CD4(+) T cells epigenetically modified by oxidative stress cause lupus-like autoimmunity in mice.

Lupus develops when genetically predisposed people encounter environmental agents such as UV light, silica, infections and cigarette smoke that cause oxidative stress, but how oxidative damage modifies the immune system to cause lupus flares is unknown. We previously showed that oxidizing agents decreased ERK pathway signaling in human T cells, decreased DNA methyltransferase 1 and caused demethylation and overexpression of genes similar to those from patients with active lupus. The current study tested whether oxidant-treated T cells can induce lupus in mice. We adoptively transferred CD4(+) T cells treated in vitro with oxidants hydrogen peroxide or nitric oxide or the demethylating agent 5-azacytidine into syngeneic mice and studied the development and severity of lupus in the recipients. Disease severity was assessed by measuring anti-dsDNA antibodies, proteinuria, hematuria and by histopathology of kidney tissues. The effect of the oxidants on expression of CD40L, CD70, KirL1 and DNMT1 genes and CD40L protein in the treated CD4(+) T cells was assessed by Q-RT-PCR and flow cytometry. H2O2 and ONOO(-) decreased Dnmt1 expression in CD4(+) T cells and caused the upregulation of genes known to be suppressed by DNA methylation in patients with lupus and animal models of SLE. Adoptive transfer of oxidant-treated CD4(+) T cells into syngeneic recipients resulted in the induction of anti-dsDNA antibody and glomerulonephritis. The results show that oxidative stress may contribute to lupus disease by inhibiting ERK pathway signaling in T cells leading to DNA demethylation, upregulation of immune genes and autoreactivity.

Association of the interferon signature metric with serological disease manifestations but not global activity scores in multiple cohorts of patients with SLE.

OBJECTIVES: The interferon (IFN) signature (IS) in patients with systemic lupus erythematosus (SLE) includes over 100 genes induced by type I IFN pathway activation. We developed a method to quantify the IS using three genes-the IS metric (ISM)-and characterised the clinical characteristics of patients with SLE with different ISM status from multiple clinical trials. METHODS: Blood microarray expression data from a training cohort of patients with SLE confirmed the presence of the IS and identified surrogate genes. We assayed these genes in a quantitative PCR (qPCR) assay, yielding an ISM from the IS. The association of ISM status with clinical disease characteristics was assessed in patients with extrarenal lupus and lupus nephritis from four clinical trials. RESULTS: Three genes, HERC5, EPSTI and CMPK2, correlated well with the IS (p>0.96), and composed the ISM qPCR assay. Using the 95th centile for healthy control data, patients with SLE from different studies were classified into two ISM subsets-ISM-Low and ISM-High-that are longitudinally stable over 36 weeks. Significant associations were identified between ISM-High status and higher titres of anti-dsDNA antibodies, presence of anti extractable nuclear antigen autoantibodies, elevated serum B cell activating factor of the tumour necrosis factor family (BAFF) levels, and hypocomplementaemia. However, measures of overall clinical disease activity were similar for ISM-High and ISM-Low groups. CONCLUSIONS: The ISM is an IS biomarker that divides patients with SLE into two subpopulations-ISM-High and ISM-Low-with differing serological manifestations. The ISM does not distinguish between high and low disease activity, but may have utility in identifying patients more likely to respond to treatment(s) targeting IFN-α. CLINICALTRIALSGOV REGISTRATION NUMBER: NCT00962832.

Oxidative stress, T cell DNA methylation, and lupus.

OBJECTIVE: Lupus develops when genetically predisposed people encounter environmental agents, such as ultraviolet light, silica, infections, and cigarette smoke, that cause oxidative stress, but how oxidative damage modifies the immune system to cause lupus flares is unknown. We previously showed that inhibiting DNA methylation in CD4+ T cells by blocking ERK pathway signaling is sufficient to alter gene expression, and that the modified cells cause lupus-like autoimmunity in mice. We also reported that T cells from patients with active lupus have decreased ERK pathway signaling, have decreased DNA methylation, and overexpress genes normally suppressed by DNA methylation. This study was undertaken to test whether oxidizing agents decrease ERK pathway signaling in T cells, decrease DNA methyltransferase levels, and cause demethylation and overexpression of T cell genes similar to that found in T cells from patients with active lupus. METHODS: CD4+ T cells were treated with the oxidizers H2 O2 or ONOO(-) . Effects on ERK pathway signaling were measured by immunoblotting, DNA methyltransferase 1 (DNMT-1) levels were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the methylation and expression of T cell genes were measured using flow cytometry, RT-PCR, and bisulfite sequencing. RESULTS: H2 O2 and ONOO(-) inhibited ERK pathway signaling in T cells by inhibiting the upstream regulator protein kinase Cδ, decreased DNMT-1 levels, and caused demethylation and overexpression of genes previously shown to be suppressed by DNA methylation in T cells from patients with active lupus. CONCLUSION: Our findings indicate that oxidative stress may contribute to human lupus flares by inhibiting ERK pathway signaling in T cells to decrease DNMT-1 and cause DNA demethylation.

Diet influences expression of autoimmune-associated genes and disease severity by epigenetic mechanisms in a transgenic mouse model of lupus.

OBJECTIVE: Lupus flares occur when genetically predisposed individuals encounter appropriate environmental agents. Current evidence indicates that the environment contributes by inhibiting T cell DNA methylation, causing overexpression of normally silenced genes. DNA methylation depends on both dietary transmethylation micronutrients and ERK-regulated DNA methyltransferase 1 (DNMT-1) levels. We used transgenic mice to study the effect of interactions between diet, DNMT-1 levels, and genetic predisposition on the development and severity of lupus. METHODS: A doxycycline-inducible ERK defect was bred into lupus-resistant (C57BL/6) and lupus-susceptible (C57BL/6 × SJL) mouse strains. Doxycycline-treated mice were fed a standard commercial diet for 18 weeks and then switched to a transmethylation micronutrient-supplemented (MS) or -restricted (MR) diet. Disease severity was assessed by examining anti-double-stranded DNA (anti-dsDNA) antibody levels, the presence of proteinuria and hematuria, and by histopathologic analysis of kidney tissues. Pyrosequencing was used to determine micronutrient effects on DNA methylation. RESULTS: Doxycycline induced modest levels of anti-dsDNA antibodies in C57BL/6 mice and higher levels in C57BL/6 × SJL mice. Doxycycline-treated C57BL/6 × SJL mice developed hematuria and glomerulonephritis on the MR and standard diets but not the MS diet. In contrast, C57BL/6 mice developed kidney disease only on the MR diet. Decreasing ERK signaling and methyl donors also caused demethylation and overexpression of the CD40lg gene in female mice, consistent with demethylation of the second X chromosome. Both the dietary methyl donor content and the duration of treatment influenced methylation and expression of the CD40lg gene. CONCLUSION: Dietary micronutrients that affect DNA methylation can exacerbate or ameliorate disease in this transgenic murine lupus model, and contribute to lupus susceptibility and severity through genetic-epigenetic interactions.

Prostaglandin E2 increases fibroblast gene-specific and global DNA methylation via increased DNA methyltransferase expression.

Although alterations in DNA methylation patterns have been associated with specific diseases and environmental exposures, the mediators and signaling pathways that direct these changes remain understudied. The bioactive lipid mediator prostaglandin E(2) (PGE(2)) has been shown to exert a myriad of effects on cell survival, proliferation, and differentiation. Here, we report that PGE(2) also signals to increase global DNA methylation and DNA methylation machinery in fibroblasts. HumanMethylation27 BeadChip array analysis of primary fetal (IMR-90) and adult lung fibroblasts identified multiple genes that were hypermethylated in response to PGE(2). PGE(2), compared with nontreated controls, increased expression and activity (EC(50)∼10(7) M) of one specific isoform of DNA methyltransferase, DNMT3a. Silencing of DNMT3a negated the ability of PGE(2) to increase DNMT activity. The increase in DNMT3a expression was mediated by PGE(2) signaling via its E prostanoid 2 receptor and the second messenger cAMP. PGE(2), compared with the untreated control, increased the expression and activity of Sp1 and Sp3 (EC(50)∼3×10(7) M), transcription factors known to increase DNMT3a expression, and inhibition of these transcription factors abrogated the PGE(2) increase of DNMT3a expression. These findings were specific to fibroblasts, as PGE(2) decreased DNMT1 and DNMT3a expression in RAW macrophages. Taken together, these findings establish that DNA methylation is regulated by a ubiquitous bioactive endogenous mediator. Given that PGE(2) biosynthesis is modulated by environmental toxins, various disease states, and commonly used pharmacological agents, these findings uncover a novel mechanism by which alterations in DNA methylation patterns may arise in association with disease and certain environmental exposures.

Environmental exposure, estrogen and two X chromosomes are required for disease development in an epigenetic model of lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease primarily afflicting women. The reason for the gender bias is unclear, but genetic susceptibility, estrogen and environmental agents appear to play significant roles in SLE pathogenesis. Environmental agents can contribute to lupus susceptibility through epigenetic mechanisms. We used (C57BL/6xSJL)F1 mice transgenic for a dominant-negative MEK (dnMEK) that was previously shown to be inducibly and selectively expressed in T cells. In this model, induction of the dnMEK by doxycycline treatment suppresses T cell ERK signaling, decreasing DNA-methyltransferase expression and resulting in DNA demethylation, overexpression of immune genes Itgal (CD11a) and Tnfsf7 (CD70), and anti-dsDNA antibody. To examine the role of gender and estrogen in this model, male and female transgenic mice were neutered and implanted with time-release pellets delivering placebo or estrogen. Doxycycline induced IgG anti-dsDNA antibodies in intact and neutered, placebo-treated control female but not male transgenic mice. Glomerular IgG deposits were also found in the kidneys of female but not male transgenic mice, and not in the absence of doxycycline. Estrogen enhanced anti-dsDNA IgG antibodies only in transgenic, ERK-impaired female mice. Decreased ERK activation also resulted in overexpression and demethylation of the X-linked methylation-sensitive gene CD40lg in female but not male mice, consistent with demethylation of the second X chromosome in the females. The results show that both estrogen and female gender contribute to the female predisposition in lupus susceptibility through hormonal and epigenetic X-chromosome effects and through suppression of ERK signaling by environmental agents.

Hypermethylation of PTGER2 confers prostaglandin E2 resistance in fibrotic fibroblasts from humans and mice.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease that is characterized by excessive proliferation of fibroblasts. The lipid mediator prostaglandin E2 (PGE2) has the capacity to limit fibrosis through its inhibition of numerous functions of these fibroblasts; however, in the setting of fibrosis, fibroblasts have been shown to be resistant to PGE2. We have linked such resistance to decreased expression levels of the E prostanoid 2 (EP2) receptor. In this study, in fibroblasts from both mice and humans with pulmonary fibrosis, we show that DNA hypermethylation is responsible for diminished EP2 expression levels and PGE2 resistance. Bisulfite sequencing of the prostaglandin E receptor 2 gene (PTGER2) promoter revealed that fibrotic fibroblasts exhibit greater PTGER2 methylation than nonfibrotic control cells. Treatment with the DNA methylation inhibitors 5-aza-2'-deoxycytidine and zebularine as well as DNA methyltransferase-specific siRNA decreased PTGER2 methylation, increased EP2 mRNA and protein expression levels, and restored PGE2 responsiveness in fibrotic fibroblasts but not in nonfibrotic controls. PTGER2 promoter hypermethylation was driven by an increase in Akt signal transduction. In addition to results described for the PTGER2 promoter, fibrotic fibroblasts also exhibited increased global DNA methylation. These findings demonstrate that the down-regulation of PTGER2 and consequent PGE2 resistance are both mediated by DNA hypermethylation; we identified increased Akt signal transduction as a novel mechanism that promotes DNA hypermethylation during fibrogenesis.

Decreased ERK and JNK signaling contribute to gene overexpression in "senescent" CD4+CD28- T cells through epigenetic mechanisms.

An inflammatory and cytotoxic CD4+CD28- T cell subset infiltrates atherosclerotic plaques and is implicated in plaque rupture and myocardial infarctions. This pathologic subset develops with replicative stress and is found in patients with chronic inflammatory diseases such as RA as well as with aging. CD4+CD28- cells overexpress genes normally suppressed by DNA methylation in CD4+CD28+ T cells, such as KIR, perforin, and CD70. How this subset over expresses methylation-sensitive genes is unknown. DNA methylation patterns are maintained in proliferating cells by Dnmts, which are up-regulated during mitosis by the ERK and JNK signaling pathways. We hypothesized that defects in these signaling pathways contribute to altered gene expression in human CD4+CD28- cells through effects on DNA methylation. We report that signaling through the ERK and JNK pathways is decreased in CD4+CD28- relative to CD4+CD28+ cells from the same individuals and that ERK and JNK pathway inhibition decreases Dnmt1 and -3a levels, which in turn, causes demethylation and overexpression of the TNFSF7 (CD70) gene. We also report that CD4+CD28- T cells overexpress PP5, a stress-induced inhibitor of the ERK and JNK signaling pathways that may contribute to the signaling defects. We conclude that decreased ERK and JNK signaling in the CD4+CD28- subset, arising with replicative stress, can lead to the overexpression of normally suppressed genes through effects on Dnmts and consequently, chromatin structure.

Stimulatory and inhibitory killer Ig-like receptor molecules are expressed and functional on lupus T cells.

T cells from lupus patients have hypomethylated DNA and overexpress genes normally suppressed by DNA methylation that contribute to disease pathogenesis. We found that stimulatory and inhibitory killer cell Ig-like receptor (KIR) genes are aberrantly overexpressed on experimentally demethylated T cells. We therefore asked if lupus T cells also overexpress KIR, and if the proteins are functional. T cells from lupus patients were found to overexpress KIR genes, and expression was proportional to disease activity. Abs to the stimulatory molecule KIR2DL4 triggered IFN-gamma release by lupus T cells, and production was proportional to disease activity. Similarly, cross-linking the inhibitory molecule KIR3DL1 prevented the autoreactive macrophage killing that characterizes lupus T cells. These results indicate that aberrant T cell KIR expression may contribute to IFN overproduction and macrophage killing in human lupus, and they suggest that Abs to inhibitory KIR may be a treatment for this disease.

Accelerated macrophage apoptosis induces autoantibody formation and organ damage in systemic lupus erythematosus.

Increased monocyte/macrophage (Mphi) apoptosis occurs in patients with systemic lupus erythematosus (SLE) and is mediated, at least in part, by an autoreactive CD4(+) T cell subset. Furthermore, autoreactive murine CD4(+) T cells that kill syngeneic Mphi in vitro induce a lupus-like disease in vivo. However, it is unclear whether increased Mphi apoptosis in SLE per se is sufficient to accelerate/promote autoimmunity. We have investigated whether increased Mphi apoptosis in vivo, induced by the administration of clodronate liposomes, can exacerbate the autoimmune phenotype in NZB x SWR (SNF(1)) lupus-prone mice, and induce autoantibody production in haplotype-matched BALB/c x DBA1 (DBF(1)) non-lupus-prone mice. Lupus-prone mice SNF(1) mice that were treated with clodronate liposomes, but not mice treated with vehicle, developed significant increases in autoantibodies to dsDNA, nucleosomes, and the idiotypically related family of nephritic Abs Id(LN)F(1), when compared with untreated SNF(1) mice. Furthermore, clodronate treatment hastened the onset of proteinuria and worsened SNF(1) lupus nephritis. When compared with vehicle-treated controls, clodronate-treated non-lupus-prone DBF(1) mice developed significantly higher levels of anti-nucleosome and Id(LN)F(1) Abs but did not develop lupus nephritis. We propose that Mphi apoptosis contributes to the pathogenesis of autoantibody formation and organ damage through both an increase in the apoptotic load and impairment in the clearance of apoptotic material. This study suggests that mechanisms that induce scavenger cell apoptosis, such as death induced by autoreactive cytotoxic T cells observed in SLE, could play a pathogenic role and contribute to the severity of the disease.

Demethylation of the same promoter sequence increases CD70 expression in lupus T cells and T cells treated with lupus-inducing drugs.

Exposing genetically predisposed individuals to certain environmental agents is believed to cause human lupus. How environmental agents interact with the host to cause lupus is poorly understood. Procainamide and hydralazine are drugs that cause lupus in genetically predisposed individuals. Understanding how these environmental agents cause lupus may indicate mechanisms relevant to the idiopathic disease. Abnormal T cell DNA methylation, a repressive epigenetic DNA modification, is implicated in procainamide and hydralazine induced lupus, as well as idiopathic lupus. Procainamide is a competitive DNA methyltransferase (Dnmt) inhibitor, hydralazine inhibits ERK pathway signaling thereby decreasing Dnmt expression, and in lupus T cells decreased ERK pathway signaling causing a similar Dnmt decrease. T cells treated with procainamide, hydralazine, and other Dnmt and ERK pathway inhibitors cause lupus in mice. Whether the same genetic regulatory elements demethylate in T cells treated with Dnmt inhibitors, ERK pathway inhibitors, and in human lupus is unknown. CD70 (TNFSF7) is a B cell costimulatory molecule overexpressed on CD4(+) lupus T cells as well as procainamide and hydralazine treated T cells, and contributes to excessive B cell stimulation in vitro and in lupus. In this report we identify a genetic element that suppresses CD70 expression when methylated, and which demethylates in lupus and in T cells treated with Dnmt and ERK pathway inhibitors including procainamide and hydralazine. The results support a model in which demethylation of specific genetic elements in T cells, caused by decreasing Dnmt expression or inhibiting its function, contributes to drug-induced and idiopathic lupus through altered gene expression.

The MAPK signalling pathways and colorectal cancer.

There are three major subfamilies of mitogen-activated protein kinases (MAPK): the extracellular-signal-regulated kinases (ERK MAPK); the c-jun N-terminal kinase or stress-activated protein kinases (JNK or SAPK); and MAPK14. The ERK MAPK pathway is one of the most important for cell proliferation. The MAPK pathways are located downstream of many growth-factor receptors, including that for epidermal growth factor. Overexpression and activation of this receptor are commonly detected in colorectal cancer, and several lines of evidence indicate that overexpression and activation of ERK MAPK play an important part in progression of this cancer. ERK MAPK could be a molecular target for treatment of the disorder. This review focuses on the ERK MAPK signal-transduction pathway, the consequences of its dysregulation in colorectal cancer, and its potential as an approach to cancer treatment. Future challenges for the assessment of these targeted agents in the clinic are also presented.

Estrogen regulates CCR gene expression and function in T lymphocytes.

Estrogen has been implicated in the observed female bias in autoimmune diseases. However, the mechanisms behind this gender dimorphism are poorly defined. We have previously reported that in vivo T cell trafficking is gender- and estrogen-dependent. Chemokine receptors are critical determinants of T cell homing and immune response. In this study, we show that the female gender is associated with increased CD4(+) T cell CCR1-CCR5 gene and protein expression in mice. The increased CCR expression correlates with enhanced in vitro chemotaxis response to MIP-1beta (CCL4). In vivo treatment of young oophorectomized and postmenopausal female mice with 17beta-estradiol also increased CD4(+) T cell CCR expression. Finally, 17beta-estradiol enhances tyrosine phosphorylation in T cells stimulated with MIP-1alpha in a time-dependent manner. Our results indicate an important role of estrogen in determining T cell chemokine response that may help explain the increased susceptibility and severity of autoimmune diseases in females.

Tissue-specific effect of estradiol on endothelial cell-dependent lymphocyte recruitment.

Estrogen profoundly affects onset and severity of many immune-mediated diseases. In our murine model of drug-induced autoimmunity, female-specific, estrogen-dependent increase in splenic lymphocyte homing was directly implicated in increased disease severity. The present study evaluated the effect of estradiol on microvascular endothelial cells from the spleen compared to endothelial cells from the dermis, which has no disease manifestation in this model. Estradiol increased spleen endothelial cell estrogen receptor (ER) alpha 2.9-fold and decreased estrogen receptor beta 2.1-fold while decreasing both receptors on dermal cells. Estradiol enhanced adhesion of D10 cells to spleen but not dermal endothelial cells 1.53-fold (P < 0.001), an increase that was inhibited by antibodies to VCAM-1 and ICAM-1, and by the estrogen receptor antagonists tamoxifen and ICI 182,780. Estradiol induced greater VCAM-1 expression on spleen than dermal endothelial cells (P < 0.05). Estradiol increased spleen endothelial cell estrogen receptor alpha 2.9-fold and decreased estrogen receptor beta 2.1-fold while decreasing both receptors on the dermal cells. Estrogen specifically and preferentially promoted spleen chemokine protein expression for MCP-1 and MCP-3, while having no effect on dermal protein expression for these chemokines. Estradiol-mediated effects on splenic chemokines were abrogated by tamoxifen and ICI 182,780. The gender-specific increase in lymphocyte homing to spleen may be attributable, at least in part, to tissue-specific estrogen-mediated effects on microvascular endothelial cells.

Attenuation of autoimmune disease in Fas-deficient mice by treatment with a cytotoxic benzodiazepine.

OBJECTIVE: Elimination of autoreactive cells relies on Fas-dependent activation-induced cell death mechanisms, an important component of peripheral tolerance. Defects in Fas or its cognate ligand lead to inefficient activation-induced cell death and are specific causes of lymphoproliferative and autoimmune diseases. The present study was undertaken to investigate a novel 1,4-benzodiazepine (Bz-423) that induces apoptosis and limits autoimmune disease in NZB/NZW mice, to determine its activity against lupus-like disease associated with defective Fas expression. We investigated the Fas-dependence of its cytotoxic actions, its therapeutic potential in mice deficient in Fas, and its therapeutic mechanism of action. METHODS: Primary lymphocytes isolated from Fas-deficient MRL/MpJ-Fas(lpr) (MRL-lpr) mice were tested for sensitivity to Bz-423. Bz-423 was administered to MRL-lpr mice for short (1-week) or long (14-week) periods, and its effects on cell survival were determined along with measures of nephritis, arthritis, antibody titers, and Th subpopulations. BALB/c mice were similarly treated to determine if Bz-423 alters normal immune functions in vivo. RESULTS: Administration of Bz-423 to MRL-lpr mice significantly reduced autoimmune disease including glomerulonephritis and arthritis. Treatment was associated with decreases in CD4+ T cells and an alteration in the Th1/Th2 balance. At the therapeutic dosage, Bz-423 did not interfere with normal T and B cell responses in BALB/c mice, suggesting that this agent is not globally immunosuppressive. CONCLUSION: Bz-423 is a novel immunomodulatory agent that is active against disease even in the context of defective Fas signaling. It is a leading compound for further investigation into the development of selective therapies for lupus.

The apoptotic ligands TRAIL, TWEAK, and Fas ligand mediate monocyte death induced by autologous lupus T cells.

Individuals with systemic lupus erythematosus show evidence of a significant increase in monocyte apoptosis. This process is mediated, at least in part, by an autoreactive T cell subset that kills autologous monocytes in the absence of nominal Ag. We have investigated the apoptotic pathways involved in this T cell-mediated process. Expression of the apoptotic ligands TRAIL, TNF-like weak inducer of apoptosis (TWEAK), and Fas ligand on lupus T cells was determined, and the role of these molecules in the monocyte apoptotic response was examined. We report that these apoptotic ligands mediate the autologous monocyte death induced by lupus T cells and that this cytotoxicity is associated with increased expression of these molecules on activated T cells, rather than with an increased susceptibility of lupus monocytes to apoptosis induced by these ligands. These results define novel mechanisms that contribute to increased monocyte apoptosis characterizing patients with lupus. We propose that this mechanism could provide a source of potentially antigenic material for the autoimmune response and interfere with normal clearing mechanisms.

Role of DNA methylation in the regulation of cell function: autoimmunity, aging and cancer.

DNA methylation plays an essential role in maintaining cellular function, and changes in methylation patterns may contribute to the development of autoimmunity, aging and cancer. Evidence for a role in autoimmunity comes from studies demonstrating that inhibiting T lymphocyte DNA methylation causes autoreactivity in vitro and a lupus-like disease in vivo. The autoimmunity is due in part to the heterodimeric beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) overexpression, and T lymphocytes from lupus patients hypomethylate the same CD11a promoter sequences, overexpress LFA-1 and demonstrate the same autoreactivity. Procainamide and hydralazine, two drugs that cause a lupus-like disease, also inhibit T cell DNA methylation, increase LFA-1 expression and induce autoreactivity in vitro and autoimmunity in vivo, supporting the association of DNA hypomethylation and autoimmunity. Methylation patterns also change with age in T lymphocytes as well as other tissues, typically with an overall decrease in methylcytosine content, but with increases in some cytosine guanine dinucleotide (CpG) islands. Age-dependent hypomethylation contributes to LFA-1 overexpression with aging, which may play a role in the development of autoimmunity in the elderly and age-dependent methylation of CpG islands in the promoters of tumor suppressor genes is an early event in the development of some cancers. DNA hypomethylation also may contribute to carcinogenesis by promoting overexpression of proto-oncogenes, chromosomal translocations and loss of imprinting. The mechanisms causing altered DNA methylation in autoimmunity, aging and carcinogenesis are incompletely characterized but include exposure to environmental agents and drugs, diet, altered signaling in pathways regulating DNA methyltransferase expression and changes in endogenous regulatory mechanisms. Other mechanisms are likely to be identified as well.