As discussed in Chapter 3, the methylation of cytosine in DNA constitutes a mechanism not only of regulating the expression of DNA but also of maintaining the normal chromosomal integrity of the cell. These two mechanisms are quite interrelated, as more recent studies have indicated (cf. Jones and Laird, 1999; Gray et al., 1999). Methylation of cytosine residues in DNA occur primarily adjacent to guanine residues, forming CpG sequences in the DNA molecule. Where multiple CpG sequences are repeated up to about 1 kb in length, the region is termed a CpG island (cf. Laird, 1997). The methyl groups of the CpG sequence do not affect base pairing but do influence protein-DNA interaction by protruding into the major groove (Razin and Riggs,1980). In general, DNA methylation in CpG islands within promoter regions of specific genes inhibits the transcription of the gene by interfering with transcription initiation through reducing the binding affinity of sequence-specific transcription factors (cf. Jones and Laird, 1999). Fur- thermore, methylation-dependent, sequence-specific DNA-binding proteins that have been de- scribed (cf. Hendrich and Bird, 1998) may act as transcriptional repressors. Particularly in relation to our consideration of the stage of progression, DNA hypomethylation of mammalian cells enhances mutation rates (Chen et al., 1998) and may lead to chromosome and genomic instability (cf. Jones and Gonzalgo, 1997; Xu et al., 1999).
Figure 15.6 is a simplistic diagram demonstrating the silencing of tumor suppressor genes and the activation of proto-oncogenes. In this diagram, tumor suppressor gene silencing involves hypermethylation of CpG islands, as has been documented in a number of instances (see below). Proto-oncogene activation, on the other hand, may occur as a result of decreased methylation of specific CpG dinucleotides rather than CpG islands. As discussed in Chapter 3, endogenous mu- tation of genes may result from oxidative deamination of 5-methylcytosine to thymine. The mis- match monitoring repair system reverses the effect of these mutations in the vast majority of instances where they occur in vivo (Chapter 3). Thus, alterations in DNA methylation may theo- retically be involved in cancer causation by a variety of mechanisms, as indicated in Figure 15.6 and mentioned in the paragraph above.
There is substantial evidence that changes in DNA methylation occur during the stage of preneo- plasia. This evidence comes from the demonstration both of global changes in DNA methylation as well as changes in CpG methylated sites in specific genes. At the moment there is little if any evidence for alterations in the methylation of CpG islands during the preneoplastic process. In addition, diets deficient in precursors of methyl groups have been shown to induce DNA hypom- ethylation quite effectively and rapidly (Wainfan and Poirier, 1992). Several such diets also in- duce neoplasia, especially in the liver, upon prolonged administration (cf. Poirier, 1994, Chapter 3). Some suggestions that alterations in DNA methylation alone are sufficient for initiation have also been made (Boehm and Drahovsky, 1983). However, other studies indicate that such is not the case (Sawada et al., 1990) but rather that diets deficient in methyl group precursors are effec- tive promoting agents at least in the liver (Yokoyama et al., 1985). This may be expected, since alteration in DNA methylation results in alterations in gene expression (see above). Further- more, the effects of methyl-deficient diets are completely reversible, both as to the lesions in- duced and the methylation status of the DNA in the liver (Christman et al., 1993). The latter process, involving global DNA methylation as well as methylation of specific genes, recovers more slowly than the restoration of the normal morphology of the liver. It is important to note, however, that these latter studies investigated only a relatively few CpG sites of specific genes by the technique of isoschizomer restriction enzyme analysis. Thus, the slower return to normal DNA methylation status with this technology may not reflect the methylation of many DNA CpG sites, since there is no substantial evidence that global DNA methylation is retarded upon return to normal dietary administration. The slow return to normal DNA methylation status of some sites may reflect cell turnover.
Table 15.8 shows examples of the relative global and specific gene alterations in DNA methylation in some putatively preneoplastic lesions. Global DNA methylation occurs quite early upon feeding severely methyl-deficient diets, as early as 1 week after initiation of the diet. Simultaneous with such global demethylation, increases in the levels of mRNAs were seen for several oncogenes and decreases in other genes such as epidermal growth factor receptors (Wainfan and Poirier, 1992). Even administration of a specific hepatocarcinogen, such as dichlo- roacetic or trichloroacetic acid, causes transient decreases in the level of 5-methylcytosine in DNA in the liver. Interestingly, determination of exposure to dichloroacetic, but not trichloroace- tic acid, resulted in an increase of 5-methylcytosine in adenomas to the level found in nonin- volved liver, suggesting different mechanisms for the two carcinogens (Tao et al., 1998). The majority of the studies on alterations in CpG methylation in specific sites with the isoschizomer restriction enzyme technique could be related to alterations in the expression of the gene. Inter- esting exceptions were noted in a specific gene, CDKN2/p16, an inhibitor of the cell cycle in which bisulfite conversion of unmethylated cytosines to uracil results in DNA sequence changes, which can be analyzed and the specific methylcytosine sites determined. This tech- nique is more accurate than the isoschizomer technology, which looks at only several specific sequences but not at many other regions containing methylated cytosines (Saluz and Jost, 1993). Similar findings were seen with the glutathione S-transferase pi gene, specifically in the pro-
moter region of animals placed on a methyl-deficient diet (Steinmetz et al., 1998). In this in- stance, specific sites in the promoter region of the gene, several involving transcription factors that might otherwise not have been noted with the isoschizomer technique were found to be hy- pomethylated. This is in congruence with the high expression of the gene both in the livers of animals fed the methyl-deficient diet and in preneoplastic and neoplastic lesions in the livers of these animals. The presence of hypomethylation of specific genes in benign neoplasms of the liver in the rodent and in the colon in the human (Table 15.8) further suggests that some changes in methylation of specific CpGs may be important in the early development of neoplasia during the stage of promotion and extending into the stage of progression.
It is becoming increasingly clear that alterations in DNA methylation in one or more genes in neoplasia are likely without exception (cf. Liang et al., 1998; Baylin et al., 1998). The examples of global hypomethylation in neoplastic cells are numerous and most definitive in rapidly grow- ing, less differentiated neoplasms (Gama-Sosa et al., 1983; Kim et al., 1994). A few examples of altered methylation of specific genes in neoplasms may be noted in Table 15.9. In many of the references given, the newer and more definitive technology of bisulfite treatment with subse- quent PCR and sequencing is utilized. Both hypo- and hypermethylation of CpG dinucleotides are seen in components of the various genes listed. In most instances, the methylation occurs in the regulatory region of the gene and in general this may be correlated with an enhanced expres- sion of the gene in the case of DNA hypomethylation or a repression of expression with DNA hypermethylation. In the case of glutathione S-transferase, π hypermethylation occurs in two different types of neoplasms in the human, whereas hypomethylation occurs in hepatomas in the
rat. In all of these instances, the correlation with expression of the gene is quite close. Hyperme- thylation of the repair enzyme methylguanine-DNA methyltransferase, with resultant repression, may be associated with alteration in response to specific drugs or carcinogens (Esteller et al.,
1999). Hypermethylation has been consistently associated with the repression of tumor suppres- sor genes, several examples of which are noted in the table. In the case of proto-oncogenes, one might suppose that hypomethylation would be the rule, as is noted in the case of c-myc in human hepatomas, where increased expression is associated with hypomethylation (Nambu et al.,
1987). However, an interesting exception to this generalization is noted in the progressive de novo DNA methylation at the bcr-abl cellular oncogene locus during the progression of chronic myelogenous leukemia (Zion et al., 1994). In this instance, patients with this condition who ex- hibit dramatic hypomethylation of this gene at the time of diagnosis invariably demonstrate in- creased methylation of the fusion gene as the disease progresses. Thus, the great majority of the information available in this field demonstrates that alterations in DNA methylation occur early during the natural history of the development of neoplasia and progressively become more devi- ant as the cell enters and continues in the stage of progression. As already noted, such changes in DNA methylation lead to dramatic alterations in the expression of specific genes as well as in genomic stability. While it is tempting to speculate that such alterations in DNA methylation may be the basis for the evolving karyotypic instability seen in the stage of progression, such a conclusion is clearly premature at this time.
Alterations in Genomic Imprinting
In Chapter 5 the phenomenon of genomic imprinting was discussed (Figure 5.7), and several neoplasms having a genetic basis to their etiology were discussed in relation to the potential importance of genomic imprinting in their neoplastic expression (Table 5.7). In neoplasms not having a genetic basis, the loss of imprinting (LOI) or alteration in the normal imprinting pattern of a specific gene may be seen in a variety of neoplasms (Table 15.10). Although the table indi- cates only five genes that have been relatively well studied in neoplasms as compared with their normal counterparts, some 20 or more imprinted genes have been described in the mouse (Kelsey and Reik, 1998), involving at least seven different chromosomes. Many of these genes
have counterparts in the human and rat, in which imprinting has also been demonstrated, al- though occasionally the imprinting pattern between species differs (Bartolomei and Tilghman, 1997). As noted in the table, the imprinted genes most extensively investigated in neoplasia are the Igf2 (insulin growth factor-2) and H19 genes. LOI of Igf2 has been seen in a variety of neo- plasms, as noted in the table; in hepatic neoplasms in the human, however, biallelic expression of the gene in this tissue occurs after birth (cf. Aihara et al., 1998). But in the rodent, the imprint- ing of this gene is maintained during adult life (cf. Bartolomei and Tilghman, 1997). While this species differential may be related to imprinting of specific promoter regions in this gene (Wutz and Barlow, 1998), the Igf2 gene lies immediately adjacent to the H19 gene in rodents and the human. Note that the H19 gene expresses the maternal allele, while Igf2, the paternal allele (Table 15.10). Thus, the expression of the two genes is closely linked, and the differential imprinting has led to a number of theories related to the mechanism of this effect (Tilghman et al., 1993; Banerjee and Smallwood, 1995). The abnormal expression of these two genes is felt to be an important factor in the phenotype of patients with the Beckwith-Wiedemann syndrome (Table 5.1), which is characterized by extensive overgrowth postnatally and neoplasms such as Wilms tumor of the kidney (Reik and Maher, 1997). A couple of recent investigations have suggested that CTCF, a specific DNA binding protein that binds in the region intermediate be- tween the two genes, may play a major role in this mechanism (Bell and Felsenfeld, 2000; Hark et al., 2000).
While the Igf2 receptor gene (mannose 6-phosphate receptor) is paternally imprinted, most of the alterations that have been described involve major mutations and deletions of the gene rather than specifically LOI. Such lack of or uncontrolled expression of the gene is felt to play a role in both the early and late stages of hepatic neoplasia (de Souza et al., 1997). The p57KIP2 cdk inhibitor is also found in the same region as the Igf2/H19 complex in mouse, human, and rat; thus one might expect to see the effects noted in the table in Wilms tumors. The actual function of p73 is not clear as yet, and thus it is difficult to relate the LOI of this gene to specific functions in neoplasia. However, it is apparent from even these few studies that LOI can play an important role in the alteration of genetic expression so characteristically seen in neoplasms in the stage of progression.