Spotlight Ni2+ Carcinogenicity The family of Fe(II)- and 2-oxoglutarate-dependent dioxygenases carries out many important functions in cells, such as gene transcription regulation. Among these enzymes are the histone H3K9 demethylases (e.g., JMJD1A) and demethylases that repair alkylated DNA bases (e.g., ABH2). Nickel ions can inhibit these enzymes, suggesting a mechanism for Ni2+ carcinogenicity. Thus, Chen, et al. [(2009) J. Biol Chem. DOI: 10.1074/jbc.M109.058503, published online Dec 30.] have investigated this phenomenon in greater detail. Chen et al. confirmed that Ni2+ inhibits purified JMJD1A and ABH2 in vitro. The proteins were more sensitive to inhibition in the absence of Fe2+, but excess concentrations of Fe2+ could not reverse Ni2+-induced inhibition. X-ray absorption spectroscopy revealed that Ni2+ binds in the ABH2 Fe2+ binding site, without
Exploring the Cancer Genome Cancer is a disease characterized by the accumulation of multiple somatic gene mutations. Some mutations, the drivers, convey a survival advantage to the cancer cell, while others, the passengers, are simply carried from one generation to the next. Although directed studies have provided substantial information on the mutation spectrum of individual cancer-related genes, only recently has the technology become available to allow the genome-wide characterization of the mutation burden of a cancer. Now, two separate reports, Pleasance, Cheetham et al. [(2009) Nature, 463, 191] and Pleasance et al. [(2009) Nature, 463, 184] provide the results of comprehensive genome sequencing of human cell lines derived from a malignant melanoma (COLO-829) and a small-cell lung carcinoma (SCLC, NCI-H209), respectively.
altering the geometry of the site. Isothermal titration calorimetry indicated that the affinity of ABH2 for Ni2+ was 2.65-fold higher than that for Fe2+. When JMJD1A was overexpressed in human embryonic kidney 293 cells, the levels of endogenous H3K9me2 decreased, an observation that was reversed by Ni2+. Isolation of the expressed enzyme revealed lower levels of demethylase activity from Ni2+-treated cells as compared to controls. Together, the data indicate that Fe-(II)- and 2-oxoglutarate dependent dioxygenases are highly susceptible to Ni2+-mediated inhibition. Such inhibition could lead to loss of control of gene transcription via histone methylation and failure of DNA methylation damage repair, effects that clearly may contribute to the carcinogenicity of Ni2+. • Carol A. Rouzer
synonymous mutations was consistent with what would be expected from chance alone. Since selection pressure should increase the relative prevalence of nonsynonymous to synonymous substitutions in driver mutations, this finding suggests that the vast majority of base substitutions detected across the genome are passenger mutations, having little effect on cancer cell survival or growth.
Both studies used high-throughput short read techniques to obtain full genome sequence data from each cancer cell line and a lymphoblastoid cell line obtained from the same patient for comparison. The data were used to identify cancer-specific mutations, including base substitutions, small insertions and/or deletions, rearrangements, and copy number alterations. In the case of COLO-829, this approach identified 33,345 base substitutions, of which 510 were double base substitutions. Of 470 newly found substitutions, 454 (97%) were confirmed by independent sequencing, and 42 out of 48 previously known substitutions in this cell line were identified (88% sensitivity). For NCI-H209, 22,910 base substitutions were identified, including 79 novel mutations of which 77 were confirmed. The approach identified 22 out of 29 known substitution mutations in this cell line.
Graphic representation of the catalog of somatic mutations in COLO-829. Reprinted by permission from Macmillan Publishers, Ltd.: Pleasance, Cheetham et al. (2009) Nature, 463, 191. Copyright 2010.
For both cell lines, an analysis of the base substitutions in protein coding regions (292 for COLO-829 and 130 in NCIH209) indicated that the distribution of the nonsynonymous mutations (those resulting in amino acid changes) versus
The predominant base substitution in COLO-829 cells was C > T/G > A transitions. In fact, 360 of the 510 double nucleotide substitutions were CC > TT/GG > AA. This finding,
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Spotlight coupled with the prevalence of these substitutions at the 3′ base of a pyrimidine or at CpG sequences, is consistent with the mutation pattern expected from ultraviolet light damage, which forms primarily pyrimidine dimers. The second most common form of base substitution in these cells was C > A/G > T, a pattern consistent with oxidative damage at dG. In contrast, HCT-H209 exhibited a more diverse pattern of base substitutions with G > T/C > A (34%), G > A/C > T (21%), and A > G/T > C (19%) predominating. The distribution of these lesions relative to CpG islands suggests that the G > T/C > A transversions likely result from polycyclic aromatic hydrocarbon (PAH) adduct formation, which is favored at methylated CpG dinucleotides, and that the G > A/C > T transitions are products of deamination of methylated cytosine bases to form uracil. These findings support the expectation that the pattern of passenger mutations should reflect the causative agent(s). In the case of melanoma, this is primarily ultraviolet light, and in the case of lung cancer, it is the >60 different carcinogens, including PAHs, present in cigarette smoke. Many base substitution mutations are repaired by nucleotide excision repair, one form of which occurs during transcription. The finding of fewer mutations in the transcribed strand than in the untranscribed strand of gene coding regions suggests an active transcription-coupled repair pathway in both cancers. However, for both strands the data also revealed fewer mutations in exons than in introns, fewer mutations at the 3′-terminus of a gene than at its 5′-terminus, and an inverse relationship between number of mutations and level of transcription. These findings suggest that additional repair mechanisms and/ or other factors are also at work. These are just some examples of the wealth of information that could be gleaned from the mutation data. For both tumors, insertions/deletions, rearrangements, and copy number alterations were also identified. Some mutations were consistent with previously identified drivers. For example, in the case of NCI-H209, mutations identified in RB1 and TP53 are consistent with known drivers of SCLC. Other mutations suggested the identities of new drivers for both melanoma and SCLC. These exciting studies confirm the value of genome-wide sequencing in cancer and pave the way for future work that will reveal new insights about the mechanism of carcinogenesis in many cancer types. • Carol A. Rouzer
Lethal Conformation The aggregation of proteins to form β-sheet-rich fibrils known as amyloid contributes to the pathogenesis of over 40 human diseases, including the neurodegenerative Alzheimer’s and Parkinson’s Diseases. Increasing evidence suggests that protein oligomers formed in the early stages of amyloid formation are the toxic species in these conditions. Thus, a clear understanding of the characteristics of protein aggregates that lead to toxicity is important for the development of better therapies of amyloid-based illnesses. Now, Published online 04/19/2010 • DOI: 10.1021/tx100057v © 2010 American Chemical Society
Campioni et al. [(2010) Nature Chem. Biol., 6, 140] address this question through their studies of two preparations of the 91 residue N-terminal fragment of the E. coli HypF protein (HypF-N). HypF-N was allowed to aggregate under two different sets of conditions, resulting in oligomer preparations A and B that were stable when transferred to aqueous solution under physiological conditions. Atomic force microscopy and staining with thioflavin T (which recognizes β-sheet structure) revealed similar morphology and tinctorial properties for the two samples. However, preparation A was toxic to SH-SY5Y cells as indicated by the MTT reduction assay and Hoechst staining, while preparation B was harmless at the same or higher concentrations.
Reprinted by permission from Macmillan Publishers, Ltd.: Campioni et al. (2010) Nature Chem. Biol., 6, 140. Copyright 2010.
To determine the basis for the difference in toxicity, Campioni et al. expressed 18 mutational variants of HypFN, each bearing a cysteine substitution at different locations along the polypeptide chain. Attachment of N-(1pyrene)maleimide (PM) to the cysteine residue provided a means to identify closely associated regions of the protein in the oligomers since the close spacial proximity of two PM molecules leads to characteristic fluorescence. The results showed that preparation B oligomers exhibited strong associations between hydrophobic regions of the protein, while the hydrophobic regions in preparation A oligomers were disordered and solvent-exposed. These conclusions were supported by the finding that 8-anilinonaphthalene-1-sulfonate, a probe of hydrophobic domains, fluoresced more intensely in the presence of A than B oligomers. Confocal microscopic observations of the interactions of oligomers with SH-SY5Y cells indicated that both A and B preparations bound to cell membranes. However, only the A oligomers were able to cross the membranes to enter the cells and induce an increase in intracellular Ca2+. Together, the data suggest that disorganized and solventexposed hydrophobic regions are structural features that convey toxicity to protein oligomers, and that these features act by increasing the oligomers’ ability to penetrate the cellular membrane and, more generally, to form aberrant interactions. Future work will reveal how these conclusions apply to other amyloid-forming proteins. • Carol A. Rouzer TX100057V Vol. 23,
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