical cell cycle genes by methylation-independent mechanism [109]. Treatment of a human lung cancer cell line by antisense oligonucleotides led to induction of p21CIP1 -independent of demethylation. Perhaps the strongest data supporting a direct transcriptional role for DNMT1 is the direct interaction with cell cycle regulatory proteins like E2F1 and Rb [110] as well as with the HDAC1 and HDAC2 [111,112]. Thus, inhibition of DNMT1 might indirectly result in a rapid induction of certain tumor suppressor genes. The observation that histone deacetylation might play a critical role in the regulation of tumor suppressors is substantiated by the fact that inhibitors of HDAC, similar to DNMT1

Table 1 Hypermethylated genes in cancer Gene

Function

Pathway affected

pRb p16I N K4a p15I N K4b ARF GSTP1 O6 -MGMT BRCA1 MLH1 APC VHL TIMP3 LKB1 E-cadherin AR RARβ RASSF1A ER DAPK1 Caspase-8 p73

Regulator of G1 /S phase transition CDK inhibitor CDK inhibitor Regulator of p53 levels Protection from DNA damage by oxygen radicals DNA repair/removal of bulky adducts from guanine DNA repair DNA mismatch repair ␤-Catenin binding protein Angiogenesis stimulation Matrix metalloproteinase inhibitor Serine/threonine protein kinase Cell adhesion Androgen receptor Retinoic acid receptor Ras association domain family 1A gene Transcriptional activation of ER responsive genes Induction of apoptosis by interferon-␥ Death receptor-associated initiator caspase p53 homologue

Cell cycle control Cell cycle control Cell cycle control Cell cycle control DNA damage repair DNA damage repair DNA damage repair DNA damage repair Invasion, tumor architecture Invasion, tumor architecture Invasion, tumor architecture Invasion, tumor architecture Invasion, tumor architecture Growth factor response Growth factor response Growth factor response Growth factor response Apoptosis Apoptosis Cell cycle control, apoptosis

According to Roundtree et al. [57].

1484

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

suppression, selectively induce the expression of tumor suppressors such as p21CIP1 [113,114]. Therefore, DNMT1 has been proposed to be a methylationindependent S phase specific suppressor of tumor suppressor genes, thereby stimulating entrance into the S phase of the cell cycle [96]. Since DNMT1 is a multifunctional protein it is not clear whether, and to what extent, the methyltransferase activity, or its regulatory repressor domains, are involved in cellular transformation. There is a growing list of methylated genes in cancer [86] (Table 1) and aberrant methylation of p16INK4a and/or O6 -methyl-guanine DNMT can be detected in sputum of squamous cell lung carcinoma patients up to 3 years before diagnosis [115]. However, although there is a clear role for aberrant methylation in tumorigenesis, the normal cell cycle regulation of DNMT1 expression is also disrupted in cancer [105,116,117]. The improper temporal regulation of DNMT1 expression during the cell cycle may lead to inappropriate silencing of tumor suppressor genes and untimely activation of cellular DNA replication [96].

8. SIR2 links chromatin silencing, aging and cancer Aging is manifested by a progressive decline in vitality over time leading to death [118]. Studies in model organism revealed that the Sir2 is a limiting component of longevity (Fig. 2). In yeast, deletions of SIR2 shortened lifespan and an extra copy extended lifespan in yeast [119]. Together with its homologs Sir3 and Sir4, Sir2 is responsible for silencing repeated DNA sequences like mating type loci, telomeres and rDNA in yeast. Whereas all homologs are required for silencing at mating type loci and telomeres, Sir2 seems to be the only component involved in rDNA silencing [118]. The Sir proteins also function in DNA repair by non-homologous end-joining (NHEJ) [120]. Sir2/Sir3/Sir4 and Ku re-localize from telomeres to DNA strand breaks sites in order to aid in their repair by NHEJ [121,122]. Telomeres are regarded as the primary reservoir for Sir proteins, from which they are mobilized for DNA damage repair functions. The function of Sir2 in promoting yeast longevity appears to be related to the rDNA silencing process. Sir2 inhibits recombination of the 100–200 tandem

copies of rDNA on chromosome 12 and mutations of Sir2 increases the frequency of recombinatorial events about 10–20-fold [123]. One of the products of this recombination is the formation of ERCs in yeast, which preferentially segregate to mother cells, thereby triggering senescence [124]. By preventing ERC formation through gene silencing Sir2 may, therefore, promote longevity. Sir2 conveys both, an ADP-ribosyl transferase and an NAD-dependent HDAC activity [125]. The HDAC function is responsible for chromatin silencing, repression of rDNA recombination and perhaps, therefore, lifespan extension. Although the targets genes of Sir2 involved in silencing of multicellular organisms that promote aging are not known, general models have been proposed [118]. Sir2 is present at silenced chromatin and can be recruited to sites of DNA damage, which occurs chronically over a lifetime. After completing DNA repair, Sir2 would return to its prior residence, i.e. silenced chromatin (Fig. 2). Assuming this “re-setting” is incomplete, a gradual erosion in the integrity of silenced chromatin over time would occur, leading to genomic instability, inappropriate gene expression and ultimately to the phenotype of aging. Sir2 function has been implicated in tumorigenesis through the regulation of tumor suppressors. In recent studies the human homologue of Sir2, SIRT1 or Sir2␣, regulated p53 function via deacetylation [126,127]. The tumor suppressor p53 exerts anti-poliferative effects, including growth arrest, apoptosis and cell senescence in response to various types of stress [128]. Alterations of the p53 pathway are found in most human tumors and mutations within the p53 gene have been well documented in more than half of all human tumors [129].The human Sir2␣-mediated deacetylation at lysine 382 antagonized p53-dependent transcriptional activation and inhibited both p21CIP1 expression and cellular apoptosis. In the case of p53, Sir2␣ may function to reverse the p53-induced cell cycle arrest following successful completion of DNA repair. Implicit in these findings, Sir2␣ overexpression may disrupt the p53 pathway promoting tumorigenesis. One could speculate that an incomplete “resetting” by altering Sir2␣ abundance not only regulates age-related process by erosion of silenced chromatin structures but may also contribute to p53 inactivation and tumorigenesis (Fig. 2). Like p53, direct acetylation regulates the function of many

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

1485

Fig. 2. Aberrant epigenetic and genetic events, likewise, may lead via inappropriate gene expression to the formation of cancer. “Incomplete resetting” of Sir2 following DNA damage repair may contribute to tumorigenesis by inactivating the p53 pathway and provides a link between aging and cancer.

different transcription factors and in some circumstances transcription factor acetylation governs cell cycle control [130–132]. Sir2 may, therefore, potentially regulate other acetylated transcription factors involved in growth control. Together these studies raise the possibility that altered Sir2 abundance and function in aging may regulate several different signaling pathways regulated by acetylation and thereby dysregulate normal cellular growth control.

9. Summary Replicative senescence of human cells occurs as a consequence of the progressive loss of TTAGGG repeats at the chromosomal ends. This causes a change in the protected status of the shortened telomeric complex and forces the cell to enter a state of invariable growth arrest. Replicative senescence can also be induced by either the p53 or the p16–Rb pathways, but

1486

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

how these pathways are activated in cells with critically shortened telomeres is not known. It seems that a failure in the protective function rather than a complete loss of telomeric DNA is responsible for senescence induction. While senescence is considered to be a mainly genetically driven tumor preventing process, epigenetic regulation of gene expression and maintenance of genomic instability in tumor formation have received increasing attention. Epigenetic gene regulation is accomplished through the modulation of chromatin packaging. Cytosine methylation establishes a compact, inaccessible chromatin through binding methyl-DNA binding proteins and recruitment of HDACs. This process is central in cellular differentiation and development. Epigenetic silencing of tumor suppressor genes by hypermethylation is a common event in many human tumors, but the mechanism underlying locus-specific or global methylation patterns in human cancer remain largely unknown. However, DNMTs seem to play a crucial role, since DNMT1 and DNMT3b double knockout cancer cell lines reduced genomic DNA methylation by 95% and was associated with growth suppression and re-expression of tumor suppressor genes. The potentially reversible state of hypermethylated tumor suppressor gene promoters in cancer creates a powerful target for new strategies in the treatment and diagnosis of cancer.

Acknowledgements P.N. is supported by an Erwin Schroedinger fellowship of the Austrian Fond Wissenschaftlicher Forschung (FWF). This work was supported by grants from NIH (R01CA70896, RO1CA75503, RO1CA86072), the Pfeiffer Foundation, The Susan Komen Breast Cancer Foundation (RO1CA093596) (to R.G.P.). Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core National Institute of Health grant 5-P30CA13330-26. J.M.G. is supported by NIH grants DK02567 and DK56768.

References [1] L. Hayflick, P.S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res. 25 (1961) 585–621.

[2] L. Hayflick, The limited in vitro lifetime of human diploid strains, Exp. Cell Res. 37 (1965) 614–636. [3] L. Hayflick, Mortality and immortality at the cellular level: a review, Biochemistry 62 (1997) 1180–1190. [4] R. Sager, Senescence as a mode of tumor suppression, Environ. Health Perspect. 93 (1991) 59–62. [5] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [6] A.P. Wolffe, M.A. Matzke, Epigenetics: regulation through repression, Science 286 (1999) 481–486. [7] E. Wang, Senescent human fibroblasts resist programmed cell death and failure to suppress bcl2 is involved, Cancer Res. 55 (1995) 2284–2292. [8] J. Campisi, Replicative senescence: an old live’s tale? Cell 84 (1996) 497–500. [9] K.H. Bayreuther, H.P. Rodemann, R. Hommel, K. Dittmann, M. Albiez, P.I. Francz, Human skin fibroblasts in vitro differentiate along a terminal cell lineage, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 5112–5116. [10] G.P. Dimri, X. Lee, G. Basile, M. Acosta, G. Scott, C. Roskelley, E.E. Medrano, M. Linsken, I. Rubelj, O.M. Pereira-Smith, M. Peacocke, J. Campisi, A biomarker that identifies senescent human cells in culture and in aging skin in vivo, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 9363–9367. [11] E.L. Schneider, Y. Mitsui, The relationship between in vitro cellular aging and in vivo human age, Proc. Natl. Acad. Sci. U.S.A. 73 (1976) 3584–3588. [12] W.E. Wright, O.M. Pereira-Smith, J.W. Shay, Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts, Mol. Cell. Biol. 9 (1989) 3088–3092. [13] A.H. Wyllie, J.F. Kerr, A.R. Currie, Cell death: the significance of apoptosis, Int. Rev. Cytol. 68 (1980) 251–306. [14] G. Bergers, D. Hanahan, L.M. Croussens, Angiogenesis and apoptotic rates are cellular parameters of neoplastic progression in transgenic mouse models of tumorigenesis, Int. J. Dev. Biol. 42 (1998) 995–1002. [15] M.A. Shibata, I.G. Maroulakou, C.L. Jorcyk, L.G. Gold, J.M. Ward, J.E. Green, p53-independent apoptosis during mammary tumor progression in C3 (1)/SV40 large T antigen transgenic mice: suppression of apoptosis during the transition from pre-neoplasia to carcinoma, Cancer Res. 56 (1996) 2998–3003. [16] J.W. Shay, E.W. Wright, When do telomeres matter? Science 291 (2001) 839–840. [17] C.J. Sherr, R.A. DePinho, Cellular senescence: mitotic clock or culture shock? Cell 102 (2000) 407–410. [18] A.M. Olovnikov, A theory of marginotomy, J. Theor. Biol. 41 (1973) 181–190. [19] R.R. Reddel, The role of senescence and immortalization in carcinogenesis, Carcinogenesis 21 (2000) 477–884. [20] S.A. Stewart, R.A. Weinberg, Telomerase and human tumorigenesis, Semin. Cancer Biol. 10 (2000) 399–406. [21] C.W. Greider, Telomere length regulation, Ann. Rev. Biochem. 65 (1996) 337–365. [22] V. Urquidi, D. Tarin, S. Goodison, Role of telomerase in cell senescence and oncogenesis, Ann. Rev. Med. 51 (2000) 65–79.

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490 [23] L.L. Sandell, Loss of a yeast telomere: arrest, recovery and chromosomal loss, Cell 75 (1993) 729–739. [24] J. Lingner, J.P. Cooper, T.R. Cech, Telomerase and DNA end replication: no longer a lagging strand problem? Science 269 (1995) 1533–1534. [25] C.B. Harley, A.B. Futcher, C.W. Greider, Telomeres shorten during aging of human fibroblasts, Nature 345 (1990) 458– 460. [26] C.M. Counter, A.A. Avilion, C.E. LeFeuvre, Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity, EMBO J. 11 (1992) 1921–1929. [27] N.D. Hastie, M. Dempster, M.G. Dunlop, Telomere reduction in human colorectal carcinoma and with aging, Nature 346 (1990) 866–868. [28] M. Meyerson, C.M. Counter, E.N. Eaton, L.W. Ellisen, P. Steiner, S.D. Caddle, L. Ziaugra, R.L. Beijersbergen, M.J. Davidoff, Q. Liu, S. Bacchetti, D.A. Haber, R.A. Weinberg, hEST2, the putative human telomerase catalytic subunit gene, is upregulated in tumor cells and during immortalization, Cell 90 (1997) 785–795. [29] A.G. Bodnar, M. Ouelette, M. Frolkis, S.E. Holt, C.P. Chiu, G.B. Morin, C.B. Harley, J.W. Shay, S. Lichtsteiner, W.E. Wright, Extension of lifespan by introduction of telomerase into normal human cells, Science 279 (1989) 349–352. [30] J. Yang, E. Chang, A.M. Cherry, C.D. Bangs, Y. Oei, A. Bodnar, A. Bronstein, C.P. Chiu, G.S. Herron, Human endothelial cell life extension by telomerase expression, J. Biol. Chem. 274 (1999) 26141–26148. [31] J.W. Shay, S. Bacchetti, A survey of telomerase activity in human cancer, Eur. J. Cancer 33 (1997) 787–791. [32] T.M. Bryan, A. Engelzou, J. Gupta, S. Bacchetti, R.R. Reddel, Telomere elongation in immortal human cells without detectable telomerase activity, EMBO J. 14 (1995) 4240–4248. [33] J. Karlseder, A. Smogorzewsk, T. de Lange, Senescence induced by altered telomere state, not telomere loss, Science 295 (2002) 2446–2449. [34] M.A. Dickson, W.C. Hahn, Y. Ino, V. Ronfard, J.Y. Wu, R.A. Weinberg, D.N. Luis, F.. P Li, J.G. Rheinwald, Human keratinocytes that express hTERT and also bypass a p16INK4a/INK4a -enforced mechanism that limits lifespan become immortal yet retain normal growth and differentiation characteristics, Mol. Cell. Biol. 20 (2000) 1436–1447. [35] T. Kiyono, S.A. Foster, J.I. Koop, J.K. McDougall, D.A. Galloway, A.J. Klingelhutz, Both Rb7p16INK4a/INK4a inactivation and telomerase activity are required to immortalize human epithelial cells, Nature 396 (1998) 84–88. [36] I. Russo, A.R. Silver, A.P. Cuthbert, D.K. Griffin, D.A. Troo, R.F. Newbold, A telomere independent senescence mechanism is the sole barrier to Syrian hamster cell immortalization, Oncogene 17 (1998) 3417–3426. [37] S. Wei, J.M. Sedivy, Expression of catalytically active telomerase does not prevent premature senescence caused by overexpression of oncogenic Ha-Ras in normal human fibroblasts, Cancer Res. 59 (1999) 1539–1543.

1487

[38] B.T. Wakimoto, Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila, Cell 93 (1998) 321–324. [39] J.A. Baur, Y. Zou, J.W. Shay, W.E. Wright, Telomere position effect in human cells, Science 292 (2001) 2075–2077. [40] K.A. Won, Y. Xiong, D. Beach, M.Z. Gilman, Growthregulated expression of D-type cyclin genes in human diploid fibroblasts, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 9910– 99914. [41] G.H. Stein, L.F. Drullinger, A. Soulard, V. Dulic, Differential roles for cyclin-dependent kinase inhibitors p16INK4a and p21CIP1 in the mechanism of senescence and differentiation in human fibroblasts, Mol. Cell. Biol. 19 (1999) 2109–2117. [42] M. Serrano, A.W. Lin, M.E. McCurrach, D. Beach, S.E. Lowe, Oncogenic Ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a , Cell 88 (1997) 593–602. [43] D.A. Alcorta, Y. Xiong, D. Phelps, G. Hannon, D. Beach, J.C. Barret, Involvement of the cyclin-dependent kinase inhibitor p16INK4a in replicative senescence of normal human fibroblasts, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 13742– 13747. [44] J. Zhu, D. Woods, M. McMahon, J.M. Bishop, Senescence of human fibroblasts induced by oncogenic Raf, Genes Dev. 12 (1998) 2997–3007. [45] A.W. Lin, M. Barradas, J.C. Stone, L. vanAelst, M. Serrano, S.W. Lowe, Premature senescence involving p53 and p16INK4a is activated in response to constitutive MEK7MAPK mitogenic signaling, Genes Dev. 12 (1998) 3008–3019. [46] G. Ferbeyre, E. de Stanchina, E. Querido, N. Baptiste, C. Prives, S.W. Lowe, PML is induced by oncogenic Ras and promotes premature senescence, Genes Dev. 14 (2000) 2015–2027. [47] M. Pearson, R. Carbone, C. Sebastiani, M. Cioce, M. Fagiola, S. Saito, Y. Higashimoto, E. Appella, S. Minucci, P.P. Pandolfi, P.G. Pelicci, PML regulates p53 acetylation and premature senescence by oncogenic Ras, Nature 13 (2000) 207–210. [48] N. Ohtani, Z. Zebedee, T.J.G. Huot, J.A. Stinson, M. Sugimoto, Y. Ohashi, A.D. Sharrocks, G. Peters, E. Hara, Opposing effects of Ets and Id proteins on p16INK4a/INK4a expression during cellular senescence, Nature 409 (2001) 1067–1070. [49] H.J. Xu, Y. Zhou, W. Ji, G.S. Perng, R. Kruzelock, C.T. Kong, R.C. Bast, G.B. Mills, J. Li, S.X. Hu, Re-expression of the retinoblastoma protein in tumor cells induces senescence and telomerase inhibition, Oncogene 15 (1997) 2589–2596. [50] K. Alexander, P.W. Hinds, Requirement for p27KIP1/KIP1 in retinoblastoma protein mediated senescence, Mol. Cell. Biol. 21 (2001) 3616–3631. [51] B.K. Duncan, J.H. Miller, Mutagenic deamination of cytosine residues in DNA, Nature 287 (1980) 560–561. [52] E.S. Lander, L.M. Linton, et al., Initial sequencing and analysis of the human genome, Nature 409 (2001) 860–921. [53] L. Ponger, L. Duret, D. Mouchiroud, Determinants of CpG islands: expression in early embryo and isochore structure, Genome Res. 11 (2001) 1854–1860.

1488

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490

[54] S.B. Baylin, J.G. Herman, J.R. Graff, P.M. Vertino, J.P. Issa, Alterations in DNA methylation: a fundamental aspect of neoplasia, Adv. Cancer Res. 72 (1997) 141–196. [55] S.U. Kass, N. Landsberger, A.P. Wolffe, DANN methylation directs a time-dependent repression of transcription initiation, Curr. Biol. 7 (1997) 157–165. [56] L. Piko, M.D. Hammons, K.D. Taylor, Amounts, synthesis, and some properties of intracisternal A particle-related RNA in early mouse embryos, Proc. Natl. Acad. Sci. U.S.A. 81 (1984) 488–492. [57] M.R. Roundtree, K.E. Bachmann, J.G. Hermann, S.B. Baylin, DNA methylation, chromatin inheritance and cancer, Oncogene 20 (2001) 3156–3165. [58] J.A. Yoder, R.C. Yen, P.M. Vertino, T.H. Bestor, S.B. Baylin, New 5 -regions of the murine and human genes for DNA (cytosine-5)-methyltransferase, J. Biol. Chem. 271 (1996) 31092–31097. [59] A. Bird, DNA methylation de novo, Science 286 (1999) 2287–2288. [60] M.R. Rountree, K.E. Bachman, S.B. Baylin, DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci, Nat. Genet. 25 (2000) 269–277. [61] K.E. Bachman, M.R. Rountree, S.B. Baylin, Dnmt3a and Dnmt3b are transcriptional repressors that exhibit unique localization properties to heterochromatin, J. Biol. Chem. 276 (2001) 32282–32287. [62] A. Hasse, W.A. Schulz, Enhancement of reporter gene de novo methylation by DNA fragments from the ␣-fetoprotein control region, J. Biol. Chem. 269 (1994) 1821–1826. [63] P.A. Yates, R.W. Burman, P. Mummaneni, S. Krussel, M.S. Turker, Tandem B1 elements located in a mouse methylation center provide a target for de novo DNA methylation, J. Biol. Chem. 274 (1999) 36357–36361. [64] P. Arnaud, C. Goubely, T. Pelissier, J.M. Deragon, SINE retroposons can be used in vivo as nucleation centers for de novo methylation, Mol. Cell. Biol. 20 (2000) 3434–3441. [65] H. Tamaru, E.U. Selker, A histone H3 methyltransferase controls DNA methylation in Neurospora crassa, Nature 414 (2001) 277–283. [66] A.H. Peters, D. O’Carroll, H. Schertan, K. Mechtler, S. Sauer, C. Schofer, K. Weipoltshammer, M. Pagani, M. Lachner, A. Kohlmaier, S. Opravil, M. Doyle, M. Sibilia, T. Jenuwein, Loss of the suv39h histone methyltransferases impairs Mammalian heterochromatin and genome stability, Cell 107 (2001) 323–337. [67] T.H. Bestor, The DNA methyltransferases of mammals, Hum. Mol. Genet. 9 (2000) 2395–2402. [68] B. Hendrich, A. Bird, Mammalian methyltransferases and methyl-CpG binding domains: proteins involved in DNA methylation, Curr. Top. Microbiol. Immunol. 249 (2000) 55–74. [69] H.H. Ng, A. Bird, DNA methylation and chromatin modification, Curr. Opin. Genet. Dev. 9 (1999) 158–163. [70] J.A. Yoder, C.P. Walsh, T.H. Bestor, Cytosine methylation and the ecology of intragenomic parasites, Trends Genet. 13 (1997) 335–340.

[71] C. Grunau, S.J. Clark, A. Rosenthal, Bisulfite genomic sequencing: systematic investigation of critical experimental parameters, Nucleic Acids Res. 29 (2001) E65–65. [72] A. Bird, M. Taggart, R.D. Nicholls, D.R. Higgs, A fraction of the mouse genome that is derived from islands of non-methylated, CpG-rich DNA, Cell 40 (1985) 91–99. [73] G.A. Deng, A. Chen, E. Pong, Y.S. Kim, Methylation in hMLH1 promoter interferes with its binding to transcription factor CBF and inhibits gene expression, Oncogene 20 (2001) 7120–7127. [74] H. Cui, E.L. Niemitz, J.D. Ravenel, P. Onyango, S.A. Brandenburg, V.V. Lobanenkov, A.P. Feinberg, Loss of imprinting of insulin-like growth factor II in Wilm’s tumor commonly involves altered methylation but not mutations of CTCF or its binding site, Cancer Res. 61 (2001) 4947–4950. [75] H. Nakagawa, R.B. Chadwick, P. Peltomaki, C. Plass, Y. Nakamura, A. De La Chapelle, Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in a core region of H19-associated CTCF-binding sites in colorectal cancer, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 591–596. [76] R.Z. Chen, U. Pettersson, C. Beard, L. Jackson-Grusby, R. Jaenisch, DNA hypomethylation leads to elevated mutation rates, Nature 395 (1998) 89–93. [77] C.M. Tuck-Muller, A. Narayan, F. Tsien, D.F. Smeets, J. Sawyer, E.S. Fiala, O.S. Sohn, M. Ehrlich, DNA hypomethylation and unusual chromosome instability in cell lines from ICF syndrome patients, Cytogenet. Cell Genet. 89 (2000) 121–128. [78] G.L. Xu, T.H. Bestor, D. Bourchis, C.L. Hsieh, N. Tommerup, M. Bugge, M. Hulten, X. Qu, J.J. Russo, E. Viegas-Pequignot, Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene, Nature 402 (1999) 187–191. [79] P.J. Lee, L.L. Washer, D.J. Law, C.R. Boland, I.L. Horon, A.P. Feinberg, Limited upregulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 10366–10370. [80] K.D. Robertson, E. Uzvolgyi, G. Liang, C. Talmadge, J. Sumegi, F.A. Gonzales, P.A. Jones, The human DNA methyltransferases (DNMTs) 1, 3a and 3b coordinate mRNA expression in normal tissues and overexpression in tumors, Nucleic Acids Res. 27 (1999) 2291–2298. [81] S.A. Belinsky, S.A. Belinsky, K.J. Nikula, S.B. Baylin, J.P. Issa, Increased cytosine DNA methyltransferase activity is target cell-specific and an early event in lung cancer, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 4045–4050. [82] K.D. Robertson, E. Uzvolgyi, G. Liang, C. Talmadge, J. Sumegi, F.A. Gonzales, P.A. Jones, The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors, Nucleic Acids Res. 27 (1999) 2291–2298. [83] J. Wu, J.P. Issa, J. Herman, D.E. Bassett Jr., B.D. Nelkin, S.B. Baylin, Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH3T3 cells, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 8891–8895.

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490 [84] P.M. Vertino, R.W. Yen, J. Gao, S.B. Baylin, De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5)-methyltransferase, Mol. Cell. Biol. 16 (1996) 4555–4565. [85] A.R. MacLeod, M. Szyf, Expression of antisense to DNA methyltransferase mRNA induces DNA demethylation and inhibits tumorigenesis, J. Biol. Chem. 270 (1995) 8037– 8043. [86] J.P. Issa, Aging, DNA methylation and cancer, Crit. Rev. Oncol. Hematol. 32 (1999) 31–43. [87] P.W. Laird, R. Jaenisch, The role of DNA methylation in cancer genetic and epigenetics, Annu. Rev. Genet. 30 (1996) 441–464. [88] I. Rhee, K.E. Bachman, B.H. Park, K.W. Jair, R.W. Chiu Yen, K.E. Schuebel, H. Cui, A.P. Feinberg, C. Lengauer, K.W. Kinzler, S.B. Baylin, B. Vogelstein, DNMT1 and DNMT3b cooperate to silence genes in human cancer cells, Nature 416 (2002) 552–556. [89] L. Di Croce, V.A. Raker, M. Corsaro, F. Fazi, M. Fanelli, M. Faretta, F. Fuks, F. Lo Coco, T. Kouzarides, C. Neri, S. Minucci, P.G. Pelicci, Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor, Science 295 (2002) 1079–1082. [90] J.P. Issa, Y.L. Ottaviano, P. Celano, S.R. Hamilton, N.E. Davidson, S.B. Baylin, Methylation of the estrogen receptor CpG island links aging and neoplasia in human colon, Nat. Genet. 7 (1994) 536–540S. [91] Y.L. Ottaviano, J.P. Issa, F.F. Parl, H.S. Smith, S.B. Baylin, N.E. Davidson, Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells, Cancer Res. 54 (1994) 2552– 2555. [92] R.G. Lapidus, A.T. Ferguson, Y.L. Ottaviano, F.F. Parl, H.S. Smith, S. Weitzman, S.B. Baylin, J.P.J. Issa, N.E. Davidson, Methylation of estrogen and progesterone receptor gene of CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors, Clin. Cancer Res. 2 (1996) 805–810. [93] J.P.J. Issa, P.M. Vertino, C.D. Boehm, I.F. Newsham, S.B. Baylin, Switch from mono- to bi-allelic human IGF2 promoter methylation during aging and carcinogenesis, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 11757–11762. [94] Z. Siegfried, S. Eden, M. Mendelsohn, X. Feng, B.Z. Tsuberi, H. Cedar, DNA methylation represses transcription in vivo, Nat. Genet. 22 (1999) 203–206. [95] A. Razin, R. Shemer, Epigenetic control of gene expression, Results Probl. Cell. Differ. 25 (1999) 189–204; B. Baylin, J.G. Herman, DNA hypermethylation in tumorigenesis: epigenetics joins genetics, Trends Genet. 16 (2000) 168–174. [96] M. Szyf, The role of DNA methyltransferase 1 in growth control, Front. Biosci. 6 (2001) 599–609. [97] H.H. Ng, Y. Zhang, B. Hendrich, C.A. Johnson, B.M. Turner, H. Erdjument-Bromage, P. Tempst, D. Reinberg, A. Bird, MBD2 is a transcriptional repressor belonging to the MeCP1–histone deacetylase complex, Nat. Genet. 23 (1999) 58–61.

1489

[98] P.A. Wade, A. Gegonne, P.L. Jones, E. Ballestar, F. Aubry, A.P. Wolffe, Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation, Nat. Genet. 23 (1999) 62–66. [99] S.K. Bhattacharya, S. Ramchandani, N. Cervoni, M. Szyf, A mammalian protein with specific demethylase activity for mCpG DNA, Nature 39 (1999) 7579–7583. [100] S. Ramchandani, S.K. Bhattacharya, N. Cervoni, M. Szyf, DNA methylation is a reversible biological signal, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 6107–6112. [101] M. Szyf, D.J. Knox, S. Milutinovic, A.D. Slack, F.D. Araujo, How does DNA methyltransferase cause oncogenic transformation? Ann. N. Y. Acad. Sci. 910 (2000) 156–174 (Discussion, pp. 175–177). [102] A.R. MacLeod, J. Rouleau, M. Szyf, Regulation of DNA methylation by the Ras signaling pathway, J. Biol. Chem. 270 (1995) 11327–11337. [103] J. Rouleau, A.R. MacLeod, M. Szyf, Regulation of the DNA methyltransferase by the Ras–AP-1 signaling pathway, J. Biol. Chem. 270 (1995) 1595–1601. [104] F.D. Araujo, J.D. Knox, M. Szyf, G.B. Price, M. Zannis-Hadjopoulos, Concurrent replication and methylation at mammalian origins of replication, Mol. Cell. Biol. 18 (1998) 3475–3482. [105] K.D. Robertson, K. Keyomarsi, F.A. Gonzales, M. Velicescu, P.A. Jones, Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G0 /G1 to S phase transition in normal and tumor cells, Nucleic Acids Res. 28 (2000) 2108–2113. [106] S. Ramchandani, A.R. MacLeod, M. Pinard, E. von Hofe, M. Szyf, Inhibition of tumorigenesis by a cytosine-DNA, methyltransferase, antisense oligodeoxy nucleotide, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 684–689. [107] C.M. Bender, M.M. Pao, P.A. Jones, Inhibition of DNA methylation by 5-aza-2 -deoxycytidine suppresses the growth of human tumor cell lines, Cancer Res. 58 (1998) 95–101. [108] J.D. Knox, F.D. Araujo, P. Bigey, A.D. Slack, G.B. Price, M. Zannis-Hadjopoulos, M. Szyf, Inhibition of DNA methyltransferase inhibits DNA replication, J. Biol. Chem. 275 (2000) 17986–17990. [109] S. Milutinovic, J.D. Knox, M. Szyf, DNA methyltransferase inhibition induces the transcription of the tumor suppressor p21CIP1(WAF1/CIP1/sdi1) , J. Biol. Chem. 275 (2000) 6353– 6359. [110] K.D. Robertson, S. Ait-Si-Ali, T. Yokochi, P.A. Wade, P.L. Jones, A.P. Wolffe, DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters, Nat. Genet. 25 (2000) 338–342. [111] F. Fuks, W.A. Burgers, A. Brehm, L. Hughes-Davies, T. Kouzarides, DNA methyltransferase Dnmt1 associates with histone deacetylase activity, Nat. Genet. 24 (2000) 88–91. [112] M.R. Rountree, K.E. Bachman, S.B. Baylin, DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci, Nat. Genet. 25 (2000) 269–277. [113] Y. Sowa, T. Orita, S. Hiranabe-Minamikawa, K. Nakano, T. Mizuno, H. Nomura, T. Sakai, Histone deacetylase inhibitor activates the p21CI P 1/W AF 1/CI P 1 gene promoter through

1490

[114]

[115]

[116]

[117]

[118] [119]

[120]

[121]

[122]

P. Neumeister et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 1475–1490 the Sp1 sites, Ann. N.Y. Acad. Sci. 886 (1999) 195–199; A. Bird, The essentials of DNA methylation, Cell 70 (1992) 5–8. W. Wharton, J. Savell, W.D. Cress, E. Seto, W.J. Pledger, Inhibition of mitogenesis in BALB/c-3t3 cells by trichostatin A: multiple alterations in the induction and activation of cyclin/cdk complexes, J. Biol. Chem. 275 (2000) 33981– 33987. W.A. Palmisano, K.K. Divine, G. Saccomanno, F.D. Gilliland, S.B. Baylin, J.G. Herman, S.A. Belinsky, Predicting lung cancer by detecting aberrant promoter methylation in sputum, Cancer Res. 60 (2000) 5954–5958. A.M. De Marzo, V.L. Marchi, E.S. Yang, R. Veeraswamy, X. Lin, W.G. Nelson, Abnormal regulation of DNA methyltransferase expression during colorectal carcinogenesis, Cancer Res. 59 (1999) 3855–3860. S.J. Nass, A.T. Ferguson, D. El-Ashry, W.G. Nelson, N.E. Davidson, Expression of DNA methyltransferase (DMT) and the cell cycle in human breast cancer cells, Oncogene 18 (1999) 7453–7461. L. Guarente, Sir2 links chromatin silencing, metabolism and aging, Genes Dev. 14 (2000) 1021–1026. M.M. Kaeberlein, Mc. Vey, L. Guarente, The SIR2/SIR3/ SIR4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms, Genes Dev. 13 (1999) 2570–2580. Y. Tsukamoto, J. Kato, H. Ikeda, Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae, Nature 388 (1997) 900–903. S.G. Martin, T. Laroche, N. Suka, M. Grunstein, S.M. Gasser, Re-localization of telomeric Ku and SIR proteins in response to DNA double strand breaks in yeast, Cell 97 (1999) 631–633. K.D. Mills, D.A. Sinclair, L. Guarente, MEC-1 dependent redistribution of the Sir3 silencing protein from telomeres to DNA double strand breaks, Cell 97 (1999) 609–620.

[123] S. Gottlieb, R.E. Esposito, A new role for yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA, Cell 56 (1989) 771–776. [124] D.A. Sinclair, L. Guarente, Extrachromosomal rDNA circles: a cause of aging in yeast, Cell 91 (1997) 1–20. [125] S. Imai, C. Armstrong, L. Guarente, Silencing and aging protein Sir2 is an NAD-dependent histone deacetylase, Nature 403 (2000) 795–800. [126] J. Luo, A.Y. Nikolaev, S. Imai, D. Chen, F. Su, A. Shiloh, L. Guarente, W. Gu, Negative control of p53 by Sir2␣ promotes cell survival under stress, Cell 107 (2001) 137–148. [127] H. Vaziri, S.K. Dessain, E.N. Eaton, S. Imai, R.A. Frey, T.K. Pandita, L. Guarente, R.A. Weinberg, hSIR2SIRT1 functions as an NAD-dependent p53 deacetylase, Cell 107 (2001) 149–159. [128] B. Vogelstein, D. Lane, A.J. Levine, Surfing the p53 network, Nature 408 (2000) 307–310. [129] M. Hollstein, K. Rice, M.S. Greenblatt, T. Soussi, R. Fuchs, T. Sorlie, E. Hovig, B. Smith-Sorensen, R. Montesano, C.C. Harris, Database of p53 gene somatic mutations in human tumors and cell lines, Nucleic Acids Res. 22 (1994) 3551– 3555. [130] C. Wang, M. Fu, S. Mani, S. Wadler, A.M. Senderowicz, R. Pestell, Histone acetylation and the cell cycle in cancer, Front. Biosci. 6 (2001) 610–629. [131] C. Wang, M. Fu, R.H. Angeletti, L. Siconolfi-Baez, A.T. Reutens, C. Albanese, M.P. Lisanti, B.S. Katzenellenbogen, S. Kato, T. Hopp, S.A. Fuqua, G.N. Lopez, P.J. Kushner, R.G. Pestell, Direct acetylation of the estrogen receptor alpha hinge region by p300 regulates transactivation and hormone sensitivity, J. Biol. Chem. 276 (2001) 18375–18383. [132] M. Fu, C. Wang, A.T. Reutens, J. Wang, R.H. Angeletti, L. Siconolfi-Baez, V. Ogryzko, M.L. Avantaggiati, R.G. Pestell, p300 and p300/cAMP-response element-binding protein-associated factor acetylate the androgen receptor at sites governing hormone-dependent transactivation, J. Biol. Chem. 275 (2000) 20853–20860.