Barbara J. Miller

Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat

Chronic dietary methyl deficiency in F344 rats was used as an in vivo mammalian model in which to evaluate the gene-specific alterations in DNA methylation patterns during multistage hepatocarcinogenesis. Using bisulfite mapping, the site-specific methylation profile within exons 6-7 of the 53 gene was determined in control liver, preneoplastic nodules (after 36 weeks of folate/methyl deficiency) and in hepatocellular carcinoma (after 54 weeks of deficiency). A progressive loss of methyl groups was observed at most CpG sites on both coding and non-coding strands during the first 36 weeks of folate/methyl deficiency, with the greatest loss occurring on the coding strand. When the same sequence was evaluated in tumor DNA after 54 weeks of deficiency, the majority of cytosines were unexpectedly found to have become remethylated. CpG sites that had previously lost methyl groups on both strands during preneoplasia as well as CpG sites that had been constitutively non-methylated, had undergone de novo methylation in tumor DNA. Maintenance methyltransferase and de novo methyltransferase activity in nuclear extracts were assessed using hemimethylated and non-methylated DNA substrates, respectively. In tumor, de novo methyltransferase capacity was increased approximately 4-fold relative to control or preneoplastic liver and associated with a relative increase in both p53 and genome-wide methylation density. In the preneoplastic nodules, the level p53 mRNA was increased and associated with hypomethylation in the coding region of the gene, whereas in tumor tissue, p53 mRNA was decreased and associated with relative hypermethylation. Taken together, these results provide additional insights into the dysregulation and instability in DNA methylation that accompanies the transition to tumor.

Mechanisms of DNA Damage, DNA Hypomethylation, and Tumor Progression in the Folate/Methyl-Deficient Rat Model of Hepatocarcinogenesis

Using the folate/methyl-deficient rat model of hepatocarcinogenesis, we obtained evidence that may provide new insights into a major unresolved paradox in DNA methylation and cancer research: the mechanistic basis for genome-wide hypomethylation despite an increase in DNA methyltransferase activity and gene-specific regional hypermethylation. Previous studies revealed that the methyltransferase binds with higher affinity to DNA strand breaks, gaps, abasic sites, and uracil than it does to its cognate hemimethylated CpG sites, consistent with its ancestral function as a DNA repair enzyme. These same DNA lesions are an early occurrence in models of folate and methyl deficiency and are often present in human preneoplastic cells. We hypothesized that the high-affinity binding of the maintenance DNA methyltransferase to unrepaired lesions in DNA could sequester available enzyme away from the replication fork and promote passive replication-dependent demethylation. In support of this possibility, we found that lesion-containing DNA is less efficiently methylated than lesion-free DNA from folate/methyl-deficient rats and that an increase in DNA strand breaks precedes DNA hypomethylation. Despite an adaptive increase in DNA methyltransferase activity, hemimethylated DNA from folate/methyl-deficient rats is progressively replaced by double-stranded unmethylated DNA that is resistant to remethylation with dietary methyl repletion. In promoter regions, the inappropriate binding of the DNA methyltransferase to unrepaired lesions or mispairs may promote local histone deacetylation, methylation, and regional hypermethylation associated with tumor suppressor gene silencing. These insights in an experimental model are consistent with the possibility that DNA lesions may be a necessary prerequisite for the disruption of normal DNA methylation patterns in preneoplastic and neoplastic cells.

Uracil misincorporation, DNA strand breaks, and gene amplification are associated with tumorigenic cell transformation in folate deficient/repleted Chinese hamster ovary cells☆

Clinical and experimental evidence has linked nutritional folic acid status to both anti- and procarcinogenic activity. Folate supplementation of normal cells appears to have a protective effect; however, folate supplementation of initiated cells may promote neoplastic progression. Given these considerations, the present series of experiments examines alterations in DNA metabolism and cumulative DNA lesions using an in vitro model of folate deprivation and repletion. DNA repair-deficient CHO-UV5 cells were cultured in Hams F-12 medium or in custom-prepared Hams F-12 medium lacking in folic acid, thymidine and hypoxanthine for a period of 18 days without cell passage. The results indicated that progressive folate and nucleotide depletion leads to a significant increase in the ratio of dUTP/dTTP and to the misincorporation of uracil into DNA. These alterations were accompanied by growth inhibition, DNA strand breaks, abasic sites and phenotypic abnormalities. After 14 days in culture, there was significant increase in gene amplification potential in the chronically folate-deficient cells, but no significant increase in anchorage-independent growth or in neoplastic transformation. Acute folate repletion of the deficient cells was used as a proliferative stimulus under conditions of dNTP pool imbalance and multiple lesions in DNA. A further increase in gene amplification was accompanied by anchorage-independent growth and neoplastic cell transformation as evidenced by aggressive tumor growth in Balb/c nu/nu mice. Using a sensitive in vitro model system, these results emphasize the essentiality of folic acid for de novo nucleotide synthesis and the integrity of the DNA. However, the in vivo relevance, especially in terms of tumorigenic potential, is not clear.

Determination of N-nitrosamines and N-nitrosamine precursors in rubber nipples from baby pacifiers by gas chromatography-thermal energy analysis

N-Nitrosamines and precursors are present in rubber products in which the accelerators and stabilizers used in the vulcanization process were derived from dialkylamines. Research was performed to develop data concerning the presence of N-nitrosamines and precursors so that the health significance of the exposure problem related to infant ingestion of these chemicals could be properly assessed. Volatile N-nitrosamines were determined in cut-up pacifier nipples by extraction with dichloromethane followed by concentration in a Kuderna-Danish evaporator, high-temperature mineral oil purge and trap, and analysis by gas chromatography--thermal energy analysis (GC-TEA). N-nitrosodibutylamine (NDBA) was the principal N-nitrosamine found, with concentrations up to 427 ppb. N-Nitrosamines and precursors in cut-up and intact nipples were determined by GC-TEA after a single extraction with artificial saliva. NDBA was the principal nitrosamine found, at levels up to 1040 ppb, while dibutylamine (DBA) was the principal precursor found, at levels up to 3890 ppb. The persistence of these compounds in intact nipples was determined by multiple artificial saliva extractions. Amounts of NDBA and DBA found after 15 artificial saliva extractions of intact pacifier nipples totalled 824 ppb and 15.6 ppm, respectively. N-Nitrosamine levels generally showed a gradual decrease in concentration with each extraction, whereas no consistent trend could be determined for concentrations of precursors.

Simulated solar light‐induced p53 mutagenesis in SKH‐1 mouse skin: A dose–response assessment

Sunlight and ultraviolet‐induced mutation of the p53 gene is a frequent, possibly obligate step in skin cancer development, making quantitative measurement of p53 mutation an ideal biomarker for sunlight‐induced skin carcinogenesis. To understand how the appearance of p53 mutation relates to skin tumor development, SKH‐1 hairless mice were exposed 5 d per week to one of four different doses of simulated solar light (SSL; 0, 6.85, 13.70, 20.55 mJ · CIE/cm2) previously characterized for their tumorigenic potential. Allele‐specific competitive blocker‐PCR (ACB‐PCR) was used to measure levels of p53 codon 270 CGT to TGT mutation within DNA isolated from dorsal skin of exposed mice. For each dose, p53 mutant fraction (MF) was measured after 4, 16, and 28 wk of exposure. Significant dose‐ and time‐dependent increases in p53 MF were identified. All p53 MF measurements were integrated by relating the observed p53 MF to the cumulative dose of SSL. The increase in the logarithm of p53 MF was described by the linear function: log10 MF = α + 0.0016 · d, where α is the spontaneous log10 MF after a particular time point and d is the dose of SSL in mJ · CIE/cm2. The p53 MF induced in nontumor bearing skin by 28 wk of exposure at the high dose of SSL was significantly lower than that found in skin tumors induced by ∼32 wk of exposure to the same dose of SSL. p53 MF showed a strong negative correlation with tumor latency, suggesting this quantitative biomarker has the potential to predict tumorigenicity.

Populations of p53 codon 270 CGT to TGT mutant cells in SKH-1 mouse skin tumors induced by simulated solar light.

The p53 codon 270 CGT to TGT mutation was investigated as a biomarker of sunlight‐induced mutagenesis and carcinogenesis. The relationship between tumor development and abundance of this hotspot mutation was analyzed in mouse skin tumors induced by chronic exposure to simulated solar light (SSL). The 24 tumors analyzed had similar growth kinetics, with an average doubling time of ∼16.4 d. Levels of the p53 codon 270 mutation were quantified in the 24 mouse skin tumors using allele‐specific competitive blocker‐polymerase chain reaction (ACB‐PCR). All tumors contained measurable amounts of the mutation. The p53 codon 270 CGT to TGT mutant fraction (MF) ranged from 2.29 × 10−3 to 9.42 × 10−2, with 3.26 × 10−2 as the median. These p53 MF measurements are lower than expected for an initiating mutation involved in the development of tumors of monoclonal origin. There was no evidence of a correlation between p53 codon 270 MF and either tumor area or an estimate of tumor cell number. Thus, the data do not support the idea that p53 mutation accumulates linearly during tumor development. To investigate how p53 mutation was distributed within tumors, 19 needle biopsies from seven different tumors were analyzed by ACB‐PCR. This analysis demonstrated that p53 codon 270 mutation is heterogeneously distributed within tumors. The long‐term goal of this research is to combine morphological and p53 MF measurements from tissues corresponding to the various stages of tumor development, in order to derive mathematical models relating the p53 codon 270 mutation to the development of SSL‐induced skin tumors. Published 2008 Wiley‐Liss, Inc.

Comparison of cell proliferation following partial hepatectomy in rats fed NIH-31 or semipurified AIN-76A diets: Effects of nucleic acid supplementation

Abstract Male Fischer 344 rats, were administered NIH-31, AIN-76A, or AIN-76A plus 0.25% yeast RNA diets for 3 weeks, partially hepatectomized (PH), and groups of 6 rats from each diet group were sacrificed at 0, 2, 7 and 14 days following PH. Liver weight, total DNA, total lipids, levels of thiobarbaturic acid reactive substances (TBARS), and levels of intracellular deoxyribonucleotides were determined. In addition, the number of hepatocytes in mitosis and/or positive for nuclear proliferating cell nuclear antigen (PCNA) were determined. Liver regeneration, measured as liver weight or total liver DNA, was equal in the three diet groups and was essentially complete by the end of 14 days. However, there was a 53% reduction in the mitotic index and a two-fold increase in the ratio of proliferating cells (G1 + S + G2 cells) to mitoses at 48 hours in animals of the AIN-76A diet group compared to those fed NIH-31 diet, suggesting that the cells were arrested or delayed in S-phase. At the time of PH, liver deoxyuridine monophosphate (dUMP) and deoxythymidine triphosphate (dTTP) levels in the AIN-76A group were decreased 29% and 57%, respectively, compared to those of the NIH-31 group. Addition of yeast RNA to the AIN-76A diet partially prevented the decrease in mitosis and maintained deoxyribonucleotide triphosphates (dNTP) levels to those of the NIH-31 group. These results indicate that the lack of dietary nucleotides in semipurified diets can alter dNTP levels and have adverse effects on cell proliferation. Consideration should be given to supplementing purified diets with a dietary source of nucletides.