1 Department of Plant Sciences, University of Tennessee, 2431 Center Drive Knoxville, TN 37996-4500, USA and 2 Department of Physics, Wright State University, 3640 Colonel Glenn Hwy, Dayton, OH 45435, USA
* To whom correspondence should be addressed. Tel: +1 865 974 0710; Fax: +1 865 974 5365; Email: tkarpine{at}utk.edu
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Abstract |
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Abbreviations: EA, epigenetic alternations; EP, epigenetically reprogrammed; MR, mutator response; SSE, sustained stress environment
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Introduction |
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Epigenetic control of gene activity is widespread in the genome of eukaryotic cells and leads to persistent gene silencing or gene expression. This control is implemented by changes in the methylation status of DNA (the methylation of cytosine residues at 5' carbon) and by chromatin modifications (e.g. histone acetylation, phosphorylation, methylation and ubiquitination). The epigenetic modifications efficiently control the metastable state of gene expression in the genome by inducing stable silencing of some genes and promoting activation of others (3,4). The epigenetic control of gene expression is heritable through cell division, but reversible, because it does not involve DNA sequences. DNA methylation is the best studied EA. It is commonly implicated in the programming of the genome during embryonic development providing its stability by silencing genes of inactive X chromosome, ensuring parent-of-origin specific expression of imprinted genes, regulating tissue specific expression, and silencing of interspersed repetitive retrotransposons and heterochromatic satellite repeat sequences (5).
Recent studies reveal the involvement of DNA methylation and histone modifications in the reprogramming of the genome of mammalian cells during many age-related human diseases including cancer. These EA have been mainly elucidated by comparing cellular genomes in healthy and pathological states and therefore, terms hypermethylation and hypomethylation refer to the degree of methylation when compared with the healthy state. Well known changes in methylation related to cancer are summarized in several recent reviews (3,4,68). They include (i) tumor or age related hypermethylation of specific CpG islands and coding regions resulting in silencing of previously active genes, (ii) changes in methylation of differentially methylated regions leading to loss of imprinting and (iii) hypomethylation of previously silent genes and repetitive sequences resulting in chromosomal instability, mitotic recombination, chromosomal loss and aneuploidy.
The exact cellular mechanism that provides epigenetic transformations is not established, but many observations indicate a relationship between the level of gene activity in the genome, the methylation state of related DNA and chromatin modifications. Several lines of evidence suggest that gene expression is inversely correlated with DNA methylation (9,10). Mammalian cells tend to methylate promoter sequences of those genes in the genome that cease their participation in cellular activity. It is proposed that chromatin remodeling as a result of histone deacetylation and methylation is the primary event in initiating gene silencing, whereas DNA methylation, a subsequent event, establishes a permanent state of gene inactivation (11). It was also postulated that methyl-binding proteins, a component of the promoter methylation mechanism, have to compete with transcription factor abundance for the establishment of gene repression (12). The existence of this competition may explain why steady cessation of a cellular function and a concomitant lack of transcription factors steadily decreases gene activity related to this function. Conversely, a steady presence of the transcription factors shifts the balance to demethylation of the promoter. Therefore, while hypermethylation of gene promoters relates to transcriptional silencing of unwanted genes, hypomethylation indicates an increase in the level of gene activity (4).
We present a hypothesis that considers EA in cells imposed by stress-related proliferative and survival signaling in a SSE as starting events in cancer development. The hypothesis describes potential epigenetic mechanisms involved in priming cells for the generation of beneficial mutations in the genome and quick adaptation to SSE. We provide support of the hypothesis from the published literature and discuss its potential implications.
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Hypothesis |
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Mutator phenotype of the ER cells. Continuing stress-induced replication of the ER cells leads to their senescence. In normal cells replicative senescence is accomplished by cell-cycle arrest, i.e. cells remain viable, but cannot subsequently divide. ER cells cannot activate the cell-cycle arrest, apoptosis or DNA repair, because these processes are silenced epigenetically in the cells. Therefore, they eventually continue the proliferation in an error-prone mode, regardless of replicative senescence or DNA damage in the genome.
Tumor-related mutations in the ER cells. We hypothesize that mutations generated in the error-prone replications are not random. They match EA in the genome of the ER cells. Hypermethylation of CpG dinucleotide predisposes the ER cells to point mutation, frameshift mutation, missense mutation and deletions. These dinucleotides are often dominated in gene promoters. Therefore, hypermethylation of a promoter will lead to a loss of gene function. Hypomethylation predisposes cells to chromosomal mutations, rearrangements and aneuploidy. These mutations usually confer a gain of gene function. In addition, global demethylation imposed by SSE leads to a variety of mutational effects and to a diversification of the genome through the activity of DNA repeats. In this way, EA in the genome of the ER cells (hypermethylation and hypomethylation imposed by SSE) predispose to generation of certain stress-associated adaptive mutations in the error-prone replications. As a result, the ER cells give rise to mutant cells bearing the tumor-related mutations. The natural selection of the mutant cells is accelerated by their continuing proliferation in the stressful environment. It favors the survival of mutant cells harboring mutations that are most suitable for this environment, i.e. confer immortality and high proliferative activity. Multiple rounds of error-prone replication fix these mutations in the genome of progenies and lead to the emergence of transformed cells. In this way, EA in the genome imposed by proliferative and survival signaling in SSE drive the tumor development.
The following experimental findings support the model and will be discussed in more detail in the following sections.
Experimental studies that support mechanisms underlying the activation of the MR and the natural selection via apoptosis are presented elsewhere (2).
EA in the genome play a leading role in the origin of cancer and its progression
According to the proposed hypothesis, the EA induced by continuing replication in the cellular genome (epigenetic reprogramming) are indispensable preliminary events for cancer-related genetic mutation. Two stress-induced epigenetic events are a requisite for cancer development in the hypothesis. They are (i) epigenetic silencing/activation of oncogenes involved in proliferation, cell-cycle control and DNA repair, and (ii) global hypomethylation of the genome. The epigenetic silencing/activation of oncogenes is a necessary introduction to adaptive genetic mutation in the cell, because it primes the genome for the error-prone cell division and provides epigenetic tags to genes that are under selection pressure in the cellular microenvironment. Global hypomethylation is important for subsequent adaptation of the cells to a new environment and for a quick acquisition of new cellular functions, because demethylation of the genome leads to activation of transposable elements and facilitates chromosomal mutations in the cells including a chromosome loss or a gain.
In support of a leading role of these mechanisms in tumorigenesis, many studies draw attention to hypermethylation and hypomethylation taking place at precancerous and early stages of cancer. Some of these studies are summarized in Tables I and II. Given a role of EA in the control of a stable state of gene expression, the early onset of hypermethylation and hypomethylation of the genes found in the studies indicates that persistent changes in the related cellular functions occur before tumorigenic transformations. In concordance with the hypothesis, studies show early epigenetic silencing of genes involved in the activation of cell-cycle arrest and apoptosis including cyclin-dependent kinase inhibitors p16, p14, p15, p73, APC gene and DAP-kinase (Table I). The other processes subjected to early hypermethylation in different tissues include the regulation of cell-to-cell adhesion (E-cadherin and H-cadherin, MINT31, SYK, TIMP-3, Snail), suppression of cytokine signaling (SOCS1, TGF-beta, SHP1, HIN-1), DNA repair (BRCA1, BRCA2, MGMT, hMLH1, hMLH2) and regulation of cell differentiation (SFRP, APC, RARbeta2, TGF-beta). Early hypomethylation (Table II) is found for transcription factors involved in activation of proliferation (c-myc, c-fos) and transduction of proliferative and survival signals (EGFR, Ras). Interestingly, when one of the two alleles is mutated in the germ line of a patient with a familial form of cancer, hypermethylation is commonly seen as the second inactivation event (48). Specifically, this epigenetic effect was reported for p16 gene and E-cadherin (49,50). Additional evidence on a leading role for DNA methylation at the preneoplastic stages of cancer development was reviewed by Jones and Laird (51) including the fact that reverse demethylation of methylated genes suppresses the formation of some tumors.
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Global hypomethylation of the genome was also reported as an early epigenetic event in several cancers (Table II). Though, if hypermethylation and hypomethylation of oncogenes are more often considered in the studies as important events in the origin of tumors, increased demethylation or hypomethylation strongly relates to progression of cancers and increased malignancy. A positive correlation between the degree of global hypomethylation, the grade of malignancy in tumors and chromosomal alterations are found in many cancers (5255). Hypomethylation is very often observed in DNA repeats (4). The repeats that display tumor-associated hypomethylation include endogenous retrotransposons, LINE-1 repeats, Alu repeats, endogeneous retroviruses and moderately repeated non-viral DNA sequences. It is believed that hypomethylation is necessary for metastasis and invasion in cancer development. Several examples of this role of hypomethylation in breast cancer are given in the review of Szyf et al. (56). These experimental observations indicate a leading role of EA not only in the origin of cancer, but also in its progression.
EA in cells exposed to SSEs are similar to those indicated at premalignant and early stages of cancer development
According to the model the EA indicated in cells at premalignant stages result from their epigenetic reprogramming in SSE. The reprogramming may be induced by continuing production of cytokines in this environment because they activate proliferative and stress-related survival signals in the cells. Proliferative signaling activates genes and pathways involved in cellular replication. Survival signaling suppresses the activation of cell-cycle arrest and apoptosis. Inactivation of DNA repair pathways may follow persistent suppression of genes involved in the cell-cycle control (like APC gene and p53) that mediate DNA repair pathways (57,58). In long-term, these changes in gene expression lead to tumor-related EA in the genome.
In support of the role of SSEs in epigenetic reprogramming, EA in oncogenes (similar to reviewed above), global hypomethylation and activation of DNA repeats are reported in non-malignant mammalian cells, if they are exposed to well-known carcinogens (gamma and ultraviolet radiation, tobacco smoke, toxicants with potent carcinogenic effects, etc.) or to oncogenic cellular stresses (chronic inflammation, wounding, oxidative stress, genotoxic stress, etc.) (Table III). These stresses usually impose continuing cellular assault and death and are therefore accompanied by persistent production of cytokines. Although studies on EA after exposure to carcinogens are not as extensive as studies in tumor cells, they also indicate (i) hypermethylation of crucial regulators of cell-cycle arrest and apoptosis (p16, p53, APC); (ii) hypomethylation of oncogenes involved in proliferation and stress-related signaling (c-myc and raf); (iii) global hypomethylation of the genome and activation of DNA repeats. These changes are similar to changes in the cellular processes at premalignant and early stages of cancers indicated in the previous section.
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Thus, we conclude that EA induced by exposure to carcinogens and by oncogenic cellular stresses are similar to those observed at premalignant and early stages of cancer. Similar to cancer cells, these EA indicate continuing proliferation of the cells accompanied by silencing of cell cycle arrest and apoptosis, and activation of DNA repeats. These observations are consistent with a central feature of the hypothesis that SSEs have a leading promotional role in the epigenetic reprogramming of the cells and their subsequent tumorigenic transformations.
Activity of cell-cycle inhibitors is essential to accomplish stress-induced replicative senescence and their inactivation in the senescent cells can lead to tumorigenic transformations
Further support for the hypothesis is provided by studies of replicative senescence. This process depends on the number of cell divisions and is triggered by short telomeres (the ends of linear chromosomes) that lose 50200 bp per round of replication. SSE is accompanied by cell death and renewal, and therefore, inevitably speeds up the cellular senescence. Many studies support the idea in the hypothesis that a stressful environment favors replicative senescence (82). In the recent review of Ben-Porath and Weinberg (83), cellular senescence is considered as a general stress response program of the cell with important biological functions including the onset of arrest on proliferation. Stress-related survival signal induced by Ras/Raf/MEK/ERK pathway caused cellular senescence in several cell types, and the activity of cell-cycle inhibitors p53 and p16 was indispensable to accomplish senescence by cell-cycle arrest. Inactivation of these genes prevents cell-cycle arrest and results in tumorigenic transformation (7,8486).
According to the studies in human fibroblasts and mammary epithelial cells, cell-cycle arrest response in senescent cells is maintained primarily by p53. P16 provides a dominant second barrier to the unlimited growth of human cells (86,87). Analysis of the methylation status of cancer-related genes in non-malignant senescent human cells from different tissues was reported by Yuasa (88). This study demonstrates that though the methylation of the promoters of cancer-related genes may also take place in normal senescence, age-related methylation never includes hypermethylation of p53 and p16 genes, which are crucial for the establishment of cell-cycle arrest in senescent cells. A different output may be observed in senescent mammalian cells subjected to continuing proliferative and survival signaling, because this environment leads to epigenetic silencing of genes involved in the onset of cell-cycle arrest and apoptosis (Tables I III). This silencing disables the onset of cell-cycle arrest in the senescent cells and leads to malignancy. Given the experimental observations, we can hypothesize that continuing replication of some cells (ER cells in the model) in SSE leads to their senescence in a way that cannot be accomplished by cell-cycle arrest, because, in addition to the replicative senescence, these cells have epigenetically silenced genes involved in cell-cycle control. This silencing in combination with persistent proliferative signals predisposes the ER cells to accomplish their divisions in an error-prone manner, resulting in the generation of mutations and subsequent malignant transformations of the cells.
Known molecular mechanisms may underlie the causative effects of EA on succeeding mutations in ER cells
Although mechanisms underlying the causative effects of EA on the succeeding mutations are poorly understood, some of them have been demonstrated in both hypermethylation and hypomethylation. A correlation between the methylation status of oncogenes and their mutations in tumor cells is also well documented (53,89,90).
The most prominent evidence of the link between EA and specific mutations is provided by studies of CpG dinucleotides. Mutagenic mechanisms involving 5-methylcytosine in CpG dinucleotides are particularly common. Methylated CpG dinucleotides are mutational hot spots in human genes including well-known cancer-relevant genes, such as p53 and retinoblastoma gene (90,91). The best characterized mechanism of G:CA:T transitions at CpG sites is deamination of 5-methylcytosine at CpG sites, resulting in substitution of 5-methylcytosine by thymine. This occurs spontaneously or is factor-mediated, e.g. by cyclobutane pyrimidine dimers after exposure to UVB and sunlight (92), by some environmental pollutants (93) or through the action of nitric oxide produced under conditions of chronic inflammation (94). The factor-mediated C to T transition mutations are particularly frequent in some codons of p53 gene at sequences containing 5-methylcytosines. It was also suggested that error-prone DNA polymerases play an important role in the mutagenic bypass of the lesions and generation of the mutations (92). Methylated CpG dinucleotides are mutation hot spots in the retinoblastoma gene (91). Mutational abnormalities indicated in Rb gene include G
A substitution in the promoter region (95). Germline mutations in this gene include frameshift mutations, missense mutation and in-frame deletions (96). Deletions (either homozygous or heterozygous) are a major mechanism of p16 and p15 genes inactivation (97). Loss of function mutations in these genes are well documented in tumors. Frequency of mutations in p16 is second only to mutation of p53. As it was reviewed above (Table I), hypermethylation of cell-cycle inhibitor p16 takes place at premalignant and early stages of many cancers and also may be imposed by exposure with carcinogens (Table III).
The following mechanisms may underlie the induction of matched mutations in genes with methylated CpG dinucleotides. It is generally agreed that C T mutations in CpG dinucleotides may be caused by spontaneous hydrolytic deamination. Cytosine deamination produces uracil, which can be removed by glycosylase, whereas deamination of 5-methylcytosine generates a GT mispair that cannot be processed by glycosylase and escapes proofreading, because it is not obvious which strand carries the correct genetic information (98). This is a common phenomenon, and in healthy cells, this type of replication error is repaired by the DNA mismatch repair system that is directed specifically to the newly synthesized strand and in this way increases the fidelity of DNA replication by up to three orders of magnitude (99). According to the model and experimental evidence considered above (Tables I
III), epigenetic silencing of cell-cycle inhibitors imposed by SSE activates an error-prone cellular division without participation of the repair machinery. As a result, mispairs at methylated CpG dinucleotides cannot be repaired and therefore produce point mutations or deletions and frameshift mutations in the case of replication slippage of error-prone DNA polymerase (100). In this way, hypermethylation status of CpG dinucleotides, which often characterizes TS genes in the earliest stage of cancer development, may predispose them to certain mutations, namely point mutation, frameshift mutations, missense mutation and deletions.
In addition to increased endogenous mutability of 5-methylcytosine, its interaction with carcinogens may also contribute to certain types of mutations (101). For example, many chemical carcinogens including aromatic hydrocarbons in tobacco smoke preferentially react with guanines flanked by 5-methylcytosine inducing the GT transversion mutations. The majority of mutations observed in skin cancer are C
T transitions or CC
TT mutations at dipyrimidine sequences resulting from UV-light-induced cyclobutane pyrimidine dimer formation. Methylation of cytosine increases this formation by up to 15-fold owing to higher energy absorption of 5-methylcytosine (102). These results suggest that hypermethylation of tumor suppressor genes, besides contributing to endogenous mutability, can also make these genes vulnerable to environmental mutagens and carcinogens.
The causative effect of hypomethylation on succeeding mutations is also well documented in tumors. If hypermethylation of CpG islands predisposes affected genes to point mutations, deletions and frameshift mutations, leading to loss of gene function, hypomethylation predisposes to chromosomal instability and to reshuffling of the mammalian genome by the activation of DNA repeats. Hypomethylated repetitive sequences are mainly located in the vicinity of the centromeres of chromosomes, comprised of heterochromatin (4). It predisposes cells to increased chromosomal rearrangements in this region, as takes place in many types of cancer. This type of mutation in tumors often provides a gain of gene function. Chromosomal instability has been linked to DNA hypomethylation in the heterochromatin regions of chromosomes in several papers (52,53,103). For example, correlation of DNA methylation status in heterochromatin regions with tumor histology and with chromosome alterations was found in a study of human breast carcinomas. Hypomethylation of these regions was associated with the accumulation of a large number of chromosome alterations and promoted the development of breast carcinomas with aggressive histological features (53). Likewise, genomewide DNA hypomethylation is associated with alterations on chromosome 8 in prostate carcinoma (52). A high percentage of Wilms tumors, breast adenocarcinomas and ovarian epithelial carcinomas have cancer-associated hypomethylation of the main satellite DNAs in the centromere-adjacent heterochromatin of chromosomes 1 and 16 (satellites Chr1 and Chr16), and it is associated with the recurrent rearrangements of the satellites in the vicinity of the centromere. These rearrangements usually lead to 1q gains or 16q losses (104).
The following mechanisms may underlie the effect of hypomethylation on chromosome instability. It was shown that in lymphoblastoid cell lines hypomethylation in the heterochromatin regions of chromosomes induced DNA uncoiling and rearrangements at these regions (103). In a study of human hepatocellular carcinoma samples, hypomethylation altered the interaction between the CpG-rich satellite DNA and chromatin proteins, resulting in heterochromatin decondensation, breakage and aberrant 1q formation (55). We may hypothesize that heterochromatin decondensation and DNA uncoiling in the centromeric regions of chromosomes lead to abnormalities in normal chromosome segregation during error-prone cell-cycle progression in the MR, resulting in karyotypic abnormalities during mitosis. In this way, the alteration in chromatin induced by hypomethylation may underlie chromosomal mutations in the ER cells.
Mobility of DNA repeats including retrotransposons and endogenous retroviruses imposed by their hypomethylation may also induce a variety of mutational effects. All retroelements are able to form an RNA transcript that may be reverse-transcribed and inserted into a new location in the genome. Different classes of retroelements may have their mechanisms for RNA formation, reverse transcription and integration (105), but in all instances they contribute to insertional mutagenesis that may modify promoter or coding gene sequences. Retrotransposition of long terminal repeats, like LINE1, may remodel the genome through the transduction of 3' flanking sequences. Such events have the potential to shuffle, e.g. exons and promoters, creating new genes or altering the function of old genes (106). LINE1 is also able to generate processed pseudogenes and chimeric retrotranscripts (107). Alu elements generate mutations by the insertion into mature mRNAs via a splicing-mediated process termed exonization (108). Repetitive human Alu elements may cause deleterious mutations both by retrotranspositional insertion and by unequal, homologous recombination (109). The variety of ways in which activated DNA repeats appear to affect genomic diversity in tumor cells lends additional support to the hypothesis, i.e. hypomethylation of DNA repeats in SSE prepare the genome for mutational modifications that are important for subsequent adaptation of the disseminated tumor cells to new environments.
Changes in methylation status of genes occur throughout the genome and involve many different genes and different codons within the gene so that the epigenetic tagging system cannot guarantee the generation of only beneficial mutations in the error-prone replication. Rather, this system facilitates the emergence of beneficial mutations in accord with the EA in the genome. Subsequent natural selection of the mutants by apoptosis adjusts the mutational spectra to the environment. Therefore, a continuing proliferation of the cells, a requisite of SSE, is very important not only for the epigenetic reprogramming of the cells but also for a quick shaping of the mutational spectra in the genome to the needs of the cellular environment. In support of this mechanism, several studies present evidence that the cytotoxicity of some carcinogens (like UVB light or tobacco smoke), rather than their mutagenicity, may drive tumorigenesis (110,111).
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Discussion |
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The mechanisms that allow cells to increase their mutation rates under conditions of sustained stress and produce the mutations in beneficial genes are especially important in evolutionary terms. Therefore, it is plausible that eukaryotic organisms retained these mechanisms in their cells and made them even more sophisticated. According to our hypothesis such an adaptive strategy is employed in the somatic cells of mammals in response to sustained stress and plays an important role in the development of malignances. In this case, a continuing proliferative and survival signaling in the tissue microenvironment indicates a poor adjustment of the cellular genome for the environment and hastens cellular adaptation. These signals are responsible for the epigenetic reprogramming of some cells leading to an increased rate of tumor-related mutations in the genome. Numerous studies support the fundamental role of the cellular environment in the transformations. This role is especially evident when we consider in vitro culture of multicellular organisms. Rodent cells can easily undergo spontaneous neoplastic transformation in culture and are widely used as in vitro models of cancer (119,120). A well-known example is the rodent fibroblast. In a primary culture of mouse embryo cells, a small number of cells, survived after apoptotic crises by forming immortalized cell lines, can overgrow the culture. These cells invariably display abnormal karyotypes and may undergo spontaneous transformations (120). This output is consistent with the proposed hypothesis. Culturing of the cells mimics SSE in a tissue, because of cellular assault and continuing proliferation of some cells. This signaling epigenetically reprograms the cells and primes them for malignant modifications in the genome. This transformation represents an adaptive response, which is similar to a bacterial one, to constraints on growth. Although human cells rarely undergo spontaneous transformation in culture, they also may be transformed by a stepwise process. For example, the transformation may be accomplished by immortalization of the cells by DNA viruses, followed by conversion of the immortalized cells to tumorigenic ones by Ras oncogenes or by using chemicals or chemical agents (121). DNA virus in this transformation provides sustained activation of c-myc that reproduces a persistent proliferative signal in the SSE. Ras-activation reproduces a stress-related survival signal that is necessary to silence genes involved in the cell-cycle arrest and to activate a global demethylation of the genome.
Delayed effects of environmental factors on cancer development may be explained in terms of the proposed hypothesis
An important inference of the proposed mechanism is that the nature of the mutations in cancer cells is not quite stochastic. The mutations are not just caused by DNA damaging agents in the cellular genome; the cell itself creates conditions to the generation of proper mutations by epigenetic modification of the genome in SSE. From this standpoint, mutations observed in tumors are not the reason for the disease, but a far-reaching consequence of cellular exposure to SSE. The mutations accomplish epigenetic changes in genes imposed by this environment. This mechanism may explain the delayed effects of toxicant exposure and the bystander effect of radiation on tumor developments, which are inconsistent with the accepted mechanism of direct DNA damage. It was shown, for example, that perinatal exposure of diethylstilbesterol (a drug with estrogenic activity) changed the methylation pattern of the promoters of several estrogen-responsive genes associated with the development of reproductive organs and resulted in a high incidence of epithelial cancers of the uterus in mice at 1824 months of age (122). Long-term effects were also revealed after the short-term exposure to arsenic. In this study, progeny of the exposed Chinese hamster cells showed an increased level of chromosomal rearrangements that were not seen in the parents after the acute exposure. Moreover, the mutations were related to genome DNA hypomethylation induced by arsenic after treatment (123). In a study of ionizing radiation, non-irradiated cells acquired mutagenesis through direct contact with cells whose nuclei had previously been irradiated with alpha-particles (124). It was also shown that the delayed effects of the ionizing radiation (delayed reproductive death or lethal mutation, chromosomal instability and mutagenesis) may be transmitted over many generations after irradiation through the progeny of surviving cells (125). Molecular mechanisms underlying these experimental findings are not known, but it is believed that it may be a consequence of bystander interactions involving intercellular signaling and production of cytokines (126), i.e. factors in the hypothesis that induce sustained proliferative and stress-related survival signals and epigenetically reprogram cells. In terms of the presented hypothesis, the exposure with carcinogens lead to accumulation of the reprogrammed cells with tumor-related EA. But their transformation may be triggered later by cellular senescence or another SSE. The same epigenetic mechanism may underlie the causative effect of parental exposure with carcinogens (pesticides, smoking and so on) on the development of childhood cancers (127).
Etiology of human cancers in terms of the proposed hypothesis
The proposed model of tumorigenesis is based on the analysis of principal common characteristics of different cancers. According to the model, these common characteristics arise as a result of sustained proliferative and survival signaling in a SSE. This signaling may be imposed by continuing production of cytokines in a tissue subjected to continuing cellular assault, for example as a result of the exposure with carcinogens. A similar stress-associated signaling may be induced by some other environmental and physiological factors including such well known inducers of cancer as inflammation, hormones and viral infection. The effect of these factors on cancer development also may be considered in terms of the proposed model.
The implication of hormones in the origin and development of many cancers is well documented. It is also known that many hormones activate both proliferative and survival signaling in cells inducing their proliferation on the one hand and inhibiting their apoptosis on the other (128). This signaling recapitulates the SSE characterized by continuing cellular assault. Although different pathways and genes may be involved in the signal transduction, both environments will impose similar cellular responses that include continuing proliferation of the exposed cells and silencing of cell-cycle arrest and apoptosis. In the long term, it will be accomplished by epigenetic reprogramming of some cells and their subsequent malignant transformation. Recent studies support the plausibility of this mechanism in the origin of breast and prostate cancers. The implication of estrogens in the development of breast cancer is well established. It is also known that this hormone activates the estrogen receptor and stimulates cellular proliferation by activation of AP-1 transcription factors (129) and cellular survival by activation of MAP kinase signaling (130). The other example is the development of prostate cancer. This cancer initially requires androgen for the growth, and responds to the hormone deprivation therapy. This responsiveness indicates that cellular apoptotic machinery is functional at this stage of cancer progression. It was found that activation of Ras-mediated stress-related survival signaling is sufficient, and may be necessary for progression of the disease; it may also induce the development of its androgen independent form (131). These examples indicate the involvement of proliferative and survival signals, which are crucial components of SSE, in breast and prostate cancers development.
The effect of transforming viruses on cellular processes also mimics many aspects of SSE. The expression of oncoproteins encoded by some viruses changes cellular processes in the host in the same way as continuing proliferative and survival signaling. Pathways involved in activation proliferation, cell-cycle arrest and DNA repair are common targets of the viral infection. A detailed summary of these effects for three well-studied oncogenic viruses (human T-cell leukemia virus type 1, hepatitis B virus and human papilloma virus) are given in the review of Gatza et al. (132). Although viral oncoproteins do not necessarily affect the same pathways, the overall mechanisms are highly analogous among the viruses. They activate/inactivate critical cellular processes to promote proliferation, suppress cell-cycle control and DNA repair. Moreover, they directly facilitate the activation of error-prone replication stimulating bypass of G1/S and G2/M checkpoints in the cell-cycle progression.
Approximately one-quarter of all malignancies are thought to arise in part to chronic inflammation (133). Similar to SSE imposed by carcinogens, this cellular environment leads to production of reactive oxygen species as an important element of cellular defense system. Therefore, inflammation, regardless of etiology, always causes cellular assault, DNA damage, cell death and renewal. It creates a tissue microenvironment rich in cytokines and growth factors that induce proliferative and stress-related survival signaling. Recent studies on the role of inflammation in cancer development are considered by Brower (134). It is believed that nuclear factorkappa B (a key transcription factor in infection and inflammation) can provide a link between chronic inflammation and cancer. It was shown that in the premalignant inflammatory environment, this transcription factor induces epithelial cell proliferation by upregulating macrophage-produced cytokines and also provides a survival signal suppressing cellular apoptosis. In summary, the effect of well-known inducers of tumorigenesis associated with the etiology of many different cancers may be described in terms of the proposed hypothesis.
Implications of the hypothesis
The proposed hypothesis may be helpful in the explanation of some common characteristics of tumor cells associated with their high adaptive potential. According to the hypothesis, the increased rate of mutations and their beneficial nature are crucial components of this potential acquired after epigenetic reprogramming of the cells in SSE. They allow the cells to easily overcome subsequent stresses imposed by tumor progression or medication. Metastasis and drug resistance are examples of implementations of this adaptive potential. Epigenetically reprogrammed and then transformed cells faced with a shortage of oxygen or nutrients, induced by overcrowding, can easily express those genes in the genome that increase their mobility and allow them to migrate and proliferate in new environments. In this way, the tumor cells acquire the sequential specialized adaptations that confer metastatic capabilities including the expression of metalloproteinases, angiogenesis, lymphangiogenesis and adhesion molecules (135). Ability to metastasize overcomes the environmental stress imposed by overcrowding. Similar survival strategies, which are based on increased mobility, are implemented by some unicellular organisms, like yeasts and slime mould colonies deprived of nutrients (136). A quick development of drug resistance, which is typical for cancer cells, is the other illustration of their high adaptability. A well-known example is a variety of mutational effects employed in the advanced prostate cancer in response to androgen ablation therapy (137).
The proposed insight into tumorigenesis may improve and extend our approaches to diagnostics, prevention and treatment of cancer. Screening of EA in genes involved in cell-cycle control, DNA repair and repetitive sequences in heterochromatin may provide a generalized picture of the exposure of cells to sustained stress and predisposition to cancer development. It opens the prospect of early diagnosis of cancer and its prevention, because EA are reversible and correction of cellular environment at this stage by proper treatment or by changing a lifestyle may be the most efficient way to prevent cancer development. Studies of the effect of environmental factors on EA and cancer development give indirect medical support for this conclusion (138). It was shown that dietary deficiencies, chemicals and genetics may alter methylation processes and cause tumor formation. Even environmental exposures in early life (e.g. estrogenic, xenobiotic, nutritional factors, stress) may alter DNA methylation and in this way modulate the risk of cancer and other chronic diseases in adulthood (139).
Although many experimental observations reviewed in the paper support the proposed model of tumorigenesis, direct evidence of the adaptation of mammalian cells to SSE by EA and succeeding matched mutations postulated in the model as the main driving force of tumorigenesis is actually absent. Additional studies are necessary to validate and refine the model.
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Acknowledgments |
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Conflict of Interest Statement: None declared.
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