Frederick E. Domann

Role for DNA methylation in the control of cell type−specific maspin expression

The nucleotide 5-methylcytosine is involved in processes crucial in mammalian development, such as X-chromosome inactivation and gene imprinting. In addition, cytosine methylation has long been speculated to be involved in the establishment and maintenance of cell type–specific expression of developmentally regulated genes; however, it has been difficult to identify clear examples of such genes, particularly in humans. Here we provide evidence that cytosine methylation of the maspin gene (SERPINB5) promoter controls, in part, normal cell type–specific SERPINB5 expression. In normal cells expressing SERPINB5, the SERPINB5 promoter is unmethylated and the promoter region has acetylated histones and an accessible chromatin structure. By contrast, normal cells that do not express SERPINB5 have a completely methylated SERPINB5 promoter with hypoacetylated histones, an inaccessible chromatin structure and a transcriptional repression that is relieved by inhibition of DNA methylation. These findings indicate that cytosine methylation is important in the establishment and maintenance of cell type–restricted gene expression.

Disruption of an AP-2α binding site in an IRF6 enhancer is associated with cleft lip

Previously we have shown that nonsyndromic cleft lip with or without cleft palate (NSCL/P) is strongly associated with SNPs in IRF6 (interferon regulatory factor 6). Here, we use multispecies sequence comparisons to identify a common SNP (rs642961, G>A) in a newly identified IRF6 enhancer. The A allele is significantly overtransmitted (P = 1 × 10−11) in families with NSCL/P, in particular those with cleft lip but not cleft palate. Further, there is a dosage effect of the A allele, with a relative risk for cleft lip of 1.68 for the AG genotype and 2.40 for the AA genotype. EMSA and ChIP assays demonstrate that the risk allele disrupts the binding site of transcription factor AP-2α and expression analysis in the mouse localizes the enhancer activity to craniofacial and limb structures. Our findings place IRF6 and AP-2α in the same developmental pathway and identify a high-frequency variant in a regulatory element contributing substantially to a common, complex disorder.

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here, we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Epigenetic silencing of maspin gene expression in human breast cancers

Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re‐arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non‐methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin‐negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin‐negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue‐restricted gene expression. Chromatin accessibility studies with MCF‐7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re‐activated in MCF‐7 cells by treatment with 5‐aza‐2`‐deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer. Int. J. Cancer 85:805–810, 2000.

The Redox Basis of Epigenetic Modifications: From Mechanisms to Functional Consequences

Epigenetic modifications represent mechanisms by which cells may effectively translate multiple signaling inputs into phenotypic outputs. Recent research is revealing that redox metabolism is an increasingly important determinant of epigenetic control that may have significant ramifications in both human health and disease. Numerous characterized epigenetic marks, including histone methylation, acetylation, and ADP-ribosylation, as well as DNA methylation, have direct linkages to central metabolism through critical redox intermediates such as NAD(+), S-adenosyl methionine, and 2-oxoglutarate. Fluctuations in these intermediates caused by both normal and pathologic stimuli may thus have direct effects on epigenetic signaling that lead to measurable changes in gene expression. In this comprehensive review, we present surveys of both metabolism-sensitive epigenetic enzymes and the metabolic processes that may play a role in their regulation. To close, we provide a series of clinically relevant illustrations of the communication between metabolism and epigenetics in the pathogenesis of cardiovascular disease, Alzheimer disease, cancer, and environmental toxicity. We anticipate that the regulatory mechanisms described herein will play an increasingly large role in our understanding of human health and disease as epigenetics research progresses.

Expression of p53 enhances selenite-induced superoxide production and apoptosis in human prostate cancer cells.

Although the anticancer effects of selenium have been shown in clinical, preclinical, and laboratory studies, the underlying mechanism(s) remains unclear. Our previous study showed that sodium selenite induced LNCaP human prostate cancer cell apoptosis in association with production of reactive oxygen species, alteration of cell redox state, and mitochondrial damage. In the present study, we showed that selenite-induced apoptosis was superoxide mediated and p53 dependent via mitochondrial pathways. In addition, we also showed that superoxide production by selenite was p53 dependent. Our study showed that wild-type p53-expressing LNCaP cells were more sensitive to selenite-induced apoptosis than p53-null PC3 cells. Selenite treatment resulted in high levels of superoxide production in LNCaP cells but only low levels in PC3 cells. LNCaP cells also showed sequential increases in levels of phosphorylated p53 (serine 15), total p53, Bax, and p21(Waf1) proteins following selenite treatment. The effects of selenite were suppressed by pretreatment with a synthetic superoxide dismutase mimic or by knockdown of p53 via RNA interference. LNCaP cells treated with selenite also showed p53 translocation to mitochondria, cytochrome c release into the cytosol, and activation of caspase-9. On the other hand, restoration of wild-type p53 expression in PC3 cells increased cellular sensitivity to selenite and resulted in increased superoxide production, caspase-9 activation, and apoptosis following selenite treatment. These results suggest that selenite induces apoptosis by producing superoxide to activate p53 and to induce p53 mitochondrial translocation. Activation of p53 in turn synergistically enhances superoxide production and apoptosis induced by selenite.

Manganese superoxide dismutase suppresses hypoxic induction of hypoxia-inducible factor-1|[alpha]| and vascular endothelial growth factor

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that governs cellular responses to reduced O2 availability by mediating crucial homeostatic processes. HIF-1 is composed of an HIF-1α subunit and an HIF-1β subunit. HIF-1α is degraded following enzyme-dependent hydroxylation of prolines of HIF-1α in the presence of molecular oxygen, Fe2+, α-ketoglutarate, and ascorbate. These cofactors contribute to the redox environment of cells. The antioxidant enzyme manganese superoxide dismutase (MnSOD) also modulates the cellular redox environment. Here we show that MnSOD suppressed hypoxic accumulation of HIF-1α protein in human breast carcinoma MCF-7 cells. This suppression was biphasic depending on MnSOD activity. At low levels of MnSOD activity, HIF-1α protein accumulated under hypoxic conditions. At moderate levels of MnSOD activity (two- to six-fold increase compared to parent cells), these accumulations were blocked. However, at higher levels of MnSOD activity (>6-fold increase), accumulation of HIF-1α protein was again observed. This biphasic modulation was observed under both 1 and 4% O2. Coexpression of mitochondrial hydrogen peroxide-removing proteins prevented the accumulation of HIF-1α protein in cells with high levels of MnSOD; this effect demonstrates that the restabilization of HIF-1α observed in high MnSOD overexpressors is probably due to hydrogen peroxide, most likely produced from MnSOD. Hypoxic induction of vascular endothelial growth factor (VEGF) protein was also suppressed by elevated MnSOD activity and its levels reflected HIF-1α protein levels. These observations demonstrated that HIF-1α accumulation and VEGF expression could be modulated by the antioxidant enzyme MnSOD.

Epigenetic silencing of DSC3 is a common event in human breast cancer

IntroductionDesmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens.MethodsWe used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines.ResultsDSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure.ConclusionThese results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.

Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression.

p53 is an important transcriptional regulator that is frequently mutated in cancer. Gene-profiling experiments of breast cancer cells infected with wt p53 revealed both MASPIN and desmocollin 3 (DSC3) to be p53-target genes, even though both genes are silenced in association with aberrant cytosine methylation of their promoters. Despite the transcriptional repression of these genes by aberrant DNA methylation, restoration of p53 resulted in the partial reactivation of both genes. This reactivation is a result of wt p53 binding to its consensus DNA-binding sites within the MASPIN and DSC3 promoters, stimulating histone acetylation, and enhancing chromatin accessibility of their promoters. Interestingly, wt p53 alone did not affect the methylation status of either promoter, suggesting that p53 itself can partially overcome the repressive barrier of DNA methylation. Pharmacologic inhibition of DNA methylation with 5-aza-2′-deoxycytidine in combination with restoration of wt p53 status resulted in a synergistic reactivation of these genes to near-normal levels. These results suggest that cancer treatments that target both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.

Superoxide generation in v-Ha-ras–transduced human keratinocyte HaCaT cells

The oncogenic ras protein controls signal‐transduction pathways that are critical for cell proliferation and tumorigenesis. Here, we demonstrate that v‐Ha‐ras–transduced human keratinocyte HaCaT cells produced significantly larger amounts of superoxide than did control cell lines. The superoxide generation was mediated by the transduced ras protein, because superoxide generation was modified by an inhibitor, lovastatin, that inhibits ras farnesylation during ras protein maturation. Superoxide generation was also inhibited by diphenylene iodonium, an inhibitor of flavoproteins, including NADPH oxidase, but not by rotenone, an inhibitor of the respiratory chain of the mitochondria. Those observations suggested that a phagocytic‐like NADPH oxidase exists in keratinocytes that could be activated by the dominant activated v‐Ha‐ras and produce superoxide. Overexpression of manganese‐containing superoxide dismutase and copper and zinc–containing superoxide dismutase cDNA via adenovirus infection also attenuated superoxide generation. Previous work has demonstrated that extracellular superoxide dismutase (SOD) can lower superoxide generation; this is the first report that intracellular SOD could also modify the amount of superoxide production from the cells. This report implies that superoxide radical may act as a second messenger molecule of oncogenic ras. Mol. Carcinog. 26:180–188, 1999.