Cytotoxicity, crosslinking and biological activity of three mitomycins.

While interstrand crosslinks (ICLs) have been considered as one type of DNA damage in the past, there is mounting evidence suggesting that these highly cytotoxic lesions are processed differently by the cellular machinery depending upon the ICL structure. In this study, we examined the crosslinking ability of three mitomycins, the structure of the ICLs they produce and the cytotoxicity of the drugs toward three different cell lines. The drugs are: mitomycin C (1), decarbamoylmitomycin C (2), and a mitomycin-conjugate (3) whose mitosane moiety is linked to a N-methylpyrrole carboxamide. We found that, overall, both MC and compound 3 show strong similarities regarding their alkylation of DNA, while DMC alkylating behavior is markedly different. To gain further insight into the mode of action of these drugs, we performed high throughput gene expression and gene ontology analysis to identify gene expression and cellular pathways most impacted by each drug treatment in MCF-7 cell lines. We observed that the novel mitomycin derivative (3) specifically causes changes in the expression of genes encoding proteins involved in cell integrity and tissue structure. Further analysis using bioinformatics (IPA) indicated that the new derivative (3) displays a stronger downregulation of major signaling networks that regulate the cell cycle, DNA damage response and cell proliferation when compared to MC and DMC. Collectively, these findings demonstrate that cytotoxic mechanisms of all three drugs are complex and are not solely related to their crosslinking abilities or the structure of the ICLs they produce.

Dietary modifications, weight loss, and changes in metabolic markers affect global DNA methylation in Hispanic, African American, and Afro-Caribbean breast cancer survivors.

BACKGROUND: Lower levels of global DNA methylation in tissue and blood have been associated with increased cancer risk. Conversely, cross-sectional analyses of healthier lifestyle patterns have been associated with higher levels of global DNA methylation. OBJECTIVE: In this trial, we explored the associations between changes in lifestyle modifications (diet, weight loss), metabolic markers, and global epigenetic biomarkers in white blood cells. METHODS: Study participants were Hispanic, African American, and Afro-Caribbean overweight and sedentary female breast cancer survivors (n = 24) who participated in a larger randomized, crossover, pilot study of a 6-mo weight loss intervention and who had available blood specimens. Anthropometric measures, a food-frequency questionnaire, and peripheral blood were collected at baseline, 6 mo, and 12 mo. Plasma samples were analyzed for metabolic markers (insulin, glucose). We measured DNA methylation of long interspersed nucleotide element 1 (LINE-1) and satellite 2 by pyrosequencing and MethyLight, respectively, and global DNA methylation by the luminometric methylation assay (LUMA). RESULTS: DNA methylation of LINE-1 was statistically significantly elevated at 6 mo [75.5% vs. 78.5% (P < 0.0001)] and 12 mo [75.5% vs. 77.7% (P < 0.0001)], compared to baseline. Over a 12-mo period, changes in percentage body fat and plasma glucose concentrations were positively associated with LINE-1 DNA methylation (ß = 0.19, P = 0.001) and LUMA DNA methylation levels (ß = 0.24, P = 0.02), respectively. Similarly, 12-mo changes in dietary measures such as vegetable (ß = 0.009, P = 0.048), protein (ß = 0.04, P = 0.001), and total caloric (ß = 0.05, P = 0.01) intake were positively associated with changes in LUMA DNA methylation, as was intake of fruit positively associated with changes in LINE-1 DNA methylation (ß = 0.004, P = 0.02). CONCLUSIONS: Our hypothesis-generating results suggest that lifestyle modifications may be associated with changes in global DNA methylation detectable at 6 and 12 mo. These biomarkers may be useful intermediate biomarkers to use in future intervention trials. This trial was registered at clinicaltrials.gov as NCT00811824.

DNA double-strand break repair genotype and phenotype and breast cancer risk within sisters from the New York site of the Breast Cancer Family Registry (BCFR).

PURPOSE: We previously observed that poor DNA repair phenotype is associated with increased breast cancer (BC) risk within families. Here, we examined whether genetic variation in double-strand break repair (DSBR) genes is associated with BC risk and if genotypes are related to phenotype in unaffected women. METHODS: Using data from the New York site of the Breast Cancer Family Registry, we investigated 25 single-nucleotide polymorphism (SNPs) involved in DSBR using biospecimens from 337 BC cases and 410 unaffected sister controls. RESULTS: Genotypes in XRCC4 were associated with BC risk, with ORs of 1.67 (95 % CI 1.01-2.76) for the combined GA/AA of rs1805377 and 1.69 (95 % CI 1.03-2.77) for rs1056503 TG/GG; these associations were no longer statistically significant in multivariable conditional logistic regression models. When examining the association of SNPs with phenotype, we found that genotypes of XRCC5 rs3834 and rs1051685, which were highly correlated with each other, were associated with end-joining (EJ) capacity; women with the XRCC5 rs3834 GA genotype had better DNA repair as measured by higher levels of EJ capacity (37.8 ± 14.1 % for GA vs. 27.9 ± 11.8 % for GG carriers p = 0.0006). Women with the AA genotype of BRCA1 rs799917 also had higher EJ capacity (35.1 ± 9.2 %) than those with GG (26.4 ± 10.1 %, p = 0.02). CONCLUSIONS: Overall, we found that selected DSBR genotypes were associated with phenotype, although they were not associated with BC risk itself, suggesting that phenotypic measures are influenced by endogenous and exogenous factors across the life course and may be better markers than genotypic measures for ascertaining BC risk.

Repetitive element DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the Breast Cancer Family Registry.

Global decreases in DNA methylation, particularly in repetitive elements, have been associated with genomic instability and human cancer. Emerging, though limited, data suggest that in white blood cell (WBC) DNA levels of methylation, overall or in repetitive elements, may be associated with cancer risk. We measured methylation levels of three repetitive elements [Satellite 2 (Sat2)], long interspersed nuclear element-1 (LINE-1) and Alu) by MethyLight, and LINE-1 by pyrosequencing in a total of 282 breast cancer cases and 347 unaffected sisters from the New York site of the Breast Cancer Family Registry (BCFR) using DNA from both granulocytes and total WBC. We found that methylation levels in all markers were correlated between sisters (Spearman correlation coefficients ranged from 0.17 to 0.55). Sat2 methylation was statistically significantly associated with increased breast cancer risk [odds ratio (OR) = 2.09, 95% confidence interval (CI) = 1.09-4.03; for each unit decrease in the natural log of the methylation level, OR = 2.12, 95% CI = 0.88-5.11 for the lowest quartile compared with the highest quartile]. These associations were only observed in total WBC but not granulocyte DNA. There was no association between breast cancer and LINE-1 and Alu methylation. If replicated in larger prospective studies, these findings support that selected markers of epigenetic changes measured in WBC, such as Sat2, may be potential biomarkers of breast cancer risk.

Differences in MB-COMT DNA methylation in monozygotic twins on phenotypic indicators of impulsivity.

Epigenetic modifications of the membrane bound catechol-O-methyltransferase (MB-COMT) gene may affect the enzymatic degradation of dopamine, and consequently, human behavior. This study investigated the association between membrane bound catechol-O-methyltransferase DNA methylation (DNAm) differences in 92 monozygotic (MZ) twins with phenotypic manifestations of cognitive, behavioral, and personality indicators associated with reward-related behaviors and lack of control. We used pyrosequencing to determine DNAm of the regulatory region of membrane bound catechol-O-methyltransferase in saliva DNA. Results of intrapair differences in the percentage of membrane bound catechol-O-methyltransferase DNAm at each of five CpG sites show that there are associations between phenotypic indicators of lack of control and membrane bound catechol-O-methyltransferase DNAm differences on CpG1, CpG2 and CpG4, suggesting the common epigenetic patterns for personality traits, cognitive functions, and risk behaviors.

The severe slow growth of Deltasrs2 Deltarqh1 in Schizosaccharomyces pombe is suppressed by loss of recombination and checkpoint genes.

Our interest in the Schizosaccharomyces pombe RecQ helicase, rqh1+, led us to investigate the function of a related putative DNA helicase, srs2+. We identified the srs2+ homolog in S.pombe, and found that srs2+ is not essential for cell viability. A Deltasrs2 Deltarqh1 double mutant grows extremely slowly with aberrant shaped cells and low viability. This slow growth does not appear to be related to stalled replication, as Deltasrs2 Deltarqh1 cells showed higher survival rates, compared with Deltarqh1, when stalled forks were increased by UV irradiation or hydroxy urea treatment. Consistent with this result, we found that Deltasrs2 Deltarqh1 cells progress through S-phase with a slight delay, but undergo a checkpoint-dependent arrest presumably at G2/M. Further, we found that Deltasrs2 Deltarqh1 slow growth is related to recombination, as loss of either the rhp51+ or rhp57+ recombination genes improves cell growth in the double mutant. Deltasrs2 is also synthetic lethal with Deltarhp54, another homologous recombination gene. This lethality is suppressed in a Deltarhp51 background. Together, these results demonstrate a clear genetic interaction between rqh1+, srs2+ and the genes of the homologous recombination pathway.

Mismatch Repair Polymorphisms as Markers of Breast Cancer Prevalence in the Breast Cancer Family Registry.

BACKGROUND: Major breast cancer susceptibility genes involved in DNA repair, including BRCA1 and BRCA2, have been identified. However, mutations in these genes account for only 5-10% of identified breast cancer cases. Additional DNA repair pathway genes may also contribute to susceptibility. MATERIALS AND METHODS: We investigated the association between 12 single nucleotide polymorphisms (SNPs) in mismatch repair (MMR) genes and breast cancer risk among 313 sister-sets enrolled in the New York site of the Breast Cancer Family Registry (BCFR) (n=744) using conditional logistic regression analysis. RESULTS: An increase in breast cancer risk was observed for women with the MUTYH_rs3219489 variant allele (odds ratio (OR)=2.23, 95% confidence interval (CI)=1.10-4.52) and for women with the MSH2_rs2303428 variant allele (OR=1.73, 95% CI=1.00-2.99). CONCLUSION: Deficiencies in DNA repair pathways, such as MMR, have implications for the onset of familial breast cancer.

Differences in DNA methylation by extent of breast cancer family history in unaffected women.

Breast cancer clusters within families but genetic factors identified to date explain only a portion of this clustering. Lower global DNA methylation in white blood cells (WBC) has been associated with increased breast cancer risk. We examined whether WBC DNA methylation varies by extent of breast cancer family history in unaffected women from high-risk breast cancer families. We evaluated DNA methylation levels in LINE-1, Alu and Sat2 in 333 cancer-free female family members of the New York site of the Breast Cancer Family Registry, the minority of which were known BRCA1 or BRCA2 mutation carriers. We used generalized estimated equation models to test for differences in DNA methylation levels by extent of their breast cancer family history after adjusting for age. All unaffected women had at least one sister affected with breast cancer. LINE-1 and Sat2 DNA methylation levels were lower in individuals with 3 or more (3+) first-degree relatives with breast cancer relative to women with only one first-degree relative. For LINE-1, Alu, and Sat2, having 3+ affected first-degree relatives was associated with a decrease of 23.4% (95%CI = -46.8%, 0.1%), 17.9% (95%CI = -39.5%, 3.7%) and 11.4% (95% CI = -20.3%, -2.5%), respectively, relative to individuals with only one affected first-degree relative, but the results were only statistically significant for Sat2. Individuals having an affected mother had 17.9% lower LINE-1 DNA methylation levels (95% CI = -28.8%, -7.1%) when compared with those not having an affected mother. No associations were observed for Alu or Sat2 by maternal breast cancer status. If replicated, these results indicate that lower global WBC DNA methylation levels in families with extensive cancer histories may be one explanation for the clustering of cancers in these families. Family clustering of disease may reflect epigenetic as well as genetic and shared environmental factors.

Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes.

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.

Genome-wide methylation analyses in glioblastoma multiforme.

Few studies had investigated genome-wide methylation in glioblastoma multiforme (GBM). Our goals were to study differential methylation across the genome in gene promoters using an array-based method, as well as repetitive elements using surrogate global methylation markers. The discovery sample set for this study consisted of 54 GBM from Columbia University and Case Western Reserve University, and 24 brain controls from the New York Brain Bank. We assembled a validation dataset using methylation data of 162 TCGA GBM and 140 brain controls from dbGAP. HumanMethylation27 Analysis Bead-Chips (Illumina) were used to interrogate 26,486 informative CpG sites in both the discovery and validation datasets. Global methylation levels were assessed by analysis of L1 retrotransposon (LINE1), 5 methyl-deoxycytidine (5m-dC) and 5 hydroxylmethyl-deoxycytidine (5hm-dC) in the discovery dataset. We validated a total of 1548 CpG sites (1307 genes) that were differentially methylated in GBM compared to controls. There were more than twice as many hypomethylated genes as hypermethylated ones. Both the discovery and validation datasets found 5 tumor methylation classes. Pathway analyses showed that the top ten pathways in hypomethylated genes were all related to functions of innate and acquired immunities. Among hypermethylated pathways, transcriptional regulatory network in embryonic stem cells was the most significant. In the study of global methylation markers, 5m-dC level was the best discriminant among methylation classes, whereas in survival analyses, high level of LINE1 methylation was an independent, favorable prognostic factor in the discovery dataset. Based on a pathway approach, hypermethylation in genes that control stem cell differentiation were significant, poor prognostic factors of overall survival in both the discovery and validation datasets. Approaches that targeted these methylated genes may be a future therapeutic goal.

Genomic methylation changes over time in peripheral blood mononuclear cell DNA: differences by assay type and baseline values.

BACKGROUND: Lower levels of genomic DNA methylation in blood DNA has been associated with risk of different cancers and several cancer risk factors. To understand the use of genomic methylation measures as biomarkers of cancer risk, data are needed on within-individual changes over time. METHODS: Using information from 77 subjects with blood collected at 2 visits on average 8 years apart, we examined whether levels of DNA methylation change with time and if so, whether selected cancer risk factors predict these changes. We measured DNA methylation levels in peripheral blood mononuclear cells (PBMC) using three assays that have been used in epidemiologic studies: (i) luminometric methylation assay (LUMA)(ii) LINE-1 by pyrosequencing, and (iii) Sat2 by MethyLight. RESULTS: Close to a third of all individuals had large changes over time (≥10%) in LUMA with 19.5% increasing and 13.0% decreasing. For Sat2, two-thirds of individuals had large changes with 40% increasing and 26% decreasing over time. In contrast, only 3.9% of individuals had large changes in LINE-1 over time. The degree of change in PBMC DNA methylation was statistically significantly inversely associated with methylation levels at baseline; greater decreases were observed in individuals with higher baseline values for each assay. CONCLUSIONS: These data, if replicated, suggest that changes in DNA methylation over time are highly associated with baseline values of the assay and vary by assay type. IMPACT: These findings suggest that assays that change more over time may warrant consideration for studies that measure later life exposures.

DNA methylation changes correlate with Gleason score and tumor stage in prostate cancer.

DNA methylation, a widely used epigenetic mark, has been associated with many tumors. However, few studies have addressed the role of cell-free plasma DNA methylation in discriminating aggressive prostate cancer (PCa) from indolent cases. We conducted a case series and a case-control study among histologically confirmed stage II/III cases and matched controls recruited at Columbia University Medical Center. The aim of this study was to investigate whether plasma DNA methylation levels are appropriate surrogate biomarker of PCa tumor tissue levels and whether these markers are associated with worse clinicopathological tumor characteristics, which correlate with poorer prognosis. Quantitative pyrosequencing was used to detect methylation levels of p16 (CDKN4A), APC, GSTP1, and LINE-1 in 24 pairs of prostate tumor and adjacent tissues, as well as 27 plasma samples of PCa patients and 24 of controls. DNA methylation levels were significantly higher in tumor tissue than in adjacent nontumor tissue for p16 (CDKN4A), GSTP1, and APC; GSTP1 had a higher average percentage methylation in tumor tissue (38.9%) compared with p16 (CDKN4A) (5.9%) and APC (14.5%). GSTP1, p16 (CDKN4A), and APC methylation in tumor tissue was statistically significantly higher for cases with Gleason score ≥7 compared with those with Gleason score <7 [49.0% vs. 21.9% (p=0.01), 6.6% vs. 4.5% (p=0.04), and 19.1% vs. 7.4% (p=0.02), respectively]. Plasma LINE-1 methylation levels were higher in those with higher Gleason (67.6%) than in those with Gleason's below 7 (64.6%, p=0.03). Significant plasma-tissue correlations were observed for GSTP1 and LINE-1 methylation. These data, although preliminary, suggest that aberrant methylation may be a useful marker to identify PCa patients with clinically aggressive disease.

Adult global DNA methylation in relation to pre-natal nutrition.

BACKGROUND: Exposure to a pre-natal famine environment has been associated with a persistent decrease in DNA methylation of the IGF2 gene, although study findings on other loci have been highly variable. There have been no studies to date of the relation between pre-natal famine and overall global DNA methylation in adulthood. METHODS: Our study population includes 350 births with pre-natal exposure to the Dutch famine of 1944-45 selected from three birth clinics, 290 births from these clinics born before or after the famine as unexposed time controls and 307 same-sex siblings of either birth group as unexposed family controls. All study subjects were interviewed and underwent a medical examination at a mean age of 58 years when blood samples were also collected. As measures of genomic DNA methylation, we analysed two repetitive elements, LINE-1 (long interspersed nucleotide element 1) and Sat2 (Satellite 2 DNA sequence) by pyrosequencing and MethyLight, respectively, and overall genomic DNA methylation using the Luminometric methylation assay (LUMA). RESULTS: Mean DNA methylation by LUMA was 75.2% [standard deviation (SD) 4.7], by LINE-1 was 77.1% (SD 2.5) and by Sat2 was 122.2 (SD 56.2). Pre-natal famine exposure was associated with negligible changes in all three assays {LUMA: -0.16% [95% confidence interval (95% CI) -0.49 to 0.81], P = 0.63; LINE-1: -0.05 % (95% CI -0.33 to 0.22), P = 0.70; and Sat2: -0.51% (95% CI -7.38 to 6.36), P = 0.88} relative to unexposed controls, adjusting for age at examination and within family clustering. CONCLUSION: Our results show no relation between overall global DNA methylation in adults and pre-natal famine exposure. Further work should focus on selected regions in the genome that may be differentially methylated in response to changes in early life exposures and predict adult health outcomes.

Global DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the Breast Cancer Family Registry.

Lower global DNA methylation is associated with genomic instability and it is one of the epigenetic mechanisms relevant to carcinogenesis. Emerging evidence for several cancers suggests that lower overall levels of global DNA methylation in blood are associated with different cancer types, although less is known about breast cancer. We examined global DNA methylation levels using a sibling design in 273 sisters affected with breast cancer and 335 unaffected sisters from the New York site of the Breast Cancer Family Registry. We measured global DNA methylation in total white blood cell (WBC) and granulocyte DNA by two different methods, the [ ( 3) H]-methyl acceptance assay and the luminometric methylation assay (LUMA). Global methylation levels were only modestly correlated between sisters discordant for breast cancer (Spearman correlation coefficients ranged from -0.08 to 0.24 depending on assay and DNA source). Using conditional logistic regression models, women in the quartile with the lowest DNA methylation levels (as measured by the [ ( 3) H]-methyl acceptance assay) had a 1.8-fold (95% CI = 1.0-3.3) higher relative association with breast cancer than women in the quartile with the highest DNA methylation levels. When we examined the association on a continuous scale, we also observed a positive association (odds ratio, OR = 1.3, 95% CI = 1.0-1.7, for a one unit change in the natural logarithm of the DPM/µg of DNA). We observed no association between measures by the LUMA assay and breast cancer risk. If replicated in prospective studies, this study suggests that global DNA methylation levels measured in WBC may be a potential biomarker of breast cancer risk even within families at higher risk of cancer.

Smoking and hepatocellular carcinoma mortality.

The association between cigarette smoking and mortality from hepatocellular carcinoma (HCC) is ambiguous. We analyzed the association between smoking and mortality in HCC patients seen at our center. We collected data retrospectively on patients diagnosed with HCC between 2002 and 2009. We estimated the association of smoking history with demographic, clinical and treatment factors. We then modeled these factors as predictors of mortality. Among smokers, we analyzed the effects of pack-year history and cessation times on survival. Two hundred and twenty-three out of 444 patients with HCC had a history of smoking. Smokers were more likely to be younger at diagnosis, to have α fetoprotein (AFP) values less than the median, and to have had surgery (p=0.04) compared to non-smokers. In a Cox model, younger age, lower AFP and Child's Class were all independently predictive of survival, but smoking was not. Smokers with over 20 pack-years did not have worse survival than lighter smokers, and cessation times also did not affect survival after controlling for age. We found a significant interaction between smoking and drinking. In our data, smoking was not independently associated with HCC survival in a multivariable model. Smoking was associated with favorable prognostic features which likely outweighed any independent effect of smoking.

DNA methylation in white blood cells: association with risk factors in epidemiologic studies.

Alterations in DNA methylation patterns, both at specific loci and overall in the genome, have been associated with many different health outcomes. In cancer and other diseases, most of these changes have been observed at the tissue level. Data on whether DNA methylation changes in white blood cells (WBC) can serve as a useful biomarker for different health outcomes are much more limited, but rapidly emerging. Epidemiologic studies have reported associations between global WBC methylation and several different cancers including cancers of the colon, bladder, stomach, breast and head and neck, as well as schizophrenia and myelodysplastic syndrome. Evidence for WBC methylation at specific loci and disease risk is more limited, but increasing. Differences in WBC DNA methylation by selected risk factors including demographic (age, gender, race), environmental exposures (benzene, persistent organic pollutants, lead, arsenic, and air pollution), and other risk factors (cigarette smoke, alcohol drinking, body size, physical activity and diet) have been observed in epidemiologic studies though the patterns are far from consistent. Challenges in inferences from the existing data are primarily due to the cross-sectional and small size of most studies to date as well as the differences in results across assay type and source of DNA. Large, prospective studies will be needed to understand whether changes in risk factors are associated with changes in DNA methylation patterns, and if changes in DNA methylation patterns are associated with changes in disease endpoints.

Global methylation profiles in DNA from different blood cell types.

DNA methylation measured in white blood cell DNA is increasingly being used as in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran), and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources (WBC, Gran, mononuclear (MN), and lymphoblastoid cell lines (LCL)), we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA), and [(3)H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LBC, MN, or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [(3)H]-methyl acceptance, LINE1, and Alu assays. Methylation in MN was correlated with methylation in WBC for the [(3)H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant positive correlations ranging from 0.3-0.7 for each cell type with one exception (Sat2 and Alu in MN). Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39-0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18-0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC.

Global DNA methylation levels in girls with and without a family history of breast cancer.

Lower levels of global DNA methylation in white blood cell (WBC) DNA have been associated with adult cancers. It is unknown whether individuals with a family history of cancer also have lower levels of global DNA methylation early in life. We examined global DNA methylation in WBC (measured in three repetitive elements, LINE1, Sat2 and Alu, by MethyLight and in LINE1 by pyrosequencing) in 51 girls ages 6-17. Compared to girls without a family history of breast cancer, methylation levels were lower for all assays in girls with a family history of breast cancer, and statistically significantly lower for Alu and LINE1 pyrosequencing. After adjusting for age, body mass index (BMI), and Tanner stage, only methylation in Alu was associated with family history of breast cancer. If these findings are replicated in larger studies, they suggest that lower levels of global WBC DNA methylation observed later in life in adults with cancer may also be present early in life in children with a family history of cancer.

RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex.

SGS1 encodes a DNA helicase whose homologues in human cells include the BLM, WRN, and RECQ4 genes, mutations in which lead to cancer-predisposition syndromes. Clustering of synthetic genetic interactions identified by large-scale genetic network analysis revealed that the genetic interaction profile of the gene RMI1 (RecQ-mediated genome instability, also known as NCE4 and YPL024W) was highly similar to that of SGS1 and TOP3, suggesting a functional relationship between Rmi1 and the Sgs1/Top3 complex. We show that Rmi1 physically interacts with Sgs1 and Top3 and is a third member of this complex. Cells lacking RMI1 activate the Rad53 checkpoint kinase, undergo a mitotic delay, and display increased relocalization of the recombination repair protein Rad52, indicating the presence of spontaneous DNA damage. Consistent with a role for RMI1 in maintaining genome integrity, rmi1Delta cells exhibit increased recombination frequency and increased frequency of gross chromosomal rearrangements. In addition, rmi1Delta strains fail to fully activate Rad53 upon exposure to DNA-damaging agents, suggesting that Rmi1 is also an important part of the Rad53-dependent DNA damage response.