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I I I ' M>;I'HYLATION PROFILE AND AMPLIFICATION OF PROTO-ONCOGENES IN CALORIC RESTRICTION BNF RAT PANCREAS in B* and Beverly D. Lyn-Cook** * Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China ** National Center for Toxicological Research, Jefferson, Arkansas, USA I , I I I I Methylation of specific CpG sites in cellular DNA is known to inactivate certain cellular genes. In order to examine the effects of caloric restriction on methylation and amplification of proto-oncogenes, Brown Norway X Fischer 344 (BNF) rats were fed either a 60% restricted diet (CR), or fed ad libitum (AL). The rats were sacrificed at 8 months (young), 15 months (middle), and 26 months (old). The pancreas was excised and pancreatic acinar cells were isolated. Isolated high molecular weight DNA was digested with Hpa II, Msp I and Hha I for methylation profiles. DNA was also digested with Hind III to examine amplification of specific fragments by Slot-Blot analysis. The results showed increased amplification of the H-ras oncogene in old male AL rats compared to old CR male rats. No difference was noted in overall methylation in young AL and CR rats, however, old male AL animals had a hypomethylation profile. Ethidium bromide staining revealed an increased apoptotic DNA fragment ladder in old AL animals compared to young and middle aged rats. General differences were noted in the overall methylation profile as a function of age in both the AL and CR rats. Molecular changes seen in these studies provide information on the role of diet and methylation of DNA in aging and carcinogenesis. Further studies are needed to determine if methylation is an important mechanism involved in these processes. I