Maintenance of Flap Endonucleases for Long-Patch Base Excision DNA Repair in Mouse Muscle and Neuronal Cells Differentiated In Vitro.

After cellular differentiation, nuclear DNA is no longer replicated, and many of the associated proteins are downregulated accordingly. These include the structure-specific endonucleases Fen1 and DNA2, which are implicated in repairing mitochondrial DNA (mtDNA). Two more such endonucleases, named MGME1 and ExoG, have been discovered in mitochondria. This category of nuclease is required for so-called "long-patch" (multinucleotide) base excision DNA repair (BER), which is necessary to process certain oxidative lesions, prompting the question of how differentiation affects the availability and use of these enzymes in mitochondria. In this study, we demonstrate that Fen1 and DNA2 are indeed strongly downregulated after differentiation of neuronal precursors (Cath.a-differentiated cells) or mouse myotubes, while the expression levels of MGME1 and ExoG showed minimal changes. The total flap excision activity in mitochondrial extracts of these cells was moderately decreased upon differentiation, with MGME1 as the predominant flap endonuclease and ExoG playing a lesser role. Unexpectedly, both differentiated cell types appeared to accumulate less oxidative or alkylation damage in mtDNA than did their proliferating progenitors. Finally, the overall rate of mtDNA repair was not significantly different between proliferating and differentiated cells. Taken together, these results indicate that neuronal cells maintain mtDNA repair upon differentiation, evidently relying on mitochondria-specific enzymes for long-patch BER.

Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase ß in vivo.

Free radical attack on the C1' position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase ß (Polß) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA-protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polß-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polß-DPC in vivo. In a dose-dependent fashion, Polß-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polß-DPC under 1% O2 than under 21% O2) and even more robustly with the "chemical nuclease" 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polß-DPC. Nonoxidative agents did not generate Polß-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polß-DPC depended on the Ape1 AP endonuclease, which generates the Polß lyase substrate, and they required the essential lysine-72 in the Polß lyase active site. Oxidative Polß-DPC had an unexpectedly short half-life (∼ 30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polß than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polß-DPC and the biological pressure to repair them.

Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates.

DNA2, a helicase/nuclease family member, plays versatile roles in processing DNA intermediates during DNA replication and repair. Yeast Dna2 (yDna2) is essential in RNA primer removal during nuclear DNA replication and is important in repairing UV damage, base damage, and double-strand breaks. Our data demonstrate that, surprisingly, human DNA2 (hDNA2) does not localize to nuclei, as it lacks a nuclear localization signal equivalent to that present in yDna2. Instead, hDNA2 migrates to the mitochondria, interacts with mitochondrial DNA polymerase gamma, and significantly stimulates polymerase activity. We further demonstrate that hDNA2 and flap endonuclease 1 synergistically process intermediate 5' flap structures occurring in DNA replication and long-patch base excision repair (LP-BER) in mitochondria. Depletion of hDNA2 from a mitochondrial extract reduces its efficiency in RNA primer removal and LP-BER. Taken together, our studies illustrate an evolutionarily diversified role of hDNA2 in mitochondrial DNA replication and repair in a mammalian system.

Essential role for mammalian apurinic/apyrimidinic (AP) endonuclease Ape1/Ref-1 in telomere maintenance.

The major mammalian apurinic/apyrimidinic endonuclease Ape1 is a multifunctional protein operating in protection of cells from oxidative stress via its DNA repair, redox, and transcription regulatory activities. The importance of Ape1 has been marked by previous work demonstrating its requirement for viability in mammalian cells. However, beyond a requirement for Ape1-dependent DNA repair activity, deeper molecular mechanisms of the fundamental role of Ape1 in cell survival have not been defined. Here, we report that Ape1 is an essential factor stabilizing telomeric DNA, and its deficiency is associated with telomere dysfunction and segregation defects in immortalized cells maintaining telomeres by either the alternative lengthening of telomeres pathway (U2OS) or telomerase expression (BJ-hTERT), or in normal human fibroblasts (IMR90). Through the expression of Ape1 derivatives with site-specific changes, we found that the DNA repair and N-terminal acetylation domains are required for the Ape1 function at telomeres. Ape1 associates with telomere proteins in U2OS cells, and Ape1 depletion causes dissociation of TRF2 protein from telomeres. Consistent with this effect, we also observed that Ape1 depletion caused telomere shortening in both BJ-hTERT and in HeLa cells. Thus, our study describes a unique and unpredicted role for Ape1 in telomere protection, providing a direct link between base excision DNA repair activities and telomere metabolism.

Intraluminal vesicle trafficking is involved in the secretion of base excision repair protein APE1.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW 4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.

A DNA-based nanomechanical device used to characterize the distortion of DNA by Apo-SoxR protein.

DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting proteins. Here, we have constructed a device wherein two DNA triple-crossover (TX) molecules are connected by a shaft, similar to a previous device that measured the binding free energy of integration host factor. In our case, the binding site on the shaft contains the sequence recognized by SoxR protein, the apo form of which is a transcriptional activator. Another active form is oxidized [2Fe-2S] SoxR formed during redox sensing, and previous data suggest that activated Fe-SoxR distorts its binding site by localized DNA untwisting by an amount that corresponds to ~2 bp. A pair of dyes report the fluorescence resonance energy transfer (FRET) signal between the two TX domains, reflecting changes in the shape of the device upon binding of the protein. The TX domains are used to amplify the signal expected from a relatively small distortion of the DNA binding site. From FRET analysis of apo-SoxR binding, the effect of apo-SoxR on the original TX device is similar to the effect of shortening the TX device by 2 bp. We estimate that the binding free energy of apo-SoxR on the DNA target site is 3.2-6.1 kcal/mol.

Distinct roles of Ape1 protein in the repair of DNA damage induced by ionizing radiation or bleomycin.

Ionizing radiation (IR) and bleomycin (BLM) are used to treat various types of cancers. Both agents generate cytotoxic double strand breaks (DSB) and abasic (apurinic/apyrimidinic (AP)) sites in DNA. The human AP endonuclease Ape1 acts on abasic or 3'-blocking DNA lesions such as those generated by IR or BLM. We examined the effect of siRNA-mediated Ape1 suppression on DNA repair and cellular resistance to IR or BLM in human B-lymphoblastoid TK6 cells and HCT116 colon tumor cells. Partial Ape1 deficiency (∼30% of normal levels) sensitized cells more dramatically to BLM than to IR cytotoxicity. In both cases, expression of the unrelated yeast AP endonuclease, Apn1, largely restored resistance. Ape1 deficiency increased DNA AP site accumulation due to IR treatment but reduced the number of DSB. In contrast, for BLM, there were more DSB under Ape1 deficiency, with little change in the accumulation of AP sites. Although the role of Ape1 in generating DSB was greater for IR, the enzyme facilitated removal of AP sites, which may mitigate the cytotoxic effects of IR. In contrast, BLM generates scattered AP sites, and the DSB have 3'-phosphoglycolate termini that require Ape1 processing. These DSB persist under Ape1 deficiency. Apoptosis induced by BLM (but not by IR) under Ape1 deficiency was partially p53-dependent, more dramatically in TK6 than HCT116 cells. Thus, Ape1 suppression or inhibition may be a more efficacious adjuvant for BLM than for IR cancer therapy, particularly for tumors with a functional p53 pathway.

DNA-mediated redox signaling for transcriptional activation of SoxR.

In enteric bacteria, the cellular response to oxidative stress is activated by oxidation of the iron-sulfur clusters in SoxR, which then induces transcription of soxS, turning on a battery of defense genes. Here we demonstrate both in vitro and in cells that activation of SoxR can occur in a DNA-mediated reaction with guanine radicals, an early genomic signal of oxidative stress, serving as the oxidant. SoxR in its reduced form is found to inhibit guanine damage by repairing guanine radicals. Moreover, cells treated with a DNA-binding photooxidant, which generates guanine radicals, promotes the expression of soxS. In vitro, this photooxidant, tethered to DNA 80 bp from the soxS promoter, induces transcription by activating SoxR upon irradiation. Thus, transcription can be activated from a distance through DNA-mediated charge transport. This chemistry offers a general strategy for DNA-mediated signaling of oxidative stress.

Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression.

Apurinic/apyrimidinic endonuclease 1/redox effector-1 (Ape1/Ref-1) is the major apurinic/apyrimidinic (AP) endonuclease in mammalian cells. It functions mainly in the base excision repair pathway to create a suitable substrate for DNA polymerases. Human Ape1 protein can activate some transcription factors to varying degrees, dependent on its N-terminal, unstructured domain, and some of the cysteines within it, apparently via a redox mechanism in some cases. Many cancer studies also suggest that Ape1 has potential for prognosis in terms of the protein level or intracellular localization. While homozygous disruption of the Ape1 structural gene APEX1 in mice causes embryonic lethality, and most studies in cell culture indicate that the expression of Ape1 is essential, some recent studies reported the isolation of viable APEX1 knockout cells with only mild phenotypes. It has not been established by what mechanism the Ape1-null cell lines cope with the endogenous DNA damage that the enzyme normally handles. We review the enzymatic and other activities of Ape1 and the recent studies of the properties of the APEX1 knockout lines. The APEX1 deletions in CH12F3 and HEK293 FT provide an opportunity to test for possible off-target effects of Ape1 inhibition. For this work, we tested the Ape1 endonuclease inhibitor Compound 3 and the redox inhibitor APX2009. Our results confirmed that both APEX1 knockout cell lines are modestly more sensitive to killing by an alkylating agent than their Ape1-proficient cells. Surprisingly, the knockout lines showed equal sensitivity to direct killing by either inhibitor, despite the lack of the target protein. Moreover, the CH12F3 APEX1 knockout was even more sensitive to Compound 3 than its APEX1+ counterpart. Thus, it appears that both Compound 3 and APX2009 have off-target effects. In cases where this issue may be important, it is advisable that more specific endpoints than cell survival be tested for establishing mechanism.

DNA binding shifts the redox potential of the transcription factor SoxR.

Electrochemistry measurements on DNA-modified electrodes are used to probe the effects of binding to DNA on the redox potential of SoxR, a transcription factor that contains a [2Fe-2S] cluster and is activated through oxidation. A DNA-bound potential of +200 mV versus NHE (normal hydrogen electrode) is found for SoxR isolated from Escherichia coli and Pseudomonas aeruginosa. This potential value corresponds to a dramatic shift of +490 mV versus values found in the absence of DNA. Using Redmond red as a covalently bound redox reporter affixed above the SoxR binding site, we also see, associated with SoxR binding, an attenuation in the Redmond red signal compared with that for Redmond red attached below the SoxR binding site. This observation is consistent with a SoxR-binding-induced structural distortion in the DNA base stack that inhibits DNA-mediated charge transport to the Redmond red probe. The dramatic shift in potential for DNA-bound SoxR compared with the free form is thus reconciled based on a high-energy conformational change in the SoxR-DNA complex. The substantial positive shift in potential for DNA-bound SoxR furthermore indicates that, in the reducing intracellular environment, DNA-bound SoxR is primarily in the reduced form; the activation of DNA-bound SoxR would then be limited to strong oxidants, making SoxR an effective sensor for oxidative stress. These results more generally underscore the importance of using DNA electrochemistry to determine DNA-bound potentials for redox-sensitive transcription factors because such binding can dramatically affect this key protein property.

Roles of Rev1, Pol zeta, Pol32 and Pol eta in the bypass of chromosomal abasic sites in Saccharomyces cerevisiae.

Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocking lesions are bypassed, but at the risk of increased mutagenesis. The exact in vivo role of the individual TLS enzymes in Saccharomyces cerevisiae has been difficult to determine from previous studies due to differing results from the variety of systems used. We have generated a series of S.cerevisiae strains in which each of the TLS-related genes REV1, REV3, REV7, RAD30 and POL32 was deleted, and in which chromosomal apyrimidinic sites were generated during normal cell growth by the activity of altered forms of human uracil-DNA glycosylase that remove undamaged cytosines or thymines. Deletion of REV1, REV3 or REV7 resulted in slower growth dependent on (rev3Delta and rev7Delta) or enhanced by (rev1Delta) expression of the mutator glycosylases and a nearly complete abolition of glycosylase-induced mutagenesis. Deletion of POL32 resulted in cell death when the mutator glycosylases were expressed and, in their absence, diminished spontaneous mutagenesis. RAD30 appeared to be unnecessary for mutagenesis in response to abasic sites, as deleting this gene caused no significant change in either the mutation rates or the mutational spectra due to glycosylase expression.

Oxidative DNA-protein crosslinks formed in mammalian cells by abasic site lyases involved in DNA repair.

Free radical attack on C1' of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Polß) in covalent DNA-protein crosslinks (DPC) via the enzyme's N-terminal lyase activity acting on 5'-deoxyribose-5-phosphate residues. We previously demonstrated formation of Polß-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Polß-DPC accumulation and trapped ubiquitin in the DPC, with Polß accounting for 60-70 % of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase λ (Polλ), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Polλ, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Polß: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) H2O2 gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following H2O2 treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15-60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Polλ, and OGG1 proteins. As seen for Polß, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Polß-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.

Intrinsic 5'-deoxyribose-5-phosphate lyase activity in Saccharomyces cerevisiae Trf4 protein with a possible role in base excision DNA repair.

In Saccharomyces cerevisiae, the base excision DNA repair (BER) pathway has been thought to involve only a multinucleotide (long-patch) mechanism (LP-BER), in contrast to most known cases that include a major single-nucleotide pathway (SN-BER). The key step in mammalian SN-BER, removal of the 5'-terminal abasic residue generated by AP endonuclease incision, is effected by DNA polymerase beta (Polbeta). Computational analysis indicates that yeast Trf4 protein, with roles in sister chromatin cohesion and RNA quality control, is a new member of the X family of DNA polymerases that includes Polbeta. Previous studies of yeast trf4Delta mutants revealed hypersensitivity to methylmethane sulfonate (MMS) but not UV light, a characteristic of BER mutants in other organisms. We found that, like mammalian Polbeta, Trf4 is able to form a Schiff base intermediate with a 5'-deoxyribose-5-phosphate substrate and to excise the abasic residue through a dRP lyase activity. Also like Polbeta, Trf4 forms stable cross-links in vitro to 5'-incised 2-deoxyribonolactone residues in DNA. We determined the sensitivity to MMS of strains with a trf4Delta mutation in a rad27Delta background, in an AP lyase-deficient background (ogg1 ntg1 ntg2), or in a pol4Delta background. Only a RAD27 genetic interaction was detected: there was higher sensitivity for strains mutated in both TRF4 and RAD27 than either single mutant, and overexpression of Trf4 in a rad27Delta background partially suppressed MMS sensitivity. The data strongly suggest a role for Trf4 in a pathway parallel to the Rad27-dependent LP-BER in yeast. Finally, we demonstrate that Trf5 significantly affects MMS sensitivity and thus probably BER efficiency in cells expressing either wild-type Trf4 or a C-terminus-deleted form.

Biochemical evaluation of genotoxic biomarkers for 2-deoxyribonolactone-mediated cross-link formation with histones.

Numerous environmental carcinogens involve radical formation interacting with DNA to produce 2-deoxyribonolactone (dL), a major type of oxidized abasic site, implicated in DNA strand breaks, mutagenesis, and formation of covalent DNA-protein cross-links (DPC). Studies showed major dL-specific DPC occurred due to reactions with DNA polymerase beta (Polbeta) dependent on native conformation, while other DPC formed involved nonenzymatic reactions of DNA binding proteins with dL lesions. Polbeta appeared to play a major role in alleviating the cytotoxic effects of neocarzinostatin, which was used as a dL-producing agent. When a duplex DNA containing a dL at a site-specific position was incubated with purified histones, DPC were formed between dL and each histone protein, including H1, H2A, H2B, H3, and H4. Comparative kinetic analysis of DPC formation with histones and Polbeta revealed two distinct mechanisms of dL-mediated DPC formation. The rate of DPC formation with Polbeta was approximately two orders of magnitude higher than that with various histone proteins. These results indicate that catalytic activity of Polbeta mediates rapid DPC formation between dL and this DNA repair enzyme, whereas nonenzymatic reactions of dL with histones form DPC more slowly. The abundance of histones and their constant interaction with DNA may nevertheless yield significant levels of DPC with dL, as biomarkers of dL-induced cytotoxicity. Overall, data suggest that occurrence of dL-mediated DPC with histones may contribute to the genotoxic effects of dL in DNA.

Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair.

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or "short-patch" (SP-BER), and "long-patch" BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase ß (Polß) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polß itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polß-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polß in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.

Human AP-endonuclease (Ape1) activity on telomeric G4 structures is modulated by acetylatable lysine residues in the N-terminal sequence.

Loss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberrant end-joining reactions leading to chromosomal fusions and translocations. Human telomeres contain repeated TTAGGG elements, in which the 3' exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8-oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4-telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures and that this interaction involves specific acetylatable lysine residues (i.e. K27/31/32/35) present in the unstructured N-terminal sequence of the protein. The cleavage of an abasic site located in a G4 structure by Ape1 depends on the DNA conformation or the position of the lesion and on electrostatic interactions between the protein and the nucleic acids. Moreover, Ape1 mutants mimicking the acetylated protein display increased cleavage activity for abasic sites. We found that nucleophosmin (NPM1), which binds the N-terminal sequence of Ape1, plays a role in modulating telomere length and Ape1 activity at abasic G4 structures. Thus, the Ape1 N-terminal sequence is an important relay site for regulating the enzyme's activity on G4-telomeric sequences, and specific acetylatable lysine residues constitute key regulatory sites of Ape1 enzymatic activity dynamics at telomeres.

Expression of Genes Involved in Stress, Toxicity, Inflammation, and Autoimmunity in Relation to Cadmium, Mercury, and Lead in Human Blood: A Pilot Study.

There is growing evidence of immunotoxicity related to exposure to toxic trace metals, and an examination of gene expression patterns in peripheral blood samples may provide insights into the potential development of these outcomes. This pilot study aimed to correlate the blood levels of three heavy metals (mercury, cadmium, and lead) with differences in gene expression in 24 participants from the Long Island Study of Seafood Consumption. We measured the peripheral blood mRNA expression of 98 genes that are implicated in stress, toxicity, inflammation, and autoimmunity. We fit multiple linear regression models with multiple testing correction to correlate exposure biomarkers with mRNA abundance. The mean blood Hg in this cohort was 16.1 µg/L, which was nearly three times the Environmental Protection Agency (EPA) reference dose (5.8 µg/L). The levels of the other metals were consistent with those in the general population: the mean Pb was 26.8 µg/L, and the mean Cd was 0.43 µg/L. The expression of three genes was associated with mercury, four were associated with cadmium, and five were associated with lead, although none were significant after multiple testing correction. Little evidence was found to associate metal exposure with mRNA abundance for the tested genes that were associated with stress, toxicity, inflammation, or autoimmunity. Future work should provide a more complete picture of physiological reactions to heavy metal exposure.

Assessing Toxicity and Nuclear and Mitochondrial DNA Damage Caused by Exposure of Mammalian Cells to Lunar Regolith Simulants.

Previous missions to the lunar surface implicated potential dangers of lunar soil. In future explorations, astronauts may spend weeks or months on the Moon, increasing the risk of inhaling lunar dust. In an effort to understand the biological impact of lunar regolith, cell cultures derived from lung or neuronal cells were challenged with lunar soil simulants to assess cell survival and genotoxicity. Lunar soil simulants were capable of causing cell death and DNA damage in neuronal and lung cell lines, and freshly crushed lunar soil simulants were more effective at causing cell death and DNA damage than were simulants as received from the supplier. The ability of the simulants to generate reactive oxygen species in aqueous suspensions was not correlated with their cytotoxic or genotoxic affects. Furthermore, the cytotoxicity was not correlated with the accumulation of detectable DNA lesions. These results determine that lunar soil simulants are, with variable activity, cytotoxic and genotoxic to both neuronal and lung-derived cells in culture.

Regulation of heme oxygenase-1 mRNA deadenylation and turnover in NIH3T3 cells by nitrosative or alkylation stress.

BACKGROUND: Heme oxygenase-1 (HO-1) catalizes heme degradation, and is considered one of the most sensitive indicators of cellular stress. Previous work in human fibroblasts has shown that HO-1 expression is induced by NO, and that transcriptional induction is only partially responsible; instead, the HO-1 mRNA half-life is substantially increased in response to NO. The mechanism of this stabilization remains unknown. RESULTS: In NIH3T3 murine fibroblasts, NO exposure increased the half-life of the HO-1 transcript from ~1.6 h to 11 h, while treatments with CdCl2, NaAsO2 or H2O2 increased the half-life only up to 5 h. Although poly(A) tail shortening can be rate-limiting in mRNA degradation, the HO-1 mRNA deadenylation rate in NO-treated cells was ~65% of that in untreated controls. In untreated cells, HO-1 poly(A) removal proceeded until 30-50 nt remained, followed by rapid mRNA decay. In NO-treated cells, HO-1 deadenylation stopped with the mRNA retaining poly(A) tails 30-50 nt long. We hypothesize that NO treatment stops poly(A) tail shortening at the critical 30- to 50-nt length. This is not a general mechanism for the post-transcriptional regulation of HO-1 mRNA. Methyl methane sulfonate also stabilized HO-1 mRNA, but that was associated with an 8-fold decrease in the deadenylation rate compared to that of untreated cells. Another HO-1 inducer, CdCl2, caused a strong increase in the mRNA level without affecting the HO-1 mRNA half-life. CONCLUSION: The regulation of HO-1 mRNA levels in response to cellular stress can be induced by transcriptional and different post-transcriptional events that act independently, and vary depending on the stress inducer. While NO appears to stabilize HO-1 mRNA by preventing the final steps of deadenylation, methyl methane sulfonate achieves stabilization through the regulation of earlier stages of deadenylation.

Nitric oxide-induced apoptosis in lymphoblastoid and fibroblast cells dependent on the phosphorylation and activation of p53.

When nitric oxide (NO) is produced at micromolar concentrations, as during inflammation, exposure to surrounding cells is potentially cytotoxic. The NO-dependent signaling pathways that initiate cell death are thought to involve the tumor suppressor protein p53, but the degree to which this factor contributes to NO-induced cell death is less clear. Various reports either confirm or negate a role for p53 depending on the cell type and NO donor used. In this study, we have used several pairs of cell lines whose only differences are the presence or absence of p53, and we have treated these cell lines with the same NO donor, spermineNONOate (SPER/NO). Treatment with SPER/NO induced such apoptotic markers as DNA fragmentation, nuclear condensation, poly(ADP-ribose) polymerase cleavage, cytochrome c release, and Annexin V staining. p53 was required for at least 50% of SPER/NO-induced apoptotic cell death in human lymphoblastoid cells and for almost all in primary and E1A-tranformed mouse embryonic fibroblasts, which highlights the possible importance of DNA damage for apoptotic signaling in fibroblasts. In contrast, p53 did not play a significant role in NO-induced necrosis. NO treatment also induced the phosphorylation of p53 at Ser15; pretreatment with phosphoinositide-3 kinase (PI3K) family inhibitors, wortmannin, LY294002, and caffeine, blocked such phosphorylation, but the p38 mitogen-activated protein kinase inhibitor, SB203580, did not. Pretreatment with the PI3K family inhibitors also led to a switch from NO-induced apoptosis to necrosis, which implicates a PI3K-related kinase such as ataxia telangiectasia mutated (ATM) or ATR (ATM and Rad3 related) in p53-dependent NO-induced apoptosis.