OXIDATIVE STRESS AND THE GUANOSINE NUCLEOTIDE TRIPHOSPHATE POOL: IMPLICATIONS FOR A BIOMARKER AND MECHANISM OF IMPAIRED CELL FUNCTION

Oxidation of the guanosine (G) moiety, yielding the oxidized lesion 8-hydroxy-2\u27-deoxyguanosine (oxo82dG), in DNA has become a hallmark biomarker in assessing cellular outcomes induced by oxidative stress. It is well established that the guanosine nucleotide triphosphate pool is also susceptible to oxidative stress and suggested to be more available for oxidation than DNA due to the lack of protective histones and robust repair mechanisms for reducing the levels of all the products of oxidation. The oxidation of guanosine in the nucleotide triphosphate pool, resulting in oxidized guanosine 5\u27-triphoshate (oxo8GTP), has been overlooked due to the lack of a reliable method. Oxo8GTP has been shown to precede oxidation to G incorporated into DNA and modulate cell processes such as G-protein signaling and RNA synthesis. Evidence is presented in this study of a reliable method to quantify oxo8GTP, a proposed mechanism for the oxidative modification of GTP in the presence of copper and L-ascorbic acid, and evidence of oxo8GTP as an inhibitor of soluble guanylyl cyclase (sGC). A significant induction of oxo8GTP in cell-free preparations as well as in PC12 and HEK 293T cells exposed to physiologically relevant oxidative conditions generated with 10 ?M copper sulphate and 1mM L-Ascorbic Acid (Cu/Asc) is also reported. Exposure to oxidative conditions by Cu/As leads to elevations in oxo8GTP significant enough to result in a reduction of the sGC product, cyclic guanosine monophosphate (cGMP), by as much as half in pure sGC and PC12 cells. GTP is protected from oxidation in the presence of reduced glutathione and this subsequently rescues sGC activity. This suggests that oxo8GTP is produced by free radicals in vivo and can significantly impact neuronal cell functions regulated by sGC activity in the central nervous system such as synaptic plasticity. Alterations in copper homeostasis and oxidative stress have been implicated in several neurodegenerative disorders including Alzheimer\u27s and Parkinson\u27s diseases as well as Amyotrophic Lateral Sclerosis. Based upon evaluation of the data presented herein, we hypothesize that neuronal deficiencies in such disorders might be due to oxidation of the GTP pool and the ensuing effects on neuronal function

Novel Mouse Mammary Cell Lines for \u3cem\u3ein vivo\u3c/em\u3e Bioluminescence Imaging (BLI) of Bone Metastasis

Background Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model. Results The 4T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4T1.2 luc3 cells but higher than 4T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4T1.2 luc3 cells in vivo in the bone microenvironment was also detected. Conclusions The engineered 4T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone

Oncostatin M Promotes Mammary Tumor Metastasis to Bone and Osteolytic Bone Degradation

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

Novel mouse mammary cell lines for in vivo bioluminescence imaging (BLI) of bone metastasis

Abstract Background Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model. Results The 4 T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4 T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4 T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4 T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4 T1.2 luc3 cells but higher than 4 T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4 T1.2 luc3 cells in vivo in the bone microenvironment was also detected. Conclusions The engineered 4 T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone.</p

Why “intergenerational feminist media studies”?

Feminism and generation are live and ideologically freighted issues that are subject to a substantial amount of media engagement. The figure of the millennial and the baby boomer, for example, regularly circulate in mainstream media, often accompanied by hyperbolic and vitriolic discourses and affects of intergenerational feminist conflict. In addition, theories of feminist generation and waves have been and continue to be extensively critiqued within feminist theory. Given the compelling criticisms directed at these categories, we ask: why bother examining and foregrounding issues of generation, intergeneration, and transgeneration in feminist media studies? Whilst remaining sceptical of linearity and familial metaphors and of repeating reductive, heteronormative, and racist versions of feminist movements, we believe that the concept of generation does have critical purchase for feminist media scholars. Indeed, precisely because of the problematic ways that is it used, and the prevalence of it as a volatile, yet only too palpable, organizing category, generation is both in need of continual critical analysis, and is an important tool to be used—with care and nuance—when examining the multiple routes through which power functions in order to marginalize, reward, and oppress. Exploring both diachronic and synchronic understandings of generation, this article emphasizes the use of conjunctural analysis to excavate the specific historical conditions that impact upon and create generation. This special issue of Feminist Media Studies covers a range of media forms—film, games, digital media, television, print media, as well as practices of media production, intervention, and representation. The articles also explore how figures at particular lifestages—particularly the girl and the aging woman—are constructed relationally, and circulate, within media, with particular attention to sexuality. Throughout the issue there is an emphasis on exploring the ways in which the category of generation is mobilized in order to gloss sexism, racism, ageism, class oppression, and the effects of neoliberalism

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

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