Dimethylated histone H3 lysine 9 (H3K9me2) is a critical epigenetic mark for gene repression and silencing and plays an essential role in embryogenesis and carcinogenesis. Here, we investigated the effects of hypoxic stress on H3K9me2 at both global and gene-specific level. We found that hypoxia increased global H3K9me2 in several mammalian cell lines. This hypoxia-induced H3K9me2 was temporally correlated with an increase in histone methyltransferase G9a protein and enzyme activity. The increase in H3K9me2 was significantly mitigated in G9a À/À mouse embryonic stem cells following hypoxia challenge, indicating that G9a was involved in the hypoxia-induced H3K9me2. In addition to the activation of G9a, our results also indicated that hypoxia increased H3K9me2 by inhibiting H3K9 demethylation processes. Hypoxic mimetics, such as deferoxamine and dimethyloxalylglycine, were also found to increase H3K9me2 as well as G9a protein and activity. Finally, hypoxia increased H3K9me2 in the promoter regions of the Mlh1 and Dhfr genes, and these increases temporally correlated with the repression of these genes. Collectively, these results indicate that G9a plays an important role in the hypoxia-induced H3K9me2, which would inhibit the expression of several genes that would likely lead to solid tumor progression. (Cancer Res 2006; 66(18): 9009-16)

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DMT1-Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2-transports Fe 2+ into the duodenum and out of the endosome during the transferrin cycle. DMT1 also is important in non-transferrin bound iron uptake. It plays similar roles in Mn 2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases ~50x over endogenous expression and ~10x over uninduced levels. Fe 2+ , Mn 2+ , Ni 2+ and Cu 1+ or Cu 2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb~Co~Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport.

Using human and rodent cells in vitro, we characterized a hypoxia-inducible signaling pathway as one of the pathways affected by carcinogenic nickel compounds. Acute exposure to nickel activates hypoxia-inducible transcription factor-1 (HIF-1), which strongly induces hypoxia-inducible genes, including the recently discovered tumor marker Cap43. This gene has been cloned based on its nickel inducibility and was found to be highly inducible by hypoxia. To identify other HIF-1-dependent/independent nickel-inducible genes, we used cells obtained from HIF-1α null mouse embryos and analyzed gene expression changes using the microarray technique. We found that genes coding for glycolytic enzymes, known to be regulated by HIF-1, were also induced in nickel-exposed cells. In addition, we identified a number of new genes highly induced by nickel in an HIF-dependent manner.

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