This study was undertaken to determine the effects of excess amounts of methyl donors in the diet on methylation of DNA, growth rate of tumors, efficacy of the demethylating drug 5-aza-2'-deoxycytidine (AdC) to demethylate DNA and finally on the initiation and development of prostate tumors. The diet used in this study is similar to the 3SZM diet used by Cooney et al
. and Cropley et al
. except for the amounts of zinc sulphate (4 fold higher) and choline chloride (3 fold lower)(24
). The changes were made based on the premise that the levels of choline in diets used in the published studies are responsible for the increased toxicity seen in the latter, as well as some other studies where a similar diet was used.
Exposure to excess methyl donors did not influence the growth rate of athymic nude mice on the diet or the xenografted PCa cells. We hypothesized that Me diet would affect the efficacy of demethylating drugs merely by releasing into circulation excess methyl donors that could potentially methylate newly synthesized DNA before AdC binds to that DNA strand. However, the heterogeneous loss of CpG methylation within each single allele examined (by bisulphite sequencing) strongly supports demethylation within the xenografts despite the presence of excess circulating methyl donors. These results could be a function of having stoichiometrically more AdC than Me donors in circulation. Unlike methotrexate – an antifolate drug used for the treatment of rheumatoid arthritis, psoriasis, ectopic pregnancies and cancer – whose efficacy is affected by dietary folate (13
), the current data suggest that AdC used at clinically relevant dose is not similarly affected by the presence of excess methyl donors in the diet.
In chemoprevention studies, agents are administered at a high dose that approximates the maximum tolerated dose for that agent. Because the excess methyl donors did not influence growth of preformed tumors or the ability of AdC to demethylate, it does not mean that the diet was insufficiently loaded with methyl donors or a lack of oral bioavailability. Measurements of circulating methyl donors - folate and methonine, showed that these donors were present in at least 2-fold and 3-fold greater amounts, respectively, than the levels circulating in mice on Reg diet. Thus, the levels of methyl donors were actually much higher than the recommended daily requirement of rodents. A similar methyl-proficient diet was shown to have epigenetic effects in the agouti mouse model (26
). The agouti locus may be more sensitive to methylation changes than either the AR or Reprimo loci examined here. The time frame of exposure to dietary methyl donors and the time elapsed since the exposure and analysis may be differentiating factors in measuring methyl dependent effects too. The body weights of age matched athymic nude or “Hi-myc” mice on Reg or Me diet were not different indicating that the total caloric intake of mice from either dietary group was not different. Thus, a lowered caloric intake could not have occurred, eliminating the reasoning that the protective effects of the Me diet on the growth of the cancer is due to the protective effects of caloric restriction on cancer- as has been observed in other experiments (27
The results showing lack of effect of excess methyl groups on growth of preformed cancer cells in vivo
also add to the evidence supporting the role of the methylating enzyme rather than excess dietary methyl donors in determining the methylation state. Eads et al
. have shown that DNMT-1 hypomorphic alleles reduce the frequency of CpG island methylation in the normal mucosa and intestinal polyps (28
). An increased level of DNMT-1 gene expression in some types of cancer (29
) changes in the set-and-site specificity of DNA-methyltransferases, and an appearance of new proteins with DNA-methyltransferase activities in tumor (hepatoma) cells of rats fed a methyl-deficient diet, have been observed (30
). Other demonstrated mechanisms of feedback regulation of DNMT-1 are: presence of methylation sensors in certain genetic elements (presence of unmethylated CpGs) (31
) and RNA-mediated feedback of methylation (32
). Are these mechanisms affected by excess dietary methyl donors, and if so how do they modulate DNMT-1 to cause unwarranted epigenetic changes that result in tumor suppressor gene silencing (33
)- should be the focus of future studies.
No change in methylation of AR or Reprimo promoters was noted in DU-145 tumors from mice fed the methyl proficient-diet compared to the same regions from mice fed their regular diet, may imply that a threshold of circulating methyl donors is already present in Reg diet. An increase in methyl donors beyond the levels already present does not have a noticeable effect on the promoters assayed here. The lack of noticeable differences may be a result of the fact that some of the methyl donors - L-methionine, betaine, zinc sulphate, Vitamin B12, and folic acid are water soluble. When tissue storage capacity of water soluble vitamins is saturated, the rate of excretion of these increases sharply.
The markers of methylation change used in this study (AR and Reprimo) are significant because they are known to be methylated in PCa. Since the results also show that the extent of DNA demethylation by demethylating agents is gene-specific - AR undergoes less demethylation by AdC compared to Reprimo, it lends credence to the fact that different regions of the genome are differently susceptible to (de)methylation. While it has not been explored here, it may be that the structure of the chromatin around the gene may influence the methylation state of that gene / promoter. Other X-chromosome genes have also been shown to be differentially demethylated when exposed to AdC when compared to genes on other chromosomes (34
). Therefore, overall changes in methylation state, global methylation, may not provide a comprehensive picture of the dynamics involved in achieving the final methylation state. Indeed, global methylation differences were not apparent in the third part of this study where the ratio of Methyl-Cytosine to Cytosine was measured for DNA from 5 month old “Hi-myc” mice fed Me diet when in utero
and weaned off the diet when they were 1 month old compared to DNA from Reg diet-fed “Hi-myc” mice of the same age. Further, it may be that a threshold of methylation density exists in promoters and once this threshold methylation density is reached, no further increase in methyl donors changes the methylation on these sites.
As reported in the original paper of the “Hi-myc” mice (21
), in this study too, there was an age-dependent increase in the grade of tumors in myc positive genotypes on Reg diet. In the present paper, “Hi-myc” mice were fed either Reg or Me diet while in utero
, at birth and until one month of age. At this time, Me diet-fed mice were weaned off the diet and fed Reg diet until they were sacrificed at different time points between 3 months and 7 months of age to see the effects the diets had on the pathology of prostate tumors. 5-7 month old Myc mice fed Reg diet developed higher grades of tumors as compared to age-matched mice fed Me diet. This result was contrary to our hypothesis as well as the observation in xenografts where the phenotype / proliferation / methylation of promoters of xenografts from Reg diet-fed mice was not different from xenografts from Me diet-fed mice. In the case of the “Hi-myc” mice that were fed Me diet in utero
and after birth only for an additional month, the significant differences in grades between groups of mice on different dietary regimens were only seen in the 5-7 month age group. If the different diets contributed to these differences in grade then it must be a residual effect of the exposure that is retained in some form of a “memory molecule” that manifests itself at the conducive time. In the present study, the conducive time was when the mice were of the ages of 5 months or higher. This is the period when the transition from mPIN to cancer takes place. According to the original paper describing the development of tumors in “Hi-myc” mice, at 6 months or higher, all “Hi-myc” mice developed invasive cancer. A previous exposure to Me diet may be influencing the mechanisms at play during the transitionary period from mPIN to invasive cancer. Our in vitro
studies show that exposure to methyl donors does not affect the expression of the c-myc-transgene. Further studies are warranted, including maintaining Hi-myc mice on Reg and Me diet since weaning and not in utero
and observing cancerous changes that may be occurring.
Such a delayed effect of maternal Me donor supplementation was also described by Cropley et al
). Cropley et al
.,report that maternal supplementation only during mid-gestation substantially affects offspring coat color. Importantly, they also show that this effect is inherited by the next generation, presumably through germ-line modifications during grandmaternal dietary supplementation of increased methyl donors. The genetic element in their case is the agouti allele Avy
. What genetic element(s) are at play in the present study, that confer protection by delaying the onset of higher grades of cancer is under investigation. An in vitro
study aimed at deciphering the role of remethylation of the genome in glioma cells by exposure to increased levels of folate shows that Sp1/Sp3-mediated transcriptional upregulation of DNMT3a and 3b proteins may be responsible for limiting the aggressiveness of glioma when exposed to increased folate (35
). However, this upregulation was reported in an in vitro
study where high levels of folate can be achieved and maintained, unlike in vivo
studies where an increase in the circulating levels of methyl donors does not translate into proportionately higher levels in tissues. What we have not examined in the present study is the effect of continued exposure of Me diet beyond 1 month of age. We cannot extrapolate the present data to hypothesize on what protective effects or otherwise would be seen then.
The protective effect in the developmental model of prostate cancer gives credence to the epidemiological observations stating the cancer-protective effects of increased folate consumption before the initiation of the cancer. The current study allays fears of a “methylising” effect of diets rich in methyl-donors. Future epidemiological studies may benefit from also examining total methyl-donor levels instead of focusing on folate levels alone, along with polymorphisms that may affect tissue levels of the methyl-donors, rather than the simplistic models many epidemiological studies are based on.