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Supplement: Trans-HHS Workshop: Diet, DNA Methylation Processes and Health

Cellular Vitamins, DNA Methylation and Cancer Risk^{1 ,2}

Department of Nutrition Sciences, The University of Alabama at Birmingham, Birmingham, AL 35294

³To whom correspondence should be addressed. E-mail: piyathic{at}uab.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

 
The overall goal of this research is to evaluate interactions among cellular vitamin levels and global DNA hypomethylation and the impact of these variables on human cancer risk. Global DNA methylation was determined by two methods: a radiolabeled methyl incorporation (RMI) assay and an immunohistochemical assay using an antibody to 5-methylcytosine (5-MC). The RMI assay is useful for evaluating methylation of DNA in tissue samples, whereas the 5-MC assay clearly reveals DNA methylation in specific types of cells and has minimal day-to-day variability. We have observed significant interactions among cancer-protective vitamins and global DNA methylation at the level of tissues. A significant positive association was observed between global DNA methylation in buccal mucosal cells and malignant tissues, but not between global DNA methylation in peripheral leukocytes and malignant tissues of the lung. These results suggest that changes in global methylation in buccal mucosal cells may reflect changes in tissues at high risk of developing lung cancer. With the antibody technique, we have demonstrated that alterations in global DNA methylation are associated with epigenetic differences in susceptibility for development of lung cancer, which is involved in the progression of the disease. The effect of race on these relationships also is discussed. Significant associations observed between expression of epidermal growth factor receptor and global DNA methylation, as assessed by the 5-MC assay but not by the RMI assay, indicate that evaluation of global methylation and biomarkers in specific types of cells may shed light on the associations between global DNA methylation and other intermediate endpoint biomarkers in the future.

KEY WORDS: • cellular vitamins • DNA methylation • cancer • 5-methylcytosine

	INTRODUCTION

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

 
There is very little doubt that a higher consumption of fruits and vegetables is associated with a lower incidence of human cancers (1 ). Although deficiency of micronutrients (vitamins and minerals) is a plausible explanation for much of the protective effect of fruits and vegetables (2 ), the mechanisms underlying these associations and the specific nutrients responsible for the observed effect are far from clear. A large majority of studies conducted to investigate the importance of vitamins in cancer either concentrated on dietary intake data or circulating concentrations of vitamins. These parameters may not reflect the vitamin levels in target tissues where cancers develop. The importance of quantifying vitamin concentrations in the target tissues to understand their role in cancer prevention is beginning to be appreciated. However, we still have to go beyond a mere documentation of concentrations of these vitamins in the target tissues. Future studies focused on genetic factors that may influence cellular vitamin levels at target tissues and their association with biological markers of malignancy will greatly contribute to the importance of vitamins in the process of carcinogenesis.

Studies of localized vitamin deficiency and risk of cancer began in the Department of Nutrition Sciences at The University of Alabama at Birmingham. Dr. Carlos Krumdieck in our department hypothesized that exposure to cigarette smoke resulted in folic acid and vitamin B-12 deficiency, which principally affected the bronchial epithelium, rendering it more susceptible to neoplastic transformation. Folate deficiency was hypothesized to occur through direct chemical inactivation by the carcinogenic hydrocarbons of tobacco smoke, including organic nitrites, cyanates and isocyanates, resulting in biological inactivity of folates. In a similar manner, cyanide, hydrogen sulfide, nitrites and nitrous oxide in cigarette smoke were proposed to render vitamin B-12 biologically inactive, which could create a secondary folate deficiency through the methyl folate trap (3 –11 ). This hypothesis was tested when the oral mucosal folate levels in smokers and nonsmokers were evaluated and were indeed found to be much lower in the buccal mucosa of smokers than in nonsmoker buccal mucosa (12 ). Although plasma folate levels were also lower in smokers relative to nonsmokers, the differences in tissue folate levels were much greater between the two groups. These results suggest that buccal mucosal levels may be related to direct exposure to cigarette smoke. Smokers also had more buccal micronuclei than nonsmokers in this study, but micronuclear levels were not correlated with buccal folate and vitamin B-12 levels. Later studies from our group focused on the cellular vitamins in tissues of smoking-related cancers of the head and neck and their association with global DNA methylation, a potential marker of increased risk for malignancy.

	DNA methylation

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

 
DNA methylation is a heritable epigenetic event that is mediated by transfer of methyl groups. Despite the frequently observed cancer-associated increases of regional hypermethylation, the near universality of global DNA hypomethylation in many types of human cancer (13 ,14 ) suggests that such hypomethylation plays a significant and fundamental role in tumorigenesis. The most likely mechanisms through which global DNA hypomethylation may induce neoplastic transformation include induction of genomic instability that results in abnormal chromosomal structures (15 ,16 ) and activation of oncogenes (17 ); both are biologically plausible. A causative role for cancer-associated DNA hypomethylation in oncogenic transformation or tumor progression has been suggested by results of several lines of animal experiments and cell culture studies (18 ,19 ). The treatment of low metastatic human or mouse cell lines with methylation inhibitors was shown to induce their conversion to highly metastatic cells (20 ,21 ).

	Global DNA methylation assay methods

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

 
In the radiolabeled methyl incorporation (RMI)⁴ assay, DNA is methylated with SssI methylase in the presence of ³H-labeled S-adenosylmethionine (source of methyl groups) (22 ). The methylation status of DNA within a tissue sample containing a mixed population of cells (fibroblasts, lymphocytes, etc., in addition to cancer and normal epithelial cells) is evaluated with the RMI assay. The RMI is inversely related to the degree of DNA methylation. The advantage of the RMI assay is that it allows for evaluation of methylation in the same frozen tissue specimens that were used for vitamin assays. We have observed that the RMI assay has some day-to-day variability. However, relative methylation values for groups of samples done on the same day are comparable, especially when matched cancer and normal samples are used.

In our recent studies (23 ), we have evaluated the global methylation status in specific types of cells involved in carcinogenesis by using a monoclonal antibody specific for 5-methylcytosine (5-MC), which has been well characterized (24 ) and used with success in several laboratories (25 –29 ). We have found that sections cut through the nuclei are labeled with this antibody, and there is no labeling outside of the nucleus. This observation indicates that the anti-5-MC antibodies actually reach the nucleus and bind methylated DNA. As with any nuclear marker, there may be steric hindrance to cytosine guanine dinucleotide (CpG) binding because of the large size of the anti-5-MC antibody relative to the size of the methylated CpG bases. Although we have used the appropriate antigen retrieval technique for this antibody (23 ), there may also be an impediment to antibody binding by endogenous DNA-binding proteins in the nucleus. The ultimate test of the validity of the antibody is the correlation between the intensity of labeling and other diagnostic and prognostic factors. As described in the studies that follow, we have shown this correlation in some initial studies using the anti-5-MC antibody. Because the commonly used RMI assay also has some deficiencies, as noted above, it is important to evaluate global DNA methylation by more than one approach.

	Nutrients and global DNA methylation

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

 
Folate and vitamin B-12 supply methyl groups for in vivo biological methylation reactions that include DNA methylation (Fig. 1 ). The influence of nutrition on gene-specific DNA methylation in humans is not well understood. However, several studies suggest that nutritional status influences global DNA hypomethylation. Folate supplementation increased the extent of genomic DNA methylation in subjects with adenomas of the colon (30 ), and DNA methylation was decreased in immortalized normal human colonocytes grown in the absence of folate (31 ). Two recent studies by our group illustrate the concept that cellular vitamin concentrations in a given tissue may be associated with global DNA methylation. In the first study lung squamous cell cancer (SCC) tissues showed localized deficiencies of folate and vitamin B-12. Lower concentrations of these two vitamins were associated with global DNA hypomethylation (32 ). The second study used paired samples of SCC and adjacent uninvolved mucosa of the lung and larynx. The SCC tissues accumulated ascorbic acid, and higher levels of this vitamin appeared to be associated with global methylation of DNA (33 ).

View larger version (22K): [in this window] [in a new window]   FIGURE 1 Pathway of methyl group metabolism.

	Evaluation of global DNA methylation by immunohistochemistry

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

To address these issues in lung carcinogenesis, we evaluated the status of global DNA methylation by using the 5-MC monoclonal antibody, in randomly selected lung specimens of 60 cigarette smokers who developed SCC and 30 cigarette smokers who did not. The racial composition was largely white (34 ). To obtain the 5-MC score, three observers independently graded the intensity of immunostaining on a scale of 0 (no staining) to 4+ (intense staining) in bronchial epithelial cells and SCC. The percentage of cells at each intensity was estimated and multiplied by the appropriate intensity score to obtain a weighted average of the immunostaining score. The final score reported is the average of the three observers. A low 5-MC score indicates global hypomethylation of DNA.

In this study, 5-MC immunostaining scores of normal bronchial epithelial cells in noncancer specimens [0.99 ± 0.52 (mean ± SD)] were not significantly different from 5-MC scores of uninvolved bronchial epithelial cells associated with SCC (0.85 ± 0.43) (P = 0.67). 5-MC scores of epithelial hyperplastic lesions of noncancer (1.20 ± 0.22), however, were significantly higher than scores for hyperplastic lesions associated with SCC (0.82 ± 0.27) (P = 0.02). 5-MC scores of normal bronchial epithelial cells in noncancer specimens were significantly higher (0.99 ± 0.52) than 5-MC scores of both SCC-associated epithelial hyperplasia (0.82 ± 0.27) and SCC (0.61 ± 0.42) (P < 0.0001 and 0.0002, respectively). Although 5-MC scores were not significantly different between SCC-associated uninvolved bronchial epithelial cells and epithelial hyperplasia, they were significantly different between epithelial hyperplasia and SCC and also between uninvolved bronchial epithelial cells and SCC (Wilcoxon signed rank test P-values 0.49, 0.01 and 0.0005, respectively). These observations suggest that altered global DNA methylation is an important epigenetic difference in susceptibility for lung cancer.

Since we observed a large variation in global methylation among subjects, similar to previous reports in breast carcinomas (35 ), we calculated the ratio between 5-MC scores of SCC and matched uninvolved bronchial mucosa (UBM) for each subject. A lower ratio represents a more marked hypomethylation in SCC than in adjacent uninvolved tissues. Forty-four of 60 SCC had 5-MC scores for both SCC and UBM and hence were available to calculate this ratio. The ratio was significantly lower with advanced stage and size of the tumor. Although the SCC/UBM ratio was one-third as much in subjects diagnosed with distant metastasis, this difference did not reach statistical significance, probably because a large majority of subjects presented with no distant metastasis at the time of surgery. The SCC/UBM ratio appeared to be unrelated to nodal status and grade of differentiation of the tumor. These results suggest that altered global DNA methylation is important in the progression of SCC of the lung.

	Race- and age-dependent alterations in global methylation of DNA in squamous cell carcinoma of the lung

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

 
We previously reported that alteration in global DNA methylation is related to an epigenetic difference in susceptibility for the development of SCC of the lung. In a recent study we investigated the influence of race- and age-dependent alterations in global DNA methylation on the development and progression of SCC of the lung. Global methylation status was evaluated in SCC and in the associated UBM and epithelial hyperplasia (EH) of 53 whites and 23 African Americans by using the 5-MC antibody described above. 5-MC scores of SCC (0.59 ± 0.06 (mean ± SEM)) were significantly lower than 5-MC scores of UBM (0.87 ± 0.07) and epithelial hyperplasia (0.82 ± 0.07) in whites (P < 0.05). In African Americans, 5-mc scores of SCC (0.55 ± 0.09) were not significantly different from 5-MC scores of UBM (0.60 ± 0.09) and EH (0.54 ± 0.14), suggesting an involvement of methylation in the development of SCC in whites but not in African Americans. 5-MC scores in whites were lower in younger (<65-y) subjects than in older (>65-y) subjects. Since cancers in younger subjects tend to be more aggressive than cancers in older subjects, these observations suggest that hypomethylation may have contributed to the aggressiveness of cancers of younger whites. Hypomethylation of SCC in white men was associated with shorter survival with the disease. These preliminary results suggest that the methylation status of DNA may affect the development, aggressiveness and prognosis of SCC in whites. These observations suggest that careful attention should be given to racial distribution of study populations in investigations of DNA methylation (36 ).

	Alterations in global DNA methylation in buccal mucosal cells reflect methylation status in malignant tissues of the lung

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

 
We believe that the levels of vitamins or biomarkers in cells or tissues at risk for developing cancer may be a better indicator of cancer risk than levels in the blood or other compartments at a distance from the at risk tissue. However, the invasive nature of obtaining tissues will prohibit us from using these indicators of disease in studies of cancer prevention and control. It may be possible to evaluate cancer markers in more easily obtainable tissues such as buccal mucosal cells or peripheral leukocytes. Some investigators have begun such evaluations, but it is not clear whether the status of markers in noninvasive tissues will reflect changes in tissues at risk. One unanswered question is whether the global DNA methylation status in peripheral leukocytes or any other easily accessible tissue will reflect methylation changes in tissues at risk of developing cancer. To address this issue we investigated the status of global DNA methylation in matched samples of peripheral leukocytes, buccal mucosal cells, malignant and nonmalignant tissues of 14 subjects who had developed lung cancer (9 with primary nonsmall cell and 5 with metastatic lung cancer). The status of global DNA methylation in matched samples of peripheral leukocytes, buccal mucosal cells and malignant and nonmalignant tissues of the lung by diagnosis is shown in Table 1 . The radiolabeled methyl incorporation was significantly higher in DNA from malignant tissues and buccal mucosal cells compared with matched nonmalignant lung DNA and leukocyte DNA in both primary nonsmall cell lung cancer and metastatic lung cancer (P < 0.05 for all comparisons, Wilcoxon signed rank test). There was no significant difference in radiolabeled methyl incorporation between malignant tissues and buccal mucosal cells in both groups (P > 0.05 for both comparisons, Wilcoxon signed rank test). In contrast, the status of global DNA methylation in buccal mucosal cells was positively associated with methylation status in malignant tissues only among primary nonsmall cell lung cancer subjects ([correlation coefficient (r) = 0.70, P = 0.06, 95% confidence interval = 0.19–1.0] (Fig. 2 ), but not in the metastatic lung cancer subjects (r = 0.20, P = 0.69, 95% confidence interval = -0.85–1.0). The association between methylation status in buccal mucosal cells and nonmalignant tissues, or leukocytes and malignant cells was nonsignificant in both primary and in metastatic lung cancer subjects. These results suggest that the changes in global methylation in buccal mucosal cells reflect changes in tissues at risk of developing nonsmall cell lung cancer and may be a useful marker for risk of developing the disease.

View larger version (18K): [in this window] [in a new window]   FIGURE 2 The association between global DNA methylation in buccal mucosal cells and primary nonsmall cell lung cancer. CI, confidence interval; r, correlation coefficient.

	Relationship between global DNA methylation and intermediate endpoint biomarker expression

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

As shown in Table 2 , lower methylation in malignant cells was associated with a higher expression of EGF-r in these tissues (P < 0.05). EGF-r scores reported are the EGF-r scores of cells that are methylated above and below the population median of 5-MC scores. The cytoplasmic and membrane scores of EGF-r were scored separately and then averaged to report the total expression of EGF-r in these cells. Although hyperplastic cells with low methylation scores also had higher EGF-r expression, this trend was not statistically significant. There was no association between methylation assessed by the RMI assay and the expression of EGF-r in SCC. These results suggest that evaluation of methylation in specific cells rather than in a mixed population of cells may provide more information with regard to its association with expression of intermediate endpoint biomarkers that are important in carcinogenesis.

	FOOTNOTES

 
¹ Presented at the "Trans-HHS Workshop: Diet, DNA Methylation Processes and Health" held on August 6–8, 2001, in Bethesda, MD. This meeting was sponsored by the National Center for Toxicological Research, Food and Drug Administration; Center for Cancer Research, National Cancer Institute; Division of Cancer Prevention, National Cancer Institute; National Heart, Lung and Blood Institute; National Institute of Child Health and Human Development; National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Environmental Health Sciences; Division of Nutrition Research Coordination, National Institutes of Health; Office of Dietary Supplements, National Institutes of Health; American Society for Nutritional Sciences; and the International Life Sciences Institute of North America. Workshop proceedings are published as a supplement to The Journal of Nutrition. Guest editors for the supplement were Lionel A. Poirier, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, and Sharon A. Ross, Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD.

² This work was supported by Grants CA70160, CA83094, CA91273 and CA87643 from the National Cancer Institute.

⁴ Abbreviations used: CpG, cytosine guanine dinucleotide; EGF-r, epidermal growth factor receptor; EH, epithelial hyperplasia; 5-MC, 5-methylcytosine; r, correlation coefficient; RMI, radiolabeled methyl incorporation; SCC, squamous cell cancer; UBM, uninvolved bronchial mucosa.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION DNA methylation Global DNA methylation assay... Nutrients and global DNA... Evaluation of global DNA... Race- and age-dependent... Alterations in global DNA... Relationship between global DNA... LITERATURE CITED

 

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