Maternal educational attainment in pregnancy and epigenome-wide DNA methylation changes in the offspring from birth until adolescence.

Maternal educational attainment (MEA) shapes offspring health through multiple potential pathways. Differential DNA methylation may provide a mechanistic understanding of these long-term associations. We aimed to quantify the associations of MEA with offspring DNA methylation levels at birth, in childhood and in adolescence. Using 37 studies from high-income countries, we performed meta-analysis of epigenome-wide association studies (EWAS) to quantify the associations of completed years of MEA at the time of pregnancy with offspring DNA methylation levels at birth (n = 9 881), in childhood (n = 2 017), and adolescence (n = 2 740), adjusting for relevant covariates. MEA was found to be associated with DNA methylation at 473 cytosine-phosphate-guanine sites at birth, one in childhood, and four in adolescence. We observed enrichment for findings from previous EWAS on maternal folate, vitamin-B12 concentrations, maternal smoking, and pre-pregnancy BMI. The associations were directionally consistent with MEA being inversely associated with behaviours including smoking and BMI. Our findings form a bridge between socio-economic factors and biology and highlight potential pathways underlying effects of maternal education. The results broaden our understanding of bio-social associations linked to differential DNA methylation in multiple early stages of life. The data generated also offers an important resource to help a more precise understanding of the social determinants of health.

Analysis of DNA methylation at birth and in childhood reveals changes associated with season of birth and latitude.

BACKGROUND: Seasonal variations in environmental exposures at birth or during gestation are associated with numerous adult traits and health outcomes later in life. Whether DNA methylation (DNAm) plays a role in the molecular mechanisms underlying the associations between birth season and lifelong phenotypes remains unclear. METHODS: We carried out epigenome-wide meta-analyses within the Pregnancy And Childhood Epigenetic Consortium to identify associations of DNAm with birth season, both at differentially methylated probes (DMPs) and regions (DMRs). Associations were examined at two time points: at birth (21 cohorts, N = 9358) and in children aged 1-11 years (12 cohorts, N = 3610). We conducted meta-analyses to assess the impact of latitude on birth season-specific associations at both time points. RESULTS: We identified associations between birth season and DNAm (False Discovery Rate-adjusted p values < 0.05) at two CpGs at birth (winter-born) and four in the childhood (summer-born) analyses when compared to children born in autumn. Furthermore, we identified twenty-six differentially methylated regions (DMR) at birth (winter-born: 8, spring-born: 15, summer-born: 3) and thirty-two in childhood (winter-born: 12, spring and summer: 10 each) meta-analyses with few overlapping DMRs between the birth seasons or the two time points. The DMRs were associated with genes of known functions in tumorigenesis, psychiatric/neurological disorders, inflammation, or immunity, amongst others. Latitude-stratified meta-analyses [higher (≥ 50°N), lower (< 50°N, northern hemisphere only)] revealed differences in associations between birth season and DNAm by birth latitude. DMR analysis implicated genes with previously reported links to schizophrenia (LAX1), skin disorders (PSORS1C, LTB4R), and airway inflammation including asthma (LTB4R), present only at birth in the higher latitudes (≥ 50°N). CONCLUSIONS: In this large epigenome-wide meta-analysis study, we provide evidence for (i) associations between DNAm and season of birth that are unique for the seasons of the year (temporal effect) and (ii) latitude-dependent variations in the seasonal associations (spatial effect). DNAm could play a role in the molecular mechanisms underlying the effect of birth season on adult health outcomes.

Placental multi-omics integration identifies candidate functional genes for birthweight.

Abnormal birthweight is associated with increased risk for cardiometabolic diseases in later life. Although the placenta is critical to fetal development and later life health, it has not been integrated into largescale functional genomics initiatives, and mechanisms of birthweight-associated variants identified by genome wide association studies (GWAS) are unclear. The goal of this study is to provide functional mechanistic insight into the causal pathway from a genetic variant to birthweight by integrating placental methylation and gene expression with established GWAS loci for birthweight. We identify placental DNA methylation and gene expression targets for several birthweight GWAS loci. The target genes are broadly enriched in cardiometabolic, immune response, and hormonal pathways. We find that methylation causally influences WNT3A, CTDNEP1, and RANBP2 expression in placenta. Multi-trait colocalization identifies PLEKHA1, FES, CTDNEP1, and PRMT7 as likely functional effector genes. These findings reveal candidate functional pathways that underpin the genetic regulation of birthweight via placental epigenetic and transcriptomic mechanisms. Clinical trial registration; ClinicalTrials.gov, NCT00912132.

Sex-specific placental gene expression signatures of small for gestational age at birth.

INTRODUCTION: Small for gestational age at birth (SGA), often a consequence of placental dysfunction, is a risk factor for neonatal morbidity and later life cardiometabolic diseases. There are sex differences in placental gene expression and fetal growth. Here, we investigated sex-specific associations between gene expression in human placenta measured using RNA sequencing and SGA status using data from ethnic diverse pregnant women in the NICHD Fetal Growth Studies cohort (n = 74). METHODS: Gene expression measures were obtained using RNA-Sequencing and differential gene expression between SGA (birthweight <10th percentile) and appropriate for gestational age (AGA: ≥10th and <90th percentile) was tested separately in males (12 SGA and 27 AGA) and females (9 SGA and 26 AGA) using a weighted mean of log ratios method with adjustment for mode of delivery and ethnicity. RESULTS: At 5% false discovery rate (FDR), we identified 40 differentially expressed genes (DEGs) related to SGA status among males (95% up- and 5% down-regulated) and 314 DEGs among females (32.5% up- and 67.5% down-regulated). Seven female-specific DEGs overlapped with known imprinted genes (AXL, CYP24A1, GPR1, PLAGL1, CMTM1, DLX5, LY6D). The DEGs in males were significantly enriched for immune response and inflammation signaling pathways whereas the DEGs in females were enriched for organ development signaling pathways (FDR<0.05). Sex-combined analysis identified no additional DEGs, rather 98% of the sex-specific DEGs were no longer significant and the remaining 2% were attenuated. DISCUSSION: This study revealed sex-specific human placental gene expression changes and molecular pathways associated with SGA and underscored that unravelling the pathogenesis of SGA warrants consideration of fetal sex as a biological variable. TRIAL REGISTRATION: https://www. CLINICALTRIALS: gov, Unique identifier: NCT00912132.

Ancestry-Matched and Cross-Ancestry Genetic Risk Scores of Type 2 Diabetes in Pregnant Women and Fetal Growth: A Study in an Ancestrally Diverse Cohort.

Maternal genetic variants associated with offspring birth weight and adult type 2 diabetes (T2D) risk loci show some overlap. Whether T2D genetic risk influences longitudinal fetal weight and the gestational timing when these relationships begin is unknown. We investigated the associations of T2D genetic risk scores (GRS) with longitudinal fetal weight and birth weight among 1,513 pregnant women from four ancestral groups. Women had up to five ultrasonography examinations. Ancestry-matched GRS were constructed separately using 380 European- (GRSeur), 104 African- (GRSafr), and 189 East Asian- (GRSeas) related T2D loci discovered in different population groups. Among European Americans, the highest quartile GRSeur was significantly associated with 53.8 g higher fetal weight (95% CI 19.2-88.5) over the pregnancy. The associations began at gestational week 24 and continued through week 40, with a 106.8 g (95% CI 6.5-207.1) increase in birth weight. The findings were similar in analysis further adjusted for maternal glucose challenge test results. No consistent association was found using ancestry-matched or cross-ancestry GRS in non-Europeans. In conclusion, T2D genetic susceptibility may influence fetal growth starting at midsecond trimester among Europeans. Absence of similar associations in non-Europeans urges the need for further genetic T2D studies in diverse ancestries.

Impact of depression and stress on placental DNA methylation in ethnically diverse pregnant women.

Aim: To investigate the association between placental genome-wide methylation at birth and antenatal depression and stress during pregnancy. Methods: We examined the association between placental genome-wide DNA methylation (n = 301) and maternal depression and stress assessed at six gestation periods during pregnancy. Correlation between DNA methylation at the significantly associated CpGs and expression of nearby genes in the placenta was tested. Results: Depression and stress were associated with methylation of 16 CpGs and two CpGs, respectively, at a 5% false discovery rate. Methylation levels at two of the CpGs associated with depression were significantly associated with expression of ADAM23 and CTDP1, genes implicated in neurodevelopment and neuropsychiatric diseases. Conclusion: Placental epigenetic changes linked to antenatal depression suggest potential fetal brain programming. Clinical trial registration number: NCT00912132 (ClinicalTrials.gov).

Lay abstract Our research examined 301 women at six time points during their pregnancies in regard to depression or stress. We then examined samples of the placenta after birth for epigenetic changes and explored whether they were linked to the status of depression or stress observed during pregnancy. We found that 16 epigenetic changes were linked to depression and two were linked to stress. Some of the epigenetic changes in the placenta linked to depression were located close to genes which are known to have important roles in brain development and occurrence of psychiatric disorders. Therefore maternal depression may have implications for the long-term mental health of the child.

Conception by fertility treatment and offspring deoxyribonucleic acid methylation.

OBJECTIVE: To investigate whether deoxyribonucleic acid (DNA) methylation at birth and in childhood differ by conception using assisted reproductive technologies (ART) or ovulation induction compared with those in children conceived without fertility treatment. DESIGN: Upstate KIDS is a matched exposure cohort which oversampled on newborns conceived by treatment. SETTING: New York State (excluding New York City). PATIENT(S): This analysis included 855 newborns and 152 children at approximately 9 years of age. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): DNA methylation levels were measured using the Illumina EPIC platform. Single CpG and regional analyses at imprinting genes were conducted. RESULT(S): Compared to no fertility treatment, ART was associated with lower mean DNA methylation levels at birth in 11 CpGs (located in/near SYCE1, SPRN, KIAA2013, MYO1D, GET1/WRB-SH4BGR, IGF1R, SORD, NECAB3/ACTL10, and GET1) and higher mean methylation level in 1 CpG (KLK4; all false discovery rate P<.05). The strongest association (cg17676129) was located at SYCE1, which codes for a synaptonemal complex that plays a role in meiosis and therefore infertility. This CpG remained associated with newborn hypomethylation when the analysis was limited to those conceived with ICSI, but this may be because of underlying male infertility. In addition, nine regions in maternally imprinted genes (IGF1R, PPIEL, SVOPL GNAS, L3MBTL, BLCAP, HYMAI/PLAGL1, SNU13, and MEST) were observed to have decreased mean DNA methylation levels among newborns conceived by ART. In childhood, hypomethylation of the maternally imprinted gene, GNAS, persisted. No CpGs or regions were associated with ovulation induction. CONCLUSION(S): ART but not ovulation induction was associated with hypomethylation at birth, but only one difference at an imprinting region appeared to persist in childhood.

Perfluorooctanoic acid (PFOA) or perfluorooctane sulfonate (PFOS) and DNA methylation in newborn dried blood spots in the Upstate KIDS cohort.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are persistent organic pollutants which may alter prenatal development, potentially through epigenetic modifications. Prior studies examining PFOS/PFOA and DNA methylation have relatively few subjects (n < 200) and inconsistent results. We examined relations of PFOA/PFOS with DNA methylation among 597 neonates in the Upstate KIDS cohort study. PFOA/PFOS were quantified in newborn dried blood spots (DBS) using high-performance liquid chromatography/tandem mass spectrometry. DNA methylation was measured using the Infinium MethylationEPIC BeadChip with DNA extracted from DBS. Robust linear regression was used to examine the associations of PFOA/PFOS with DNA methylation at individual CpG sites. Covariates included sample plate, estimated cell type, epigenetically derived ancestry, infant sex and plurality, indicators of maternal socioeconomic status, and prior pregnancy loss. In supplemental analysis, we restricted the analysis to 2242 CpG sites previously identified as Correlated Regions of Systemic Interindividual Variation (CoRSIVs) which include metastable epialleles. At FDR<0.05, PFOA concentration >90th percentile was related to DNA methylation at cg15557840, near SCRT2, SRXN1; PFOS>90th percentile was related to 2 CpG sites in a sex-specific manner (cg19039925 in GVIN1 in boys and cg05754408 in ZNF26 in girls). When analysis was restricted to CoRSIVs, log-scaled, continuous PFOS concentration was related to DNA methylation at cg03278866 within PTBP1. In conclusion, there was limited evidence of an association between high concentrations of PFOA/PFOS and DNA methylation in newborn DBS in the Upstate KIDS cohort. These findings merit replication in populations with a higher median concentration of PFOA/PFOS.

Early pregnancy dyslipidemia is associated with placental DNA methylation at loci relevant for cardiometabolic diseases.

Aim: To identify placental DNA methylation changes that are associated with early pregnancy maternal dyslipidemia. Materials & methods: We analyzed placental genome-wide DNA methylation (n = 262). Genes annotating differentially methylated CpGs were evaluated for gene expression in placenta (n = 64). Results: We found 11 novel significant differentially methylated CpGs associated with high total cholesterol, low-density lipoprotein cholesterol and triglycerides, and low high-density lipoprotein cholesterol. High triglycerides were associated with decreased methylation of cg02785814 (ALX4) and decreased expression of ALX4 in placenta. Genes annotating the differentially methylated CpGs play key roles in lipid metabolism and were enriched in dyslipidemia pathways. Functional annotation found cis-methylation quantitative trait loci for genetic loci in ALX4 and EXT2. Conclusion: Our findings lend novel insights into potential placental epigenetic mechanisms linked with maternal dyslipidemia. Trial Registration: ClinicalTrials.gov, NCT00912132.

DNA methylation loci in placenta associated with birthweight and expression of genes relevant for early development and adult diseases.

BACKGROUND: Birthweight marks an important milestone of health across the lifespan, including cardiometabolic disease risk in later life. The placenta, a transient organ at the maternal-fetal interface, regulates fetal growth. Identifying genetic loci where DNA methylation in placenta is associated with birthweight can unravel genomic pathways that are dysregulated in aberrant fetal growth and cardiometabolic diseases in later life. RESULTS: We performed placental epigenome-wide association study (EWAS) of birthweight in an ethnic diverse cohort of pregnant women (n = 301). Methylation at 15 cytosine-(phosphate)-guanine sites (CpGs) was associated with birthweight (false discovery rate (FDR) < 0.05). Methylation at four (26.7%) CpG sites was associated with placental transcript levels of 15 genes (FDR < 0.05), including genes known to be associated with adult lipid traits, inflammation and oxidative stress. Increased methylation at cg06155341 was associated with higher birthweight and lower FOSL1 expression, and lower FOSL1 expression was correlated with higher birthweight. Given the role of the FOSL1 transcription factor in regulating developmental processes at the maternal-fetal interface, epigenetic mechanisms at this locus may regulate fetal development. We demonstrated trans-tissue portability of methylation at four genes (MLLT1, PDE9A, ASAP2, and SLC20A2) implicated in birthweight by a previous study in cord blood. We also found that methylation changes known to be related to maternal underweight, preeclampsia and adult type 2 diabetes were associated with lower birthweight in placenta. CONCLUSION: We identified novel placental DNA methylation changes associated with birthweight. Placental epigenetic mechanisms may underlie dysregulated fetal development and early origins of adult cardiometabolic diseases. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT00912132.

Cord blood DNA methylation reflects cord blood C-reactive protein levels but not maternal levels: a longitudinal study and meta-analysis.

BACKGROUND: Prenatal inflammation has been proposed as an important mediating factor in several adverse pregnancy outcomes. C-reactive protein (CRP) is an inflammatory cytokine easily measured in blood. It has clinical value due to its reliability as a biomarker for systemic inflammation and can indicate cellular injury and disease severity. Elevated levels of CRP in adulthood are associated with alterations in DNA methylation. However, no studies have prospectively investigated the relationship between maternal CRP levels and newborn DNA methylation measured by microarray in cord blood with reasonable epigenome-wide coverage. Importantly, the timing of inflammation exposure during pregnancy may also result in different effects. Thus, our objective was to evaluate this prospective association of CRP levels measured during multiple periods of pregnancy and in cord blood at delivery which was available in one cohort (i.e., Effects of Aspirin in Gestation and Reproduction trial), and also to conduct a meta-analysis with available data at one point in pregnancy from three other cohorts from the Pregnancy And Childhood Epigenetics consortium (PACE). Secondarily, the impact of maternal randomization to low dose aspirin prior to pregnancy on methylation was assessed. RESULTS: Maternal CRP levels were not associated with newborn DNA methylation regardless of gestational age of measurement (i.e., CRP at approximately 8, 20, and 36 weeks among 358 newborns in EAGeR). There also was no association in the meta-analyses (all p > 0.5) with a larger sample size (n = 1603) from all participating PACE cohorts with available CRP data from first trimester (< 18 weeks gestation). Randomization to aspirin was not associated with DNA methylation. On the other hand, newborn CRP levels were significantly associated with DNA methylation in the EAGeR trial, with 33 CpGs identified (FDR corrected p < 0.05) when both CRP and methylation were measured at the same time point in cord blood. The top 7 CpGs most strongly associated with CRP resided in inflammation and vascular-related genes. CONCLUSIONS: Maternal CRP levels measured during each trimester were not associated with cord blood DNA methylation. Rather, DNA methylation was associated with CRP levels measured in cord blood, particularly in gene regions predominately associated with angiogenic and inflammatory pathways. TRIAL REGISTRATION: Clinicaltrials.gov, NCT00467363, Registered April 30, 2007, http://www.clinicaltrials.gov/ct2/show/NCT00467363.

Placental DNA methylation changes associated with maternal prepregnancy BMI and gestational weight gain.

BACKGROUND: Maternal obesity prior to or during pregnancy influences fetal growth, predisposing the offspring to increased risk for obesity across the life course. Placental epigenetic mechanisms may underlie these associations. We conducted an epigenome-wide association study to identify placental DNA methylation changes associated with maternal prepregnancy body mass index (BMI) and rate of gestational weight gain at first (GWG1), second (GWG2), and third trimester (GWG3). METHOD: Participants of the NICHD Fetal Growth Studies with genome-wide placental DNA methylation (n = 301) and gene expression (n = 75) data were included. Multivariable-adjusted regression models were used to test the associations of 1 kg/m2 increase in prepregnancy BMI or 1 kg/week increase in GWG with DNA methylation levels. Genes harboring top differentially methylated CpGs (FDR P < 0.05) were evaluated for placental gene expression. We assessed whether DNA methylation sites known to be associated with BMI in child or adult tissues, were also associated with maternal prepregnancy BMI in placenta. RESULTS: Prepregnancy BMI was associated with DNA methylation at cg14568196[EGFL7], cg15339142[VETZ], and cg02301019[AC092377.1] (FDR P < 0.05, P ranging from 1.4 × 10-10 to 1.7 × 10-9). GWG1 or GWG2 was associated with DNA methylation at cg17918270[MYT1L], cg20735365[DLX5], and cg17451688[SLC35F3] (FDR P < 0.05, P ranging from 6.4 × 10-10 to 1.2 × 10-8). Both prepregnancy BMI and DNA methylation at cg1456819 [EGFL7] were negatively correlated with EGFL7 expression in placenta (P < 0.05). Several CpGs previously implicated in obesity traits in children and adults were associated with prepregnancy BMI in placenta. Functional annotations revealed that EGFL7 is highly expressed in placenta and the differentially methylated CpG sites near EGFL7 and VEZT were cis-meQTL targets in blood. CONCLUSIONS: We identified placental DNA methylation changes at novel loci associated with prepregnancy BMI and GWG. The overlap between CpGs associated with obesity traits in placenta and other tissues in children and adults suggests that epigenetic mechanisms in placenta may give insights to early origins of obesity.

Maternal fatty acid concentrations and newborn DNA methylation.

BACKGROUND: Preconception nutrition sets the stage for a healthy pregnancy. Maternal fatty acids (FAs) are related to beneficial neonatal outcomes with DNA methylation proposed as a mechanism; however, few studies have investigated this association and none with preconception FAs. OBJECTIVES: We examined the relations of maternal plasma FA concentrations at preconception (n = 346) and 8 weeks of gestation (n = 374) with newborn DNA methylation. METHODS: The Effects of Aspirin in Gestation and Reproduction Trial (2006-2012) randomly assigned women with previous pregnancy loss to low dose aspirin or placebo prior to conception. We measured maternal plasma phospholipid FA concentration at preconception (on average 4 mo before pregnancy) and 8 weeks of gestation. Cord blood DNA from singletons was measured using the MethylationEPIC BeadChip. We used robust linear regression to test the associations of FA concentration with methylation ß-values of each CpG site, adjusting for estimated cell count using a cord blood reference, sample plate, maternal sociodemographic characteristics, cholesterol, infant sex, and epigenetic-derived ancestry. False discovery rate correction was used for multiple testing. RESULTS: Mean ± SD concentrations of preconception marine (20:5n-3+22:6n-3+22:5n-3) and ω-6 PUFAs, SFAs, MUFAs, and trans FAs were 4.7 ± 1.2, 38.0 ± 2.0, 39.4 ± 1.8, 11.6 ± 1.1, and 1.0 ± 0.4 % of total FA, respectively; concentrations at 8 weeks of gestation were similar. Preconception marine PUFA concentration was associated with higher methylation at GRAMD2 (P = 1.1 × 10-8), LOXL1 (P = 5.5 × 10-8), SIK3 (P = 1.6 × 10-7), HTR1B (P = 1.9 × 10-7), and MCC (P = 2.1 × 10-7) genes. Preconception SFA concentration was associated with higher methylation at KIF25-AS1 and lower methylation at SLC39A14; other associations exhibited sensitivity to outliers. The trans FA concentration was related to lower methylation at 3 sites and higher methylation at 1 site. FAs at 8 weeks of gestation were largely unrelated to DNA methylation. CONCLUSIONS: Maternal preconception FAs are related to newborn DNA methylation of specific CpG sites, highlighting the importance of examining nutritional exposures preconceptionally. This trial was registered at clinicaltrials.gov as NCT00467363.

Adenovirus early region 3 RIDα protein limits NFκB signaling through stress-activated EGF receptors.

The host limits adenovirus infections by mobilizing immune systems directed against infected cells that also represent major barriers to clinical use of adenoviral vectors. Adenovirus early transcription units encode a number of products capable of thwarting antiviral immune responses by co-opting host cell pathways. Although the EGF receptor (EGFR) was a known target for the early region 3 (E3) RIDα protein encoded by nonpathogenic group C adenoviruses, the functional role of this host-pathogen interaction was unknown. Here we report that incoming viral particles triggered a robust, stress-induced pathway of EGFR trafficking and signaling prior to viral gene expression in epithelial target cells. EGFRs activated by stress of adenoviral infection regulated signaling by the NFκB family of transcription factors, which is known to have a critical role in the host innate immune response to infectious adenoviruses and adenovirus vectors. We found that the NFκB p65 subunit was phosphorylated at Thr254, shown previously by other investigators to be associated with enhanced nuclear stability and gene transcription, by a mechanism that was attributable to ligand-independent EGFR tyrosine kinase activity. Our results indicated that the adenoviral RIDα protein terminated this pathway by co-opting the host adaptor protein Alix required for sorting stress-exposed EGFRs in multivesicular endosomes, and promoting endosome-lysosome fusion independent of the small GTPase Rab7, in infected cells. Furthermore RIDα expression was sufficient to down-regulate the same EGFR/NFκB signaling axis in a previously characterized stress-activated EGFR trafficking pathway induced by treatment with the pro-inflammatory cytokine TNF-α. We also found that cell stress activated additional EGFR signaling cascades through the Gab1 adaptor protein that may have unappreciated roles in the adenoviral life cycle. Similar to other E3 proteins, RIDα is not conserved in adenovirus serotypes associated with potentially severe disease, suggesting stress-activated EGFR signaling may contribute to adenovirus virulence.

Administration of a Nucleoside Analog Promotes Cancer Cell Death in a Telomerase-Dependent Manner.

Telomerase, the end-replication enzyme, is reactivated in malignant cancers to drive cellular immortality. While this distinction makes telomerase an attractive target for anti-cancer therapies, most approaches for inhibiting its activity have been clinically ineffective. As opposed to inhibiting telomerase, we use its activity to selectively promote cytotoxicity in cancer cells. We show that several nucleotide analogs, including 5-fluoro-2'-deoxyuridine (5-FdU) triphosphate, are effectively incorporated by telomerase into a telomere DNA product. Administration of 5-FdU results in an increased number of telomere-induced foci, impedes binding of telomere proteins, activates the ATR-related DNA-damage response, and promotes cell death in a telomerase-dependent manner. Collectively, our data indicate that telomerase activity can be exploited as a putative anti-cancer strategy.

Host cell autophagy modulates early stages of adenovirus infections in airway epithelial cells.

Human adenoviruses typically cause mild infections in the upper or lower respiratory tract, gastrointestinal tract, or ocular epithelium. However, adenoviruses may be life-threatening in patients with impaired immunity and some serotypes cause epidemic outbreaks. Attachment to host cell receptors activates cell signaling and virus uptake by endocytosis. At present, it is unclear how vital cellular homeostatic mechanisms affect these early steps in the adenovirus life cycle. Autophagy is a lysosomal degradation pathway for recycling intracellular components that is upregulated during periods of cell stress. Autophagic cargo is sequestered in double-membrane structures called autophagosomes that fuse with endosomes to form amphisomes which then deliver their content to lysosomes. Autophagy is an important adaptive response in airway epithelial cells targeted by many common adenovirus serotypes. Using two established tissue culture models, we demonstrate here that adaptive autophagy enhances expression of the early region 1 adenovirus protein, induction of mitogen-activated protein kinase signaling, and production of new viral progeny in airway epithelial cells infected with adenovirus type 2. We have also discovered that adenovirus infections are tightly regulated by endosome maturation, a process characterized by abrupt exchange of Rab5 and Rab7 GTPases, associated with early and late endosomes, respectively. Moreover, endosome maturation appears to control a pool of early endosomes capable of fusing with autophagosomes which enhance adenovirus infection. Many viruses have evolved mechanisms to induce autophagy in order to aid their own replication. Our studies reveal a novel role for host cell autophagy that could have a significant impact on the outcome of respiratory infections.

Impact of Autophagy on Chemotherapy and Radiotherapy Mediated Tumor Cytotoxicity: "To Live or not to Live".

Autophagy, a highly regulated cell "self-eating" pathway, is controlled by the action of over 34 autophagy-related proteins (collectively termed Atgs). Although they are fundamentally different processes, autophagy and apoptosis (type I programmed cell death), under certain circumstances, can be regulated by common signaling mediators. Current cancer therapies including chemotherapy and ionizing radiation are known to induce autophagy within tumor cells. However, autophagy plays a dual role of either pro-cell survival or pro-cell death in response to these cancer treatments, depending on the cellular context and the nature of the treatment. We review the current basic and translational cancer research literature on how autophagy impacts tumor cell survival ("to live") and death ("not to live") following treatment as well as the role of chemical mediators of autophagy.

BNIP3 is essential for mediating 6-thioguanine- and 5-fluorouracil-induced autophagy following DNA mismatch repair processing.

DNA mismatch repair (MMR) processes the chemically induced mispairs following treatment with clinically important nucleoside analogs such as 6-thioguanine (6-TG) and 5-fluorouracil (5-FU). MMR processing of these drugs has been implicated in activation of a prolonged G2/M cell cycle arrest for repair and later induction of apoptosis and/or autophagy for irreparable DNA damage. In this study, we investigated the role of Bcl2 and adenovirus E1B Nineteen-kilodalton Interacting Protein (BNIP3) in the activation of autophagy, and the temporal relationship between a G2/M cell cycle arrest and the activation of BNIP3-mediated autophagy following MMR processing of 6-TG and 5-FU. We found that BNIP3 protein levels are upregulated in a MLH1 (MMR(+))-dependent manner following 6-TG and 5-FU treatment. Subsequent small-interfering RNA (siRNA)-mediated BNIP3 knockdown abrogates 6-TG-induced autophagy. We also found that p53 knockdown or inhibition of mTOR activity by rapamycin cotreatment impairs 6-TG- and 5-FU-induced upregulation of BNIP3 protein levels and autophagy. Furthermore, suppression of Checkpoint kinase 1 (Chk1) expression with a subsequent reduction in 6-TG-induced G2/M cell cycle arrest by Chk1 siRNA promotes the extent of 6-TG-induced autophagy. These findings suggest that BNIP3 mediates 6-TG- and 5-FU-induced autophagy in a p53- and mTOR-dependent manner. Additionally, the duration of Chk1-activated G2/M cell cycle arrest determines the level of autophagy following MMR processing of these nucleoside analogs.

Mammalian target of rapamycin and S6 kinase 1 positively regulate 6-thioguanine-induced autophagy.

DNA mismatch repair (MMR) ensures the fidelity of DNA replication and is required for activation of cell cycle arrest and apoptosis in response to certain classes of DNA damage. We recently reported that MMR is also implicated in initiation of an autophagic response after MMR processing of 6-thioguanine (6-TG). It is now generally believed that autophagy is negatively controlled by mammalian target of rapamycin (mTOR) activity. To determine whether mTOR is involved in 6-TG-induced autophagy, we used rapamycin, a potential anticancer agent, to inhibit mTOR activity. Surprisingly, we find that rapamycin cotreatment inhibits 6-TG-induced autophagy in MMR-proficient human colorectal cancer HCT116 (MLH1(+)) and HT29 cells as measured by LC3 immunoblotting, GFP-LC3 relocalization, and acridine orange staining. Consistently, short interfering RNA silencing of the 70-kDa ribosomal S6 kinase 1 (S6K1), the downstream effector of mTOR, markedly reduces 6-TG-induced autophagy. Furthermore, we show that inhibition of mTOR by rapamycin induces the activation of Akt as shown by increased Akt phosphorylation at Ser(473) and the inhibition of 6-TG-induced apoptosis and cell death. Activated Akt is a well-known inhibitor of autophagy. In conclusion, our data indicate that mTOR-S6K1 positively regulates autophagy after MMR processing of 6-TG probably through its negative feedback inhibition of Akt.

A novel role for DNA mismatch repair and the autophagic processing of chemotherapy drugs in human tumor cells.

DNA Mismatch repair (MMR) maintains genome integrity by correcting DNA replication errors and blocking homologous recombination between divergent DNA sequences. The MMR system also activates both checkpoint and apoptotic responses following certain types of DNA damage. In a recent study, we describe a novel role for MMR in mediating an autophagic response to 6?thioguanine (6-TG), a DNA modifying chemical. Our results show that MMR proteins (MLH1 or MSH2) are required for signaling to the autophagic pathway after exposure to 6-TG. Using PFT-alpha, a p53 inhibitor, and shRNA-mediated silencing of p53 expression, we also show that p53 plays an essential role in the autophagic pathway downstream of the MMR system. This study suggests a novel function of MMR in mediating autophagy following chemical (6-TG) DNA mismatch damage through p53 activation. Here, we present the model and the clinical implications of the role of MMR in autophagy.