Oxidative damage to DNA has been associated with neurodegenerative diseases. Developmental exposure to lead (Pb) has been shown to elevate the Alzheimer's disease (AD) related beta-amyloid peptide (Abeta), which is known to generate reactive oxygen species in the aging brain. This study measures the lifetime cerebral 8-hydroxy-2'-deoxyguanosine (oxo8dG) levels and the activity of the DNA repair enzyme 8-oxoguanine DNA glycosylase (Ogg1) in rats developmentally exposed to Pb. Oxo8dG was transiently modulated early in life (Postnatal day 5), but was later elevated 20 months after exposure to Pb had ceased, while Ogg1 activity was not altered. Furthermore, an age-dependent loss in the inverse correlation between Ogg1 activity and oxo8dG accumulation was observed. The effect of Pb on oxo8dG levels did not occur if animals were exposed to Pb in old age. These increases in DNA damage occurred in the absence of any Pb-induced changes in copper/zinc-superoxide dismutase (SOD1), manganese-SOD (SOD2), and reduced-form glutathion (GSH). These data suggest that oxidative damage and neurodegeneration in the aging brain could be impacted by the developmental disturbances.

Oncostatin M (OSM) is an interleukin-6 (IL-6) family cytokine that has been implicated in a number of biological processes including inflammation, hematopoiesis, immune responses, development, and bone homeostasis. Recent evidence suggests that OSM may promote breast tumor invasion and metastasis. We investigated the role of OSM in the formation of bone metastases in vivo using the 4T1.2 mouse mammary tumor model in which OSM expression was knocked down using shRNA (4T1.2-OSM). 4T1.2-OSM cells were injected orthotopically into Balb/c mice, resulting in a greater than 97% decrease in spontaneous metastasis to bone compared to control cells. Intratibial injection of these same 4T1.2-OSM cells also dramatically reduced the osteolytic destruction of trabecular bone volume compared to control cells. Furthermore, in a tumor resection model, mice bearing 4T1.2-OSM tumors showed an increase in survival by a median of 10 days. To investigate the specific cellular mechanisms important for OSM-induced osteolytic metastasis to bone, an in vitro model was developed using the RAW 264.7 preosteoclast cell line co-cultured with 4T1.2 mouse mammary tumor cells. Treatment of co-cultures with OSM resulted in a 3-fold induction of osteoclastogenesis using the TRAP assay. We identified several tumor cell-induced factors including vascular endothelial growth factor, IL-6, and a previously uncharacterized OSM-regulated bone metastasis factor, amphiregulin (AREG), which increased osteoclast differentiation by 4.5-fold. In addition, pretreatment of co-cultures with an anti-AREG neutralizing antibody completely reversed OSM-induced osteoclastogenesis. Our results suggest that one mechanism for OSM-induced osteoclast differentiation is via an AREG autocrine loop, resulting in decreased osteoprotegerin secretion by the 4T1.2 cells. These data provide evidence that OSM might be an important therapeutic target for the prevention of breast cancer metastasis to bone.

The community members of Libby, MT, have experienced significant asbestos exposure and developed numerous asbestos-related diseases including fibrosis and lung cancer due to an asbestos-contaminated vermiculite mine near the community. The form of asbestos in the contaminated vermiculite has been characterized in the amphibole family of fibers. However, the pathogenic effects of these fibers have not been previously characterized. The purpose of this study is to determine the cellular consequences of Libby amphibole exposure in macrophages compared to another well-characterized amphibole fiber; crocidolite asbestos. Our results indicate that Libby asbestos fibers are internalized by macrophages and localize to the cytoplasm and cytoplasmic vacuoles similar to crocidolite fibers. Libby asbestos fiber internalization generates a significant increase in intracellular reactive oxygen species (ROS) as determined by dichlorofluorescein diacetate and dihydroethidine fluorescence indicating that the superoxide anion is the major contributing ROS generated by Libby asbestos. Elevated superoxide levels in macrophages exposed to Libby asbestos coincide with a significant suppression of total superoxide dismutase activity. Both Libby and crocidolite asbestos generate oxidative stress in exposed macrophages by decreasing intracellular glutathione levels. Interestingly crocidolite asbestos, but not Libby asbestos, induces significant DNA damage in macrophages. This study provides evidence that the difference in the level of DNA damage observed between Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure.

Oxidation of the guanosine moiety in DNA has become a hallmark biomarker in assessing oxidative stress. The oxidation of guanosine in the nucleotide triphosphate pool has been overlooked due to the lack of a reliable methodology. This method describes a sample processing and high performance liquid chromatography with electrochemical detection protocol for the analysis of the cellular pool of guanosine triphosphates and oxidized guanosine triphosphates. Validation of this method is demonstrated along with evaluation of these analytes in control and oxidizing conditions in vitro and in HEK 293T cells. Oxidation of this triphosphate pool occurred independently of oxidation to DNA.

BackgroundSystemic and chronic inflammatory conditions in patients with breast cancer have been associated with reduced patient survival and increased breast cancer aggressiveness. This paper characterizes the role of an inflammatory cytokine, oncostatin M (OSM), in the preintravasation aspects of breast cancer metastasis.MethodsOSM expression levels in human breast cancer tissue samples were assessed using tissue microarrays, and expression patterns based on clinical stage were assessed. To determine the in vivo role of OSM in breast cancer metastasis to the lung, we used three orthotopic breast cancer mouse models, including a syngeneic 4T1.2 mouse mammary cancer model, the MDA-MB-231 human breast cancer xenograft model, and an OSM-knockout (OSM-KO) mouse model. Progression of metastatic disease was tracked by magnetic resonance imaging and bioluminescence imaging. Endpoint analysis included circulating tumor cell (CTC) counts, lung metastatic burden analysis by qPCR, and ex vivo bioluminescence imaging.ResultsUsing tissue microarrays, we found that tumor cell OSM was expressed at the highest levels in ductal carcinoma in situ. This finding suggests that OSM may function during the earlier steps of breast cancer metastasis. In mice bearing MDA-MB-231-Luc2 xenograft tumors, peritumoral injection of recombinant human OSM not only increased metastases to the lung and decreased survival but also increased CTC numbers. To our knowledge, this is the first time that a gp130 family inflammatory cytokine has been shown to directly affect CTC numbers. Using a 4T1.2 syngeneic mouse model of breast cancer, we found that mice bearing 4T1.2-shOSM tumors with knocked down tumor expression of OSM had reduced CTCs, decreased lung metastatic burden, and increased survival compared with mice bearing control tumors. CTC numbers were further reduced in OSM-KO mice bearing the same tumors, demonstrating the importance of both paracrine- and autocrine-produced OSM in this process. In vitro studies further supported the hypothesis that OSM promotes preintravasation aspects of cancer metastasis, because OSM induced both 4T1.2 tumor cell detachment and migration.ConclusionsCollectively, our findings suggest that OSM plays a crucial role in the early steps of metastatic breast cancer progression, resulting in increased CTCs and lung metastases as well as reduced survival. Therefore, early therapeutic inhibition of OSM in patients with breast cancer may prevent breast cancer metastasis.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-0971-5) contains supplementary material, which is available to authorized users.

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-011-0811-6) contains supplementary material, which is available to authorized users.

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Onset of Parkinson's disease (PD) and Parkinson-like syndromes has been associated with exposure to diverse environmental stimuli. Epidemiological studies have demonstrated that exposure to elevated levels of manganese produces neuropathological changes localized to the basal ganglia, including neuronal loss and depletions in striatal dopamine content. However, understanding the mechanisms associated with manganese neurotoxicity has been hampered by the lack of a good rodent model. Elevated levels of 8-hydroxy-2′-deoxyguanosine (oxo 8 dG) have been found in brain areas affected in PD. Whether increased DNA damage is responsible for neuronal degeneration or is a mere epiphenomena of neuronal loss remains to be elucidated. Thus, by using mice deficient in the ability to remove oxo 8 dG we aimed to determine if dysregulation of DNA repair coupled to manganese exposure would be detrimental to dopaminergic neurons. Wild-type and OGG1 knockout mice were exposed to manganese from conception to postnatal day 30; in both groups, exposure to manganese led to alterations in the neurochemistry of the nigrostriatal system. After exposure, dopamine levels were elevated in the caudate of wild-type mice. Dopamine was reduced in the caudate of OGG1 knockout mice, a loss that was paralleled by an increase in the dopamine index of turnover. In addition, the reduction of dopamine in caudate putamen correlated with the accumulation of oxo 8 dG in midbrain. We conclude that OGG1 function is essential in maintaining neuronal stability during development and identify DNA damage as a common pathway in neuronal loss after a toxicological challenge.