Tobacco Documents Online

I I I I I I I I I I I I I I I I I I MODULATION OF MOLECULAR MECHANISMS BY DIETARY RESTRICTION IN RATS Beverly D. Lyn-Cook, Jin Bo and Ronald W. Hart Nutritional Modulators of Toxicity Program, National Center for Toxicological Research, Jefferson, Arkansas, U.S.A. Abstract Dietary restriction, which is 40% reduction in caloric intake of the ad libitum-fed animals, is known to modulate a number of pathological diseases, as well as, extend the life-span of a number of animals. Studies in our laboratory have shown that dietary restriction modulates epigenetic and genetic mechanisms which may contribute to the etiology and progression of a number of diseases, including cancer. Epigenetic mechanisms, such as the methylation status of specific cellular proto-oncogenes, are modulated by dietary restriction. The proto-oncogenes found in the ras family are known to be activated by point mutations or hypomethylation of CpG sites at critical points on the gene. Dietary restriction reduces hypomethylation and accumulation of mutations that results as a function of age in the exocrine pancreas of rats. Dietary restrictions decrease gene amplification of specific genes as a function of age. Gene amplification often occurs as a result of normal aging and metabolism. The p53 suppressor gene could become an oncogene upon mutation. It is also known to be modulated by dietary restriction. The wild-type p53 is known to suppress the growth of transformed or initiated cells. Mutated forms of the p53 gene have been associated with a number of human cancers. The mutational spectrum of the p53 gene ranges over a large area of the gene, however four known hot spots have been identified to be associated with exposure to certain classes of chemicals or carcinogens. Modulation of these mechanisms - epigenetic, point mutation frequencies or amplification by dietary restriction may play an important role in the ability of dietary restrictions to prevent or delay the formation of diseases such as cancer. Gene Expression and Dietary Restriction Cancer and other degenerative diseases are often associated with cell proliferation. Dietary restriction decreases the rate of cell proliferation in vivo and in vitro(1,2). Cell proliferation is often associated with activation of certain proto-oncogenes such as those found in the ras family(3,4). The gene product of the ras family is known to function as a G-protein and play a role in the signal transduction pathway. Activation of the signal transduction pathway generates second messengers in the cell which exert other effects that lead to proliferation. Dietary restriction is known through epigenetic and genetic means to decrease or delay ras activation which would lead to decreased cell proliferation(5). The expression of proto-oncogenes was initially linked with dietary restriction by Nakamura and co- workers(6), who showed that c-myc expression in cells from B6C3F1 mouse liver followed a circadian pattern and that the expression in dietary restricted animals was always suppressed relative to their ad libitum fed-controls. I

' ' Methylation and Dietary Restriction Epigenetic factors such as methylation may modify gene structure by modifying the interaction of transcriptional factors which bind to specific regions on the gene(7). The loss of methylation (hypomethylation) may result in a change in the fidelity of the DNA. The loss of methylation may result from a number of factors including changes in methylation metabolism. Alteration in methylation metabolism may be due to a decrease in availability of methyl donors to various macromolecules as a result of deficiencies in certain lipotropes and molecules such as methionine, choline, vitamin B12 and folic acid(8). A decreased intake of these nutrients is known to promote a hypomethylated environment(9). Hypomethylation of cellular DNA may also result from an impairment of enzymes such as methyltransferase, which is known to catalize the post-synthetic methylation of DNA. Recently, studies have shown that the activity of inethyltransferases is dependent upon the presence of the trace metal, zinc(10). Our laboratory recently has shown a correlation between the hypomethylation of the H- and K-ras genes and marginal zinc deficiency in the rat exocrine pancreas(11). Gene Amplification and Dietary Restriction Amplification of cellular genes was first encountered in mammalian cells that had acquired a resistance to chemotherapeutic agents(12). However, amplification of specific proto-oncogenes has been found as an occasional feature of a number of tumors. Our laboratory has demonstrated that dietary restriction delays or decreases amplification of the H-ras gene as a function of age and gender in the exocrine pancreas of rats. The dietary restriction effect is more pronounced in the exocrine pancreas from female rats. Conclusion Pathological diseases, such as cancer, have many causes many of which may act through a common pathway via damage to cellular DNA. The three molecular mechanisms for carcinogenesis described in this manuscript-epigenetic, gene amplification and mutations-are summarized in Tables 1 and 2. Our studies, along with others (13), have shown that dietary restriction decreases hypomethylation of proto-onogenes, particularly the H-ras gene. Figure 1 shows the effect of age, dietary restriction, and gender on H-ras gene amplification in rat pancreatic acinar cells. Amplification and expression of the H- ras gene in aged and dietary restricted animals is decreased relative to their ad libitum counterparts. Dietary restriction in animals has provided a model that suggests low intake of calories to have profound protective effects against tumor formation. Such a paradigm also contributes to our understanding of the molecular mechanisms which may contribute to the development of tumors. However, establishing dietary restriction criteria as possible measures used in human studies need further investigation. Noninvasive methods must be developed to monitor dietary restriction effects on humans if such they are to be used as a tool in cancer prevention or as cancer therapy. Investigations must be carried out to determine if dietary restriction may play a role in the initiation or progression stage of carcinogenesis. N O 00 ~ N 00 -2- W ~ A T I I I I I I I I I I I I I I I I I

I I I I I I I I I I I I I I I I References 1. Hass, B.S.; Hart, R.W.; Gaylor, D.W.; Poirier, L.A. and Lyn-Cook, B.D., 1992. An In Vitro Pancreas Acinar Model for Testing Modulation Effects of Caloric Restriction and Aging on Cellular Proliferation and Transformation. Carcinoeenesis 13:2419-2425. 2. Roebuck, B.D.; Baumgartner, K.J. and MacMillan, D.L., 1993. Caloric Restriction and Intervention in Pancreatic Carcinogenesis in the Rat. Cancer Res., 53:46-52. 3. Barbacid, M., 1987. Ras Genes. Annu. Rev. Biochem., 56:779-827. 4. Krengel, U.; Schlichting, L; Scherer, A.; Schumann, R.; Frech, M.; John, J.; Kabasch, W.; Pai, E.F. and Wittinghofer, A., 1990. Three-Dimensional Structures of H-ras p21 Mutants: Molecular Basis for Their Ability to Function as Signal Switch Molecules. Cell 62:539-548. 5. Hass, B.S.; Hart, R.W.; Lu, M.H. and Lyn-Cook, B.D., 1993. Effects of Caloric Restriction in Animals on Cellular Function, Oncogene Expression and DNA Methylation in Vitro. Mutation Research 295:281-298. 6. Nakamura, K.D.; Duffy, P. H.; Lu, M.H. and Hart, R.W., 1990. Hepatic Myc Proto-Oncogene Expression is Reduced and Possibly Correlated With Body Temperature in Fasted Peromyscus Leucopus Mice. Age 13:27-31. 7. Borrello, M.G.; Pierotti, M.A.; Tamborini, E.; Biassoni, D.; Rizzetti, M.G.; Pilotti S. and Della Porta, G. 1992. DNA Methylation of Coding and Non-Coding Regions of the Human H-ras Gene in Normal and Tumor Tissue. Oncogene 7:269-275. 8. Poirier, L.A.; Zapisek, W.F. and B.D. Lyn-Cook, 1990. Physiological methylation in carcinogenesis. In: Mutation and the Environment, Ed. Wiley-Liss, Inc. 9. Zapisek, W.F.; Cronin, G.M.; Lyn-Cook, B.D. and L.A. Poirier, 1992. The Onset of Oncogene Hypomethylation in the Livers of Rats Fed Methyl-Deficient, Amino Acid-Defined Diets. Carcinogenesis 13:1869-1972. 10. Bestor, T.H. 1992. Activation of Mammalian DNA Methltransferase by Cleavage of a Zn Binding Regulatory Domain. EMBO J. 11(7):2611-2617. 11. Lyn-Cook, B.D.; Ellwood, K.; Bo, J.; Roebuck, B.D, and Hathcock, J.N., 1994. Increased Expression of the Multidrug Resistance (MDR) Gene in Rat Pancreas of Rats Fed a Marginally Zinc-Deficient Diet. Proc. of American Association of Cancer Research. 35:3288. 12. Schimke, R.T., 1984. Gene Amplification in Cultured Animals Cells. Cell 37:705-713. N0 ' 00 ~ ~ 00 3_ ~ ~ I

I 13. Miyamma, Y.; Tawa, R.; Koizumi, A.; Uehara, Y.; Kurishita, A.; Sakurai, H.; Kamiyama, S. and Ono, T., 1993. Effects of Energy Restriction on Age-associated Changes of DNA Methylation in Mouse Liver. Mutation Res., 295:63-69. I I I I I I I I I I I I I I '

a I I I I I I  ' , ~ I I I I I Figure 1. Ha-ras Gene Amplification in the Pancreas of BNF Rats. Figure 1 demonstrates increased gene amplification of the H-ras gene in young, middle age and old rat pancreatic acinar cells. It shows the decrease in gene amplification in DNA from old dietary restricted animals as a function of age. Dietary restriction in female rats (CR-F) show a pronounced inhibitory effect on gene amplification. A=AL-M, young; B=CR-M, Middle; C=CR-F. 1200 1000 o »r 800 ~ L o CL 00 0 U a 400 N ~ 200 YOUNG MIDDLE AGE OLD N ~ ~ -4 ~ 5- ~  W I

I Table 1. Hypomethylation of H- and K-Ras Gene in Ad Libitum-Fed and Dietary Animals I AD LIB (0) DR (0) K-RAS I H-RAS increased decreased AD LIB refers to animals allowed to eat ad libitum without any restriction of calories. DR refers to animals placed on a 40% restriction diet of the ad libitum fed. O-denotes that this study was conducted on aged animals. I I I I I Table 2. 1 Gene Amplification and Gene Expression in Dietary Restricted Animals Amplification Expression H-ras H-ras AD LIB (0) increased increased DR (0) decreased decreased AD LIB refers to animals allowed to eat ad libitum without any restriction of calories. DR refers to animals placed on a 40% restriction diet of the ad libitum fed. O-denotes aged animals. -6- I I I I I I I I I I