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Dnmt1^N/+ Reduces the Net Growth Rate and Multiplicity of Intestinal Adenomas in C57BL/6-Multiple Intestinal Neoplasia (Min)/+ Mice Independently of p53 but Demonstrates Strong Synergy with the Modifier of Min 1^AKR Resistance Allele¹

McArdle Laboratory for Cancer Research [R. T. C., W. F. D.] and Laboratory of Genetics [W. F. D.], University of Wisconsin, Madison, Wisconsin 53706

	ABSTRACT

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Altered patterns of the 5-cytosine methylation of genomic DNA are associated with the development of a wide range of human cancers. We have studied the mechanisms and genetic pathways by which a targeted heterozygous deficiency in the murine 5-cytosine DNA methyltransferase gene (Dnmt1^N/+) diminishes intestinal tumorigenesis in C57BL/6-multiple intestinal neoplasia (Min)/+ mice. We found that Dnmt1^N/+ retards the net growth rate of intestinal adenomas and reduces tumor multiplicity by approximately 50%. This tumor resistance affects the entire intestinal tract and is independent of the status of modifier of Min 1 and p53, two loci that have been found to confer strong resistance to Min-induced neoplasia. Interestingly, Dnmt1^N/+ and modifier of Min 1 resistance interact synergistically, together virtually eliminating tumor incidence. This finding may provide an insight into potential combinatorial therapeutic approaches for treating human colon cancer.

	INTRODUCTION

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Altered patterns of 5-cytosine methylation at CpG islands located in the promoter region of genes are correlated with the development of a wide range of human cancers. These changes have been linked to both increases and decreases in the transcription of genes thought to be critically important for carcinogenesis. Examples of proto-oncogene activation, ostensibly via promoter hypomethylation, include BCL-2 in B-cell leukemias (1) , MYC and epidermal growth factor receptor in hepatocellular carcinomas (2, 3) , Ha-RAS in lung cancer (4) , and the biallelic expression of the imprinted insulin-like growth factor 2 locus in Wilms’ tumor (5) . Alternatively, there are also numerous reports connecting gene promoter hypermethylation to the silencing of tumor suppressor genes.

Consistent with Knudson’s "two-hit" hypothesis (6) , such events can provide one or more inactivating hits in the development and progression of cancers. Examples include RB in retinoblastoma (7) ; VHL in renal carcinoma (8) ; p15 in gliomas and leukemias (9) ; BRCA1 in breast cancer (10) ; E-CADHERIN in hepatocellular carcinoma, breast cancer, and prostate cancer (11, 12) ; GSTP1 in prostate, breast, and renal cancer (13, 14) ; and p16^INK4a in virtually all human cancers studied (15, 16) . It remains to be determined what upstream causes generate these changes in CpG-methylation status in tumor lineages.

One of the major human cancers in the United States is colorectal cancer, accounting for 130,000 new cases and more than 50,000 deaths each year (17) . It is estimated that 50% of the Western population can expect to develop at least one colorectal tumor by the age of 70 (18) . Molecular analyses of human colorectal cancers have identified a growing number of cancer-linked genes, the promoters of which are differentially hypermethylated in tumors, sometimes biallelically. This list includes p15 (19) , p16^INK4a (16) , estrogen receptor (20) , MLH1 (21–23) , WT1 (24) , and APC³ (25) in primary tumors and MUC2 (26) in colon cancer cell lines. MLH1 appears to be a special target for this silencing mechanism because this locus may be monoallelically or biallelically hypermethylated in more than 80% of the human colorectal cancers showing microsatellite instability (27) .

An extremely useful tool for understanding the etiology of human cancers is a mutant mouse model. A model of intestinal cancer is the Min mouse, which carries a germ-line mutation in the Apc gene (note: Min is used as a genotypic abbreviation for Apc^Min/+ whenever strain genotypes are indicated, and Min is used generically for mice and tumors). On the B6 genetic background, this mutation predisposes heterozygous mice to the development of as many as 300 adenomas throughout the intestinal tract (28) . Apc is the mouse homologue of APC, which has been found to be mutant in a majority of familial and sporadic human colon cancers (29) . In an important study, Laird et al. (30) bred the Min mouse to animals carrying a targeted heterozygous deficiency in the Dnmt1^S/+ gene. Dnmt1 catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to carbon 5 of cytosine residues in 5'-CpG-3' dinucleotides. Maintenance methylation occurs post-DNA replication and uses the parental strand as a template, thus maintaining genomic methylation patterns (31) . In the Laird et al. (30) study, (129/SvJ Dnmt1^S/+ x B6-Min/+) F1 progeny developed 60% fewer intestinal adenomas than F1 Dnmt1^+/+ Min/+ control animals. When global genomic methylation levels were further depressed by early administration of the pharmacological demethylating agent 5-Aza-dC, tumor incidence was reduced by a factor of nearly 60 (30) .

How does a decrease in genomic methylation cause the suppression of Min-induced intestinal tumorigenesis? Two functions of methylation were initially considered: promotion of somatic mutagenesis by 5-methylcytosine and loss of genomic stability (30) . The involvement of DNA methylation in promoting mutagenesis has been observed in bacteria, and in mammals, its importance is inferred from the low frequency of the CpG dinucleotide in the genome (32) . In cancer, it has been proposed that methylated CpG residues are mutational hot spots for the generation of transition mutations caused by the deamination of 5-meC, possibly mediated by the actions of Dnmt1. Analysis of the mutational spectrum of the tumor suppressor p53 has provided one piece of evidence for this hypothesis (33, 34) . However, subsequent investigations by Chen et al. (35) demonstrated that hypomethylation enhanced, rather than decreased, the mutation rate at a mouse test locus. This finding is consistent with the hypothesis of Smith and Crocitto (36) that mammalian DNA methyltransferases can maintain genomic stability by preventing clastic mutagenesis. Furthermore, studies of human colon cancer cell lines by Lengauer et al. (37) indicate that genomic hypomethylation is likely to be associated with an increase in genomic instability. Jackson-Grusby and Jaenisch (38) also tested the effect of hypomethylation in Min mice fed a diet deficient in choline and methionine. Min-induced tumor multiplicity was reduced in mice fed the methyl group-restricted diet. Together, these studies support the hypothesis that the reduction in methylation levels, rather than the reduction in the levels of the enzyme, is the cause of intestinal tumor resistance.

To explore hypomethylation-mediated resistance to Min-induced tumorigenesis, we have used a mouse carrying the N mutant allele of Dnmt1 (39) on an inbred B6 genetic background. The N allele of Dnmt1 has a biochemical phenotype that is comparable to but slightly weaker than the S mutant allele of Dnmt1 used by Laird et al. (30) .⁴ We have tested the hypothesis, first proposed by Balmain (40) , that DNA methylation affects a growth regulatory gene, by examining the growth kinetics of intestinal tumors arising in B6-Min Dnmt1^N/+ mice. To test for independence between the effects of distinct resistance modifier loci, we have made genetic crosses to detect interactions between Dnmt1^N/+ and Mom1 (40) or p53, two loci that confer a strong resistance to Min-induced neoplasia. Finally, we have assessed the potential effect of Dnmt1^N/+ on cellular indices of apoptosis, mitosis, and DNA synthesis in preneoplastic intestinal mucosa.

	MATERIALS AND METHODS

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Animal Care and Breeding.
Experimental mice were bred at the McArdle Laboratory for Cancer Research. Diets and husbandry were as described previously (28) .

Experimental Classes of Mice.
The B6-Min pedigree has been maintained by backcrossing B6-Apc^Min/+ males to B6 females (n > 40). The B6-Mom1^AKR congenic strain has been maintained as described (41) . B6-Dnmt1^N/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME; n > 15). The B6-p53^–/– line originated from a (129/Sv x B6) F2 female founder that carried a targeted disruption of the p53 gene (42) . This founder was obtained from Larry Donehower (Division of Molecular Virology at Baylor University, Houston, TX). The congenic line was developed by backcrossing p53^+/– females to B6 or B6-Apc^Min/+ males for a minimum of 10 generations.

Mice listed in Tables 1–5 and Figs. 1 and 2 were primarily generated from crosses between B6-Apc^+/+Dnmt1^N/+ Mom1^AKR/B6 females x B6-Apc^Min/+Dnmt1^+/+ Mom1^AKR/B6 males. Mice described in Table 6 were primarily generated from crosses between B6-Apc^+/+ Dnmt1^N/+p53^+/– females x B6-Apc^Min/+Dnmt1^+/+ p53^–/– males. Only male B6-Min p53^–/– mice survived to 90 days of age, because p53^–/– females are subject to several developmental abnormalities (43) .⁵

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. The Dnmt1N/+ class of Min tumors was deficient for larger adenomas in the small intestine. This histogram represents 600 tumors pooled from each genotype. Size is the maximum diameter of adenomas.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Dnmt1N/+ retarded the net tumor growth rate of Min-induced adenomas in the small intestine. Tumor sizes were plotted at 70, 100, and 130 days of age. Tumor sizes were not significantly different at 70 days, but became significantly different at 100 days and beyond. Because of early mortality of the B6-Min Dnmt1+/+ class, size could be plotted only at 120 days, but this value was significantly greater than B6-Min Dnmt1N/+ tumors measured at 130 days; P < 0 .05. Tumor sizes represent per-mouse averages. The numbers of mice measured were as follows. 70 days: Min control, n = 5; Dnmt1N/+, n = 5. 100 days: Min control, n = 36; Dnmt1N/+, n = 23. 120 days: Min control, n = 13. 130 days: Dnmt1N/+, n = 17. Min controls measured at 110 days (1.95 mm; not indicated on graph) were also larger than Dnmt1N/+ tumors measured at 130 days (1.70 mm).

Genotyping.
DNA was isolated from blood as described previously (44) . The Apc and p53 genotypes were determined as described previously (41, 45) . The genotype at the Mom1 locus was assigned on the basis of genotypes at the closely linked flanking markers D4Mit54 and D4Mit13, as described previously (41) . The genotype for the Dnmt1 N and Dnmt1 wild-type alleles was determined by PCR analysis using oligonucleotide primers (exon 1-1, Nae 3', and PGKPr-1) flanking the pMT(N)neo targeting vector (39) . The following primer sequences were provided by Rudi Jaenisch (Massachusetts Institute of Technology): exon 1-1, 5'-GGG CCA GTT GTG TGA CTT GG-3'; Nae 3', 5'-CTT GGG CCT GGA TCT TGG GGA TC-3'; and PGKPr-1, 5'-GGG AAC TTC CTG ACT AGG GG-3'. PCR was carried out as follows: each DNA sample (2 µl) was amplified in a 25-µl reaction containing 2 µl of primer exon 1-1 (2 µM), 1 µl of primer Nae 3' (2 µM), 1 µl of primer PGKPr-1 (2 µM), 1.5 µl of MgCl₂ (25 mM, Promega), 2.5 µl of 10x reaction buffer (Promega), 0.5 µl of dNTPs (25 mM, Promega), 0.4 µl of Taq polymerase (5 units/µl, Promega), and 14.1 µl of doubly distilled water. Samples were amplified in a PTC-200 Peltier thermal cycler (MJ Research) under the following conditions: 1 cycle at 95°C for 4 min; 25 cycles at 95°C for 1 min, 54°C for 1 min, 72°C for 1 min; 1 cycle at 72°C for 3 min; and cooling to 4°C. Agarose gel (2.0%) electrophoresis of the PCR products followed by ethidium bromide staining permits visualization of 334-bp wild-type and 472-bp N bands.

Histology.
Tissue specimens analyzed by fixed positional analysis of intestinal crypts were isolated from the medial 4-cm section of the small intestine and the distal region of the large intestine. The intestine was cut longitudinally, rinsed in 1x PBS, fixed flat overnight in 10% formalin, washed, transferred to 70% ethanol, embedded in paraffin, and serially sectioned (5 µM), with alternate sections stained with H&E. All animals were sacrificed at approximately the same time of day to reduce the influence of circadian variation in cellular proliferation and apoptosis.

Morphological Analysis for Mitosis and Apoptosis.
Quantitative analysis of apoptosis and mitosis in the intestinal crypts of histologically normal mucosa was conducted using the static positional scoring method developed by Potten et al. (46, 47) . Briefly, 100 complete half-crypt sections per mouse were analyzed from both the medial small intestine and the distal large intestine. Samples were scored with an Olympus BX-40 System microscope at x 600 magnification by one observer (R. T. C.) who was blind to the genotype. In this method, each intestinal crypt cell position is numbered and scored for either an apoptotic or mitotic event with morphological criteria. Scores were entered into the PC-Crypts program (provided by Dr. Potten) using a laptop computer. The PC-Crypts program yields crypt mitotic and apoptotic indices and the relative distribution of these events along the crypt.

DNA Synthesis Analysis.
BrdUrd (Sigma B5002) at a dose of 400 mg/kg body weight was administered by i.p. injection 40 min prior to sacrifice. Paraffinized serial sections of formalin-fixed tissues prepared for morphological analysis (described above) were processed for BrdUrd incorporation as follows. Sections were dewaxed in xylene; rehydrated through serial ethanol solutions; washed in distilled water; protease digested; blocked for endogenous peroxide, avidin, and biotin; and then incubated at room temperature with 1° BrdUrd monoclonal mouse IgG antibody (M0744, DAKO A/S, Glostrup, Denmark). After washing in 1x PBS, biotinylated 2° goat antimouse IgG antibody (Sigma B-8774) was applied, followed by a wash in 1x PBS. Samples were then treated with streptavidin peroxidase conjugate, stained with 3-amino-9-ethylcarbazole (AEC; Zymed, 00-2007) to visualize the BrdUrd antigen, and lightly counterstained with hematoxylin. Intestinal crypt cells were then scored for BrdUrd-positive staining, with the same scoring system as described above for morphological analysis.

Statistics.
One-sided Ps for tumor numbers and intestinal tumor sizes were determined by comparison of each test class with contemporaneous B6-Min control mice (often siblings) by use of the nonparametric Wilcoxon rank-sum test. One-sided Ps for the fixed positional analysis of intestinal crypt apoptosis and proliferation were determined using Student’s t test.

	RESULTS

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Dnmt1^N/+ Controls the Size of Min Adenomas.
When measured at 100 days of age, adenomas from the small intestine of B6-Min Dnmt1^N/+ mice are significantly smaller than those from B6-Min siblings (see Table 1 ). In the Dnmt1^N/+ class, no tumors greater than 3.8 mm were observed, whereas 17 larger adenomas were found in a comparable group of B6-Min control mice. Moreover, only 1.1% of Dnmt1^N/+ tumors grew to a size of 3.0 mm, whereas 7.7% of Min control tumors reached this size (see Fig. 1 ). This effect on intestinal adenoma size is not attributable to a shift in the distribution of tumors (see Table 2 ) but is strongly associated with inhibition of the net growth rate of individual adenomas (see Table 1 and Fig. 2 ). We restricted our analysis to adenomas of the small intestine because of low tumor multiplicities in the large intestine and the difficulty in accurately measuring the diameters of pedunculated colonic adenomas. However, it appears that Dnmt1^N/+ also restricts adenoma growth in the large intestine.

Dnmt1^N/+ Significantly Retards the Net Growth Rate of Min Adenomas.
To determine whether the reduction in the size of B6-Min Dnmt1^N/+ adenomas was caused by a slowing in the net tumor growth rate, the tumor sizes were scored at 70, 100, and 130 days (see Fig. 2 ). We found that B6-Min Dnmt1^N/+ and B6-Min control tumors were similar in size at 70 days of age but diverged soon after. Linear regression analysis of maximum tumor diameters demonstrated a 2.5-fold higher rate of increase in tumor diameters in Min control mice than in Dnmt1^N/+ carriers. Because it was difficult to obtain many B6-Min control mice that lived to 130 days, we used a 120-day time point for controls. At 120 days, tumors from control mice were significantly larger than adenomas obtained from B6-Min Dnmt1^N/+ mice measured at 130 days.

Dnmt1^N/+ and Mom1^AKR Act Synergistically.
On the B6 genetic background, heterozygosity for the N allele of Dnmt1 reduces Min-induced intestinal tumorigenesis by a factor of 2 (Table 3 ). A similar effect has been observed in congenic mice that are heterozygous for the Mom1 AKR-derived region of distal chromosome 4 (44) . To test for a genetic interaction between Dnmt1^N/+ and Mom1^AKR, we scored intestinal tumors from mice resulting from crosses as described in "Materials and Methods." Heterozygosity for either the N allele of Dnmt1 or the Mom1^AKR resistance allele reduced tumor multiplicity by a factor of 2. Mom1 had previously been shown to act in a semi-dominant fashion (44) . Mice homozygous for Mom1^AKR demonstrated a reduction in tumor multiplicity by a factor of 5, a phenotype similar to that observed in Dnmt1^N/+ Mom1^AKR/B6 double heterozygotes. A more striking effect was observed when Mom1 was homozygous for the AKR resistance allele. Dnmt1^N/+ Mom1^AKR/AKR mice developed fewer tumors by a factor of 44 (Table 3) , with almost half exhibiting no intestinal tumors. Furthermore, the few adenomas observed tended to be very small (Table 1) .

Dnmt1^N/+ and Mom1^AKR also demonstrate additivity in reducing adenoma size (Table 1) . Tumors from Dnmt1^N/+ Mom1^AKR/B6 mice (1.06 mm) are smaller than tumors from either Dnmt1^N/+ (1.19 mm) or Mom1^AKR/B6 (1.14 mm) by a degree that is significant for Dnmt1^N/+ alone (P = 0.004) and that is close to significant for Mom1^AKR/B6 alone (P = 0.06), whereas tumors from Dnmt1^N/+ Mom1^AKR/AKR mice (0.75 mm) are strikingly smaller than adenomas obtained from Mom1^AKR/AKR mice (1.14 mm; P = 0.006).

Dnmt1^N/+ Suppresses Tumorigenesis throughout the Intestinal Tract.
Recent studies by our group indicate that the B6 intestine consists of discrete subregions that are differentially susceptible to Min-induced tumorigenesis. Modifiers of Min can act differentially in various parts of the intestinal tract.^{6 7} Furthermore, in several mouse models of intestinal cancer, such as genetic knockouts in the Tgf-ß pathway, neoplasia is restricted to the large intestine (48) . Here, we report that Dnmt1^N/+ provides tumor resistance in all regions of the intestine, with the strongest effect in the proximal half of the small intestine (see Table 2 ) and a more modest effect in the large intestine. By contrast, Mom1^AKR exerts its strongest effect in both the distal half of the small intestine and the large intestine.

Dnmt1 Deficiency Does Not Alter Apoptotic Indices in the Intestinal Crypt and May Help Restore Rates of Proliferation.
To define further the mode of action of Dnmt1^N/+, we measured the apoptotic, mitotic and BrdUrd labeling indices in histologically normal crypts of B6-Apc^+/+, B6-Min, B6-Min Dnmt1^N/+, and Min mice heterozygous or homozygous for the Mom1 AKR allele (Tables 4 and 5). Dnmt1^N/+ had no significant impact on the apoptotic index and, instead, appears to elevate the mitotic and BrdUrd labeling indices versus B6-Min controls. The difference is statistically significant only in the BrdUrd labeling indices (P < 0.05); however, the Min mutation itself led to a significant systemic reduction in DNA synthesis compared with the marginal effects of both Dnmt1^N/+ and Mom1^AKR. Notably, B6-Min control animals also had significantly reduced mitotic indices (P < 0.05) compared with B6-Apc wild-type mice.

	DISCUSSION

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It is now well known that accumulated genetic and epigenetic alterations accompany neoplastic development in many human tissues. In the colon, changes in the expression of APC, p53, Ras, and ß-Catenin, (and surely other genes) are observed during the progressive transformation of normal mucosal tissue to adenomas and eventually malignant carcinomas. In this study, we used the Dnmt1^N/+ mutant mouse to extend the important work of Laird et al. (30) by addressing some of the functional mechanisms and genetic pathways by which Dnmt1^N/+ provides resistance to Min-induced tumorigenesis. Use of the N knockout allele of Dnmt1 permits an assessment of the Dnmt1 heterozygous knockout phenotype on a homogeneous B6 background, alone and in combination with Mom1^AKR and p53-deficiency, to determine their possible interactions.

We found that Dnmt1^N/+ slows the rate of growth of intestinal adenomas. Tumors from the small intestine of Min Dnmt1^N/+ animals and their Min control siblings were similar in size at 70 days of age, but their respective growth rates diverged significantly thereafter. This result is consistent with a model in which gene promoter hypermethylation may contribute to the epigenetic silencing of a locus that negatively regulates tumor growth. Reduction in global methylation levels by a germ-line deficiency in the maintenance 5-cytosine methylase (or by administration of 5-Aza-dC to neonates) could, therefore, attenuate such modulation of a negative tumor regulator.

An alternative mechanistic explanation might involve the hypothesized interaction between Dnmt1, PCNA, and p21^WAF1. In a model proposed by Chuang et al. (51) , Dnmt1 affects the cell cycle by competing with p21^WAF1 for PCNA binding sites, and Fournel et al. (52) have recently reported that down-regulation of human DNMT1 via antisense RNA induces p21^WAF1 protein activity in cultured human tumor cells. p21^–/– mice do not spontaneously develop cancer (53) , but sensitization via crosses to Min might be a means to test for interaction between deficiencies in Dnmt1 and p21 in tumorigenesis. Nullizygosity for p53, a key transactivator of p21 (54) , does significantly enhance Min tumorigenesis, but Dnmt1^N/+ can still exert considerable tumor resistance in the absence of p53. This observation argues against the interaction of Dnmt1 with p21^WAF1 and PCNA as being involved in its effect on Min tumor formation.

The tumor resistance of Dnmt1^N/+ is also expressed independently of the resistance allele of Mom1. The combined quantitative resistance phenotypes of Dnmt1^N/+ and Mom1^AKR/AKR are far more than additive, and each resistance modifier can act in the genetic absence of the other factor. Specifically, Dnmt1^N/+ does not require the secreted phospholipase encoded by Pla2g2a, a gene that is a strong candidate for at least one component of Mom1 (55) and that is defective in sensitive B6 mice. Regional differences in the resistance phenotypes of Dnmt1^N/+ and Mom1^AKR also support their action via independent pathways (Table 2) . In the colon, Dnmt1^N/+ has a very modest effect on tumorigenesis, whereas mice overexpressing a wild-type Pla2g2a^AKR transgene on the B6 background develop 3-fold fewer colon adenomas (55) . Interestingly, the combination of Mom1^AKR/AKR and Dnmt1^N/+ demonstrated a very strong synergy, virtually abrogating tumorigenesis, with half of these mice developing no tumors at all. Mom1^AKR has previously been shown to retard net adenoma growth rate (44) . Notably, the combination of Mom1^AKR and Dnmt1^N/+ further reduced adenoma size significantly. Thus together, Dnmt1^N/+ and Mom1^AKR/AKR significantly reduce both the growth rate and incidence of Min-induced tumors, suggesting the potential efficacy of combinatorial therapeutic protocols based on their distinct biochemical activities.

The preliminary result that Dnmt1^N/+ does not alter apoptotic indices in histologically normal intestinal mucosa of the Min mouse nominally distinguishes the biochemical mode of action of Dnmt1^N/+ from some other known classes of intestinal anticancer agents, such as the NSAIDs (56) . The ability of NSAIDs to cause intestinal tumor regression has been linked to their effect on rates of apoptosis in both normal mucosa and in tumors (57) . The finding that Dnmt1^N/+ and Mom1^AKR may act to normalize rates of proliferation in non-tumor-bearing crypts requires further investigation. We observed that Min animals have reduced indices of mitosis and DNA synthesis in histologically normal crypts. This effect does not result from an increase in crypt length (data not shown). Both Dnmt1^N+ and Mom1^AKR appear to reverse partially this reduction in crypt cell proliferation, although the difference is significant only for the BrdUrd labeling index. Mahmoud et al. (57–59) have reported that Min significantly decreases the PCNA-labeling index and slows the migration rate of enterocytes in normal intestinal tissue. They have also reported that NSAIDs, such as sulindac, can restore both cellular proliferation and enterocyte migration rates to wild-type levels (58) . Investigators in our laboratory have long observed that nonneoplastic crypts and villi surrounding Min adenomas are decidedly hyperplastic, but our current results and those of the Bertagnolli group indicate that this hyperplasia probably does not involve increased DNA synthesis rates. We are currently investigating whether Dnmt1^N/+ can modulate the maturation of migrating enterocytes within the intestinal crypt. A failure of APC mutant cells to differentiate properly has been observed previously in human familial adenomatous polyposis patients (60) , an observation that correlates with the development of preneoplastic dysplastic lesions and adenomas. In addition, Wasan et al. (61) have proposed that familial adenomatous polyposis patients have a deficiency in intestinal crypt differentiation, specifically through the crypt cycle. Differential methylation has been shown to be connected to the differentiation of certain mammalian cell types (62, 63) , and Bestor et al. (64) have shown that hypomethylation can revert the phenotype of transformed mouse cells by induction of terminal differentiation.

Very little is known about how heritable, tissue-specific methylation patterns are established during development (65) , let alone how regulated methylation might help to maintain adult tissue homeostasis in the rapidly renewing intestine, where cellular production, differentiation, and death are tightly controlled. As suggested by our study of tumor growth kinetics, if promoter silencing of growth-regulatory loci is important for Min tumorigenesis, a key question to be addressed is whether these epigenetic events occur during or prior to the clonal expansion of initiated tumor cells. This change might arise by a random embryonic de novo methylase activity in the pluripotent long-lived intestinal stem cells, or, as recently reported by Ramchandani et al. (66) and Kanai et al. (67) as a result of a demethylase activity.

Observations from the Jaenisch laboratory and our laboratory support the view that most Min intestinal tumors arise very early. In the study of Laird et al. (30) , prevention of tumorigenesis by administration of 5-Aza-dC was efficacious only when administered early, and Shoemaker et al. (68) demonstrated that tumor enhancement resulting from somatic mutagenesis was optimal prior to 14 days of age. An intriguing possibility is that in Min mice a distinct subset of intestinal stem cells might demonstrate what Toyota et al. (69) have referred to as a "CpG island methylator phenotype," possibly formed in utero. Interestingly, De Marzo et al. (70) have recently reported that human adenomatous polyps demonstrate a striking heterogeneity in Dnmt1 protein expression, and Eads et al. (71) have recently found that specific CpG island hypermethylation observed in colon cancer does not correlate with the temporal overexpression of Dnmt1.

In summary, we have found that a germ-line deficiency in maintenance 5-cytosine methylation slows Min intestinal adenoma growth rate and reduces tumor multiplicity in B6 mice while also demonstrating a strong synergy with the Mom1^AKR resistance allele. These results are consistent with the selection in intestinal tumors for random differential methylation events, occurring either developmentally or in clonal tumor cell growth. Studies with the Min mouse should continue to reveal some of the underlying biology governing regulation of genomic methylation in intestinal cancer.

	ACKNOWLEDGMENTS

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by NIH Grants CA 07075 (to the McArdle Laboratory); CA 50585 and CA 63677 (to W. F. D.), and predoctoral training grant CA 09135 (R. T. C.).

² To whom requests for reprints should be addressed, at McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Madison, WI 53706. Phone: (608) 262-4977; Fax: (608) 262-2824; E-mail: dove{at}oncology.wisc.edu

³ The abbreviations used are: APC, adenomatous polyposis coli; B6, C57BL/6; Mom1, modifier of Min 1; Min, multiple intestinal neoplasia; BrdUrd, 5-bromodeoxyuridine; Dnmt1, maintenance 5-cytosine DNA methyltransferase gene; 5-Aza-dC, 5-Aza-2-deoxycytidine; PCNA, proliferating cell nuclear antigen; NSAID, nonsteroidal anti-inflammatory drug.

⁴ R. Jaenisch, personal communication.

⁵ R. Halberg, unpublished observations.

⁶ A. Bilger, personal communication.

⁷ R. Cormier, A. Bilger, A. Lillich, R. Halberg, K. Hong, K. Gould, N. Borenstein, E. Lander, and W. Dove. The Mom1^AKR intestinal tumor resistance region consists of pla2g2a and a locus distal to D4Mit64, submitted for publication.

Received 12/ 2/99. Accepted 5/10/00.

	REFERENCES

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