Hypoxia-inducible factor-1α (HIF-1α) is a master transcription factor that is critical for the regulation of a variety of cellular functions. HIF-1α is rapidly degraded under normoxic conditions by ubiquitin-mediated proteasome pathway controlled by the tumor suppressor von Hippel Lindau (VHL). Several recent studies reveal that heat-shock proteins (Hsp) can regulate HIF-1α protein degradation by a VHL-independent pathway. Here, we demonstrate that the stress kinase c-Jun NH 2 -terminal kinase 1 ( JNK1) is required for Hspdependent regulation of HIF-1α. Stabilization of HIF-1α was impaired in JNK1−/− cells but could be rescued by JNK1 reconstitution under hypoxic conditions. These effects could be phenocopied in other cell settings by JNK1 silencing. Accordingly, HIF-1 transcriptional activity and target gene expression were dramatically reduced in JNK1−/− cells. Further, decreased levels of endogenous Hsp90/Hsp70 proteins in JNK1−/− cells affected the protective roles of these chaperones in stabilizing newly synthesized HIF-1α, whereas enforced expression of Hsp90/Hsp70 in JNK1−/− cells increased HIF-1α stability relative to parental control cells. Furthering this connection, we also found that defective expression of the Hsp90 acetyltransferase HDAC6 in JNK1−/− cells was associated with reduced Hsp90 chaperone activity. Taken together, our studies define a novel function for JNK1 in regulating HIF-1α turnover by a VHL-independent mechanism.

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Epigenetics refers to heritable patterns of gene expression that do not depend on alterations of the genomic DNA sequence. Nickel compounds have demonstrated carcinogenicity without any associated mutagenesis, suggesting that its mechanism of carcinogenesis is epigenetic in nature. One such potential mechanism is the heterochromatinization of chromatin within a region of the genome containing a gene sequence, inhibiting any further molecular interactions with that underlying gene sequence and effectively inactivating that gene. We report here the observation, by atomic force microscopy and circular dichroism spectropolarimetry, that nickel ion (Ni 2+ ) condenses chromatin to a greater extent than the natural divalent cation of the cell, magnesium ion (Mg 2+ ). In addition, we use a model experimental system that incorporates a transgene, the bacterial xanthine guanine phosphoribosyl transferase gene (gpt) differentially near to, and far away from, a heterochromatic region of the genome, in two cell lines, the Chinese hamster V79-derived G12 and G10 cells, respectively, to demonstrate by DNase I protection assay that nickel treatement protects the gpt gene sequence from DNase I exonuclease digestion in the G12 cells, but not in the G10 cells. We conclude that condensation of chromatin by nickel is a potential mechanism of nickel-mediated gene regulation. Keywords chromatin; heterochromatin; nickel; divalent cation; carcinogenesisChromatin is the natural state of protein-associated DNA in all eukaryotes. It is a dynamic polymer of subunits called nucleosomes. The nucleosome subunit consists of approximately 146 bps of DNA wrapped around an octamer of histone proteins. The histone octamer is made up of two each of histone H2A, H2B, H3, and H4. The DNA makes one and a half turns around the octamer, with the entry and exit points of the DNA at opposite sides of the disk-shaped histone octameric complex. This complex of 146 bps and the octamer of histones is known as the nucleosome core particle. In chromatin, the exiting and entering DNA duplex strands of this nucleosome core particle extend to the next nucleosome core particles on either side of it. The intervening DNA connecting each pair of nucleosomes is termed the linker DNA. The lengths of these intervening DNA duplex strands vary from species to species and from tissue types to tissues types, as well as from one stretch between two nucleosomes to another. Linker DNA length ranges from a minimum of 25 bps to upwards of 100 bps, averaging approximately 50 to 60 bps (1). A fifth histone type, the linker histone (H1), binds to the linker DNA in a stoichiometry of 1 H1 to 1 nucleosome. The complex of the core particle (the histone octamer

The effect of various metal compounds on the DNA of Chinese hamster ovary (CHO) cells was studied. Both NiCl2 and crystalline NiS caused DNA strand breaks in cultured CHO cells, whereas amorphous NiS did not. Strand breaks were quantitated by determining the number of average molecular weight of DNA following treatment with the metal compounds. Exposure of cells to crystalline NiS, CoS, CdS, AgS, CuS and Ni3S2 at 10 micrograms/ml for 24 h also induced DNA strand breaks. Similar exposure to activated charcoal, which was also actively phagocytosed, failed to cause any effect on the DNA of CHO cells. In the case of NiCl2 and NiS the effect was shown to be both time and dose dependent. Other soluble metal compounds such as HgCl2, CaCrO4, and CdCl2 also decreased the molecular weight of DNA while MnCl2, ZnCl2 and FeCl2 caused no significantly detectable change in DNA molecular weight. These effects, which occur at low metal concentrations suggest that nickel and other metals which cause cellular transformation have a very selective and specific effect upon DNA.

Raising the extracellular level of magnesium ions inhibited nickel-induced DNA strand breaks, DNA-protein crosslinks, sister chromatid exchanges, chromosomal aberrations and cell transformation. Carcinogenic nickel ions preferentially damaged centromeres and other heterochromatic regions of Chinese hamster ovary cell chromosomes. Elevation of extracellular magnesium levels prevented the effects of nickel on heterochromatin and inhibited cell transformation, but did not substantially reduce the DNA damage induced by nickel in euchromatic regions. This study suggests that heterochromatic DNA damage may be important to the nickel-induced neoplastic transformation process.