Wild-type Mouse Embryo Fibroblasts Research Articles

Abstract A033: The ING5 epigenetic reader regulates a subset of DNA repair genes to maintain genomic integrity

Abstract The ING family of epigenetic histone code readers (ING1-5) recognize the H3K4Me3 epigenetic mark to target histone acetyltransferase (HAT) or histone deacetylase (HDAC) complexes to regulate local acetylation levels and affect transcription. We and others have shown that ING5 promotes stem cell character in normal and in cancer stem cells and that loss of ING5 promotes stem cell differentiation. Examination of an ING5 knockout (KO) mouse model showed increased levels of DNA damage in different tissues, and particularly in the male germ line. ING5 mouse embryo fibroblasts (MEFs) also show a >2-fold increase in cells containing gH2AX foci in the absence of exogenous DNA damaging agents. Examining the transcriptomes of ING5 KO and wild-type littermate MEFs indicates that several genes encoding key DNA repair proteins such as those acting in homologous recombination (BRCA1 and BRCA2) and base excision repair (PARP1) are repressed several-fold and ING5 KO MEFs show increased sensitivity to the PARP inhibitor olaparib. Given that ING5 functions to help maintain stem cell character in normal and in cancer stem cells, we propose that ING5 serves a role in maintaining a high level of DNA repair capability in stem cell populations to preserve genomic integrity. Citation Format: Arthur E Dantas, Buthaina Al-Shueili, Mahbod Djamshidi, Karl Riabowol. The ING5 epigenetic reader regulates a subset of DNA repair genes to maintain genomic integrity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A033.

Dammarane-type saponins from Gynostemma pentaphyllum and their anti-aging activities via up-regulating mitochondria related proteins

The importance of mitochondria in regulation of aging has been extensively recognized and confirmed. Gynostemma pentaphyllum (Thunb.) Makino, a homology of medicine and food, has been widely utilized as dietary supplement. In this study, the transcriptome of normal cells (wild type mouse embryo fibroblasts) regulated by the 30% aqueous EtOH extract of G. pentaphyllum was firstly evaluated by RNA sequencing and the results revealed that the G. pentaphyllum could up-regulate the genes involved in oxidative phosphorylation (OXPHOS) and sirtuin (SIRT) signaling pathways, indicating its effect in promoting cell viability might be attributed to the role of improving mitochondrial functions. To further discover the bioactive compounds, sixteen undescribed dammarane-type saponins along with twenty-eight known analogues were isolated from the active extract of G. pentaphyllum. Their structures were elucidated by means of comprehensive analysis of NMR and HRMS spectroscopic data. All isolates were evaluated for the regulatory effects on SIRT3 and translocase of the outer membrane 20 (TOM20), and thirteen of them exhibited satisfactory agonist activities on both SIRT3 and TOM20 at 5 μM. Furthermore, the preliminary structure-activity relationships analysis demonstrated the additional hydroxymethyl and carbonyl groups or less sugar residues in saponins could contribute positively to the up-regulatory effect on SIRT3 and TOM20. These findings encouraged the potential roles of G. pentaphyllum and its bioactive saponins in the development of natural drugs for the treatment of aging-related diseases.

Caspase-8 is required for HSV-1-induced apoptosis and promotes effective viral particle release via autophagy inhibition

Regulated cell death (RCD) plays an important role in the progression of viral replication and particle release in cells infected by herpes simplex virus-1 (HSV-1). However, the kind of RCD (apoptosis, necroptosis, others) and the resulting cytopathic effect of HSV-1 depends on the cell type and the species. In this study, we further investigated the molecular mechanisms of apoptosis induced by HSV-1. Although a role of caspase-8 has previously been suggested, we now clearly show that caspase-8 is required for HSV-1-induced apoptosis in a FADD-/death receptor-independent manner in both mouse embryo fibroblasts (MEF) and human monocytes (U937). While wild-type (wt) MEFs and U937 cells exhibited increased caspase-8 and caspase-3 activation and apoptosis after HSV-1 infection, respective caspase-8-deficient (caspase-8−/−) cells were largely impeded in any of these effects. Unexpectedly, caspase-8−/− MEF and U937 cells also showed less virus particle release associated with increased autophagy as evidenced by higher Beclin-1 and lower p62/SQSTM1 levels and increased LC3-I to LC3-II conversion. Confocal and electron microscopy revealed that HSV-1 stimulated a strong perinuclear multivesicular body response, resembling increased autophagy in caspase-8−/− cells, entrapping virions in cellular endosomes. Pharmacological inhibition of autophagy by wortmannin restored the ability of caspase-8−/− cells to release viral particles in similar amounts as in wt cells. Altogether our results support a non-canonical role of caspase-8 in both HSV-1-induced apoptosis and viral particle release through autophagic regulation.

Procollagen C-proteinase enhancer 1 promotes physiologic retinal angiogenesis via regulating the process of collagen.

To investigate the role of procollagen C-proteinase enhancer 1 (PCPE1) in retinal angiogenesis and relevant mechanisms. The Pcolce1-knockout (KO) mice were used to explore the effect of PCPE1 on retinal angiogenesis in vivo. Pcolce1 siRNA were designed, cell count kit 8 (CCK8) assays and tube formation assays were performed to investigate the cell proliferation and tube formation abilities of retinal microvascular endothelial cells (hRMECs) in vitro. Mouse embryo fibroblasts (MEF) cells were isolated and cultured to analyze the effect of PCPE1 on enhancing procollagen cleavage. In vivo studies showed that the retinal vascular density of Pcolce1-/- mice was significantly lower than that of the control group. Furthermore, silencing of Pcolce1 inhibited cell proliferation and tube formation abilities of hRMECs in vitro. Additionally, much more pro-collagen was found in Pcolce1-/- MEF cells, compared to wild type MEF cells. PCPE1 may promote physiological retinal angiogenesis by regulating the processing of collagen, which may provide a potential therapeutic target of retinal vascular disease.

Calreticulin exploits TGF-β for extracellular matrix induction engineering a tissue regenerative process.

Topical application of extracellular calreticulin (eCRT), an ER chaperone protein, in animal models enhances wound healing and induces tissue regeneration evidenced by epidermal appendage neogenesis and lack of scarring. In addition to chemoattraction of cells critical to the wound healing process, eCRT induces abundant neo-dermal extracellular matrix (ECM) formation by 3days post-wounding. The purpose of this study was to determine the mechanisms involved in eCRT induction of ECM. In vitro, eCRT strongly induces collagen I, fibronectin, elastin, α-smooth muscle actin in human adult dermal (HDFs) and neonatal fibroblasts (HFFs) mainly via TGF-β canonical signaling and Smad2/3 activation; RAP, an inhibitor of LRP1 blocked eCRT ECM induction. Conversely, eCRT induction of α5 and β1 integrins was not mediated by TGF-β signaling nor inhibited by RAP. Whereas eCRT strongly induces ECM and integrin α5 proteins in K41 wild-type mouse embryo fibroblasts (MEFs), CRT null MEFs were unresponsive. The data show that eCRT induces the synthesis and release of TGF-β3 first via LRP1 or other receptor signaling and later induces ECM proteins via LRP1 signaling subsequently initiating TGF-β receptor signaling for intracellular CRT (iCRT)-dependent induction of TGF-β1 and ECM proteins. In addition, TGF-β1 induces 2-3-fold higher level of ECM proteins than eCRT. Whereas eCRT and iCRT converge for ECM induction, we propose that eCRT attenuates TGF-β-mediated fibrosis/scarring to achieve tissue regeneration.

ER/Golgi trafficking is facilitated by unbranched actin filaments containing Tpm4.2.

We have identified novel actin filaments defined by tropomyosin Tpm4.2 at the ER. EM analysis of mouse embryo fibroblasts (MEFs) isolated from mice expressing a mutant Tpm4.2 (Tpm4Plt53/Plt53 ), incapable of incorporating into actin filaments, revealed swollen ER structures compared with wild-type (WT) MEFs (Tpm4+/+ ). ER-to-Golgi, but not Golgi-to-ER trafficking was altered in the Tpm4Plt53/Plt53 MEFs following the transfection of the temperature sensitive ER-associated ts045-VSVg construct. Exogenous Tpm4.2 was able to rescue the ER-to-Golgi trafficking defect in the Tpm4Plt53/Plt53 cells. The treatment of WT MEFs with the myosin II inhibitor, blebbistatin, blocked the Tpm4.2-dependent ER-to-Golgi trafficking. The lack of an effect on ER-to-Golgi trafficking following treatment of MEFs with CK666 indicates that branched Arp2/3-containing actin filaments are not involved in anterograde vesicle trafficking. We propose that unbranched, Tpm4.2-containing filaments have an important role in maintaining ER/Golgi structure and that these structures, in conjunction with myosin II motors, mediate ER-to-Golgi trafficking.

Diacylglycerol kinase epsilon suppresses expression of p53 and glycerol kinase in mouse embryo fibroblasts

The incorporation of glycerol into lipid was measured using SV40 transformed mouse embryo fibroblasts (MEFs) from either wild-type (WT) mice or from mice in which the epsilon isoform of diacylglycerol kinase (DGKε) was knocked out (DGKε−/−). We present an explanation for our finding that DGKε−/− MEFs exhibited greater uptake of 3H-glycerol into the cell and a greater incorporation into lipids compared with their WT counterparts, with no change in the relative amounts of various lipids between the DGKε−/− and WT MEFs. Glycerol kinase is more highly expressed in the DGKε−/− cells than in their WT counterparts. In addition, the activity of glycerol kinase is greater in the DGKε−/− cells than in their WT counterparts. Other substrates that enter the cell independent of glycerol kinase, such as pyruvate or acetate, are incorporated into lipid to the same extent between DGKε−/− and WT cell lines. We also show that expression of p53, a transcription factor that increases the synthesis of glycerol kinase, is increased in DGKε−/− MEFs in comparison to WT cells. We conclude that the increased incorporation of glycerol into lipids in DGKε−/− cells is a consequence of up-regulation of glycerol kinase and not a result of an increase in the rate of lipid synthesis. Furthermore, increased expression of the pro-survival gene, p53, in cells knocked out for DGKε suggests that cells over-expressing DGKε would have a greater propensity to become tumorigenic.

Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study

Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling.

Accumulation of RNA-dependent protein kinase (PKR) in the nuclei of lung cancer cells mediates radiation resistance.

We have previously demonstrated that radiation induced cell death in PKR (−/−) deficient mouse embryo fibroblasts (MEFs) but not in PKR (+/+) wild type MEFs. Our study indicated that PKR can also be involved in survival pathways following radiation therapy through activation of the AKT survival pathways in these MEFs is mediated in part through PKR. The role of PKR on radiation sensitivity in cancer cells has not been evaluated. In this study, we demonstrated that radiation treatment causes nuclear translocation of PKR in human lung cancer cells. The transduction of lung cancer cells with a dominant negative adenoviral PKR vector blocks nuclear translocation of PKR and leads to the reversal of radiation resistance. Plasmid transduction of lung cancer cells with nuclear targeted wild type PKR vectors also increased radiation resistance. This effect is selectively abrogated by plasmid transduction of dominant negative PKR vectors which restore radiation sensitivity. These findings suggest a novel role for PKR in lung cancer cells as a mediator of radiation resistance possibly through translocation of the protein product to the nucleus.

Gadd45b deficiency promotes premature senescence and skin aging.

The GADD45 family of proteins functions as stress sensors in response to various physiological and environmental stressors. Here we show that primary mouse embryo fibroblasts (MEFs) from Gadd45b null mice proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. The impaired proliferation and increased senescence in Gadd45b null MEFs is partially reversed by culturing at physiological oxygen levels, indicating that Gadd45b deficiency leads to decreased ability to cope with oxidative stress. Interestingly, Gadd45b null MEFs arrest at the G2/M phase of cell cycle, in contrast to other senescent MEFs, which arrest at G1. FACS analysis of phospho-histone H3 staining showed that Gadd45b null MEFs are arrested in G2 phase rather than M phase. H2O2 and UV irradiation, known to increase oxidative stress, also triggered increased senescence in Gadd45b null MEFs compared to wild type MEFs. In vivo evidence for increased senescence in Gadd45b null mice includes the observation that embryos from Gadd45b null mice exhibit increased senescence staining compared to wild type embryos. Furthermore, it is shown that Gadd45b deficiency promotes senescence and aging phenotypes in mouse skin. Together, these results highlight a novel role for Gadd45b in stress-induced senescence and in tissue aging.

Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels

The transcription factor far upstream element binding protein (FBP) binds and activates the MYC promoter when far upstream element is via TFIIH helicase activity early in the transcription cycle. The fundamental biology and pathology of FBP are complex. In some tumors FBP seems pro-oncogenic, whereas in others it is a tumor suppressor. We generated an FBP knockout (Fubp1(-/-)) mouse to study FBP deficiency. FBP is embryo lethal from embryonic day 10.5 to birth. A spectrum of pathology is associated with FBP loss; besides cerebral hyperplasia and pulmonary hypoplasia, pale livers, hypoplastic spleen, thymus, and bone marrow, cardiac hypertrophy, placental distress, and small size were all indicative of anemia. Immunophenotyping of hematopoietic cells in wild-type versus knockout livers revealed irregular trilineage anemia, with deficits in colony formation. Despite normal numbers of hematopoietic stem cells, transplantation of Fubp1(-/-) hematopoietic stem cells into irradiated mice entirely failed to reconstitute hematopoiesis. In competitive transplantation assays against wild-type donor bone marrow, Fubp1(-/-) hematopoietic stem cells functioned only sporadically at a low level. Although cultures of wild-type mouse embryo fibroblasts set Myc levels precisely, Myc levels of mouse varied wildly between fibroblasts harvested from different Fubp1(-/-) embryos, suggesting that FBP contributes to Myc set point fixation. FBP helps to hold multiple physiologic processes to close tolerances, at least in part by constraining Myc expression.

P19(Arf) is required for the cellular response to chronic DNA damage.

The p53 tumor suppressor is a stress sensor, driving cell-cycle arrest or apoptosis in response to DNA damage or oncogenic signals. p53 activation by oncogenic signals relies on the p19Arf tumor suppressor, while p53 activation downstream of acute DNA damage is reported to be p19Arf-independent. Accordingly, p19Arf-deficient mouse embryo fibroblasts (MEFs) arrest in response to acute DNA damage. However, p19Arf is required for replicative senescence, a condition associated with an activated DNA damage response, as p19Arf−/− MEFs do not senesce after serial passage. A possible explanation for these seemingly disparate roles for p19Arf is that acute and chronic DNA damage responses are mechanistically distinct. Replicative senescence may result from chronic, low-dose DNA damage responses in which p19Arf has a specific role. We therefore examined the role of p19Arf in cellular responses to chronic, low-dose DNA damaging agent treatment by maintaining MEFs in low oxygen and administering 0.5 Gy γ-irradiation daily or 150μM hydroxyurea, a replication stress-inducer. In contrast to their response to acute DNA damage, p19Arf−/− MEFs exposed to chronic DNA damage do not senesce, revealing a selective role for p19Arf in senescence upon low-level, chronic DNA damage. We show further that p53 pathway activation in p19Arf−/− MEFs exposed to chronic DNA damage is attenuated relative to wild-type MEFs, suggesting a role for p19Arf in fine-tuning p53 activity. However, combined Nutlin3a and chronic DNA damaging agent treatment is insufficient to promote senescence in p19Arf−/− MEFs, suggesting that the role of p19Arf in the chronic DNA damage response may be partially p53-independent. These data suggest the importance of p19Arf for the cellular response to the low-level DNA damage incurred in culture or upon oncogene expression, providing new insight into how p19Arf serves as a tumor suppressor. Moreover, our study helps reconcile reports suggesting crucial roles for both p19Arf and DNA damage signaling pathways in tumor suppression.

A central role of TRAX in the ATM-mediated DNA repair.

DNA repair is critical for the maintenance of genome stability. Upon genotoxic stress, dysregulated DNA repair may induce apoptosis. Translin-associated factor X (TRAX), which was initially identified as a binding partner of Translin, has been implicated in genome stability. However, the exact role of TRAX in DNA repair remains largely unknown. Here, we showed that TRAX participates in the ATM/H2AX-mediated DNA repair machinery by interacting with ATM and stabilizing the MRN complex at double-strand breaks. The exogenous expression of wild-type (WT) TRAX, but not a TRAX variant lacking the nuclear localization signal (NLS), rescued the vulnerability of TRAX-null mouse embryo fibroblasts (MEFs). This finding confirms the importance of the nuclear localization of TRAX in the repair of DNA damage. Compared with WT MEFs, TRAX-null MEFs exhibited impaired DNA repair (for example, reduced phosphorylation of ATM and H2AX) after treatment with ultra violet-C or γ-ray irradiation and a higher incidence of p53-mediated apoptosis. Our findings demonstrate that TRAX is required for MRN complex-ATM-H2AX signaling, which optimizes DNA repair by interacting with the activated ATM and protects cells from genotoxic stress-induced apoptosis.

Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor-stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

AMPKα1 Deficiency in Fibroblasts Promotes Angiogenesis in Athymic Nude Mice

Angiogenesis is required and essential for tumor development. Increasing evidence suggests that adenosine monophosphate‐activated protein kinase (AMPK), an energy sensor and modulator of redox system, is associated with cancer development. However, the effect of AMPK on tumor development is controversial. Mouse embryo fibroblasts (MEF) were isolated from AMPKα1 knock out (AMPKα1‐KO), AMPKα2‐KO, and wild type (WT) C57BL/6J mouse embryos, and immortalized by using standard 3T3 protocol to investigate the mechanisms of fibroblast‐mediated angiogenesis. Xenograft model data revealed that knock out of AMPKα1, but not AMPKα2, stimulated the cellular proliferation of immortalized MEFs, angiogenesis, and tumor development in athymic nude mice in vivo. Consistent with this finding, the migration of human microvascular endothelial cells (hmvEC) was enhanced by co‐culturing with AMPKα1‐KO MEFs when compared to either WT or AMPKα2‐KO MEFs. Moreover, the network formation of hmvEC was stimulated by conditioned medium from AMPKα1‐KO MEFs, but not from WT or AMPKα2‐KO MEFs. Mechanistically, we found that the protein level of noncanonical nuclear factor kappa B2 (NF‐ĸB2)/p52 was elevated in AMPKα1‐KO MEFs and was responsible for induction of erythropoietin (Epo) expression. Finally, the most conclusive evidence for AMPKα1‐dependent inhibition of EC migration and consequent angiogenesis as well as tumor progression was that the xenograft growth of the inoculated AMPKα1‐KO MEFs was ameliorated by treatment with Epo neutralization antibody. This compelling evidence indicates that AMPKα1, rather than AMPKα2 deletion in fibroblasts, instigates EC migration, angiogenesis, and the resultant tumor development.

Paraoxonase 2 Serves a Proapopotic Function in Mouse and Human Cells in Response to the Pseudomonas aeruginosa Quorum-sensing Molecule N-(3-Oxododecanoyl)-homoserine Lactone

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (Δψmito) depolarized; Ca(2+) was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca(2+)] (Cacyto); and caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: Δψmito depolarized, Cacyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψmito, Ca(2+) release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12.

Abstract 4905: Targeting NPM1 for the radiosensitization of NSCLC

Abstract Lung cancer is a global scourge. In 2008 alone there were more than 1.25 million deaths from this disease worldwide. Eighty five percent of lung cancers are classified as non-small cell lung cancer (NSCLC) and for those NSCLC patients who present with locally advanced stage IIIA/B disease the standard of care is chemo/radiation therapy. Yet even with this approach the median duration of survival is only 17 months. Thus, there is an urgent need for the development of new strategies that improve therapeutic outcomes. We addressed this challenge by employing a forward chemical genetics screen designed to identify compounds that could be used to exploit DNA repair pathways in tumor cells burdened with oncogenesis-mediated replication stress. Using this approach we identified YTR107, a substituted 5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H) trione, that amplifies replication stress and inhibits repair of DNA DSBs. Using biochemical approaches we identified NPM1 as a potential target and found that YTR107 impairs NPM1 oligomerization, a requirement for NPM1's biochemical activity. However, direct genetic evidence demonstrating that the specific targeting of NPM1 is a radiosensitizing event remains to be established. Therefore we employed a defined genetic model consisting of NPM1 null and wild type mouse embryo fibroblasts (MEFs). We found that proliferating NPM1-null mouse embryo fibroblasts (MEFs), but not wild type MEFs, are burdened with high levels of γH2AX foci, a characteristic of replication stress. NPM1-null MEFs exhibit a defect in Rad51-mediated repair of DNA DSBs that increases radiation sensitivity. Use of this model allowed us to demonstrate that YTR107-mediated radiosensitization requires NPM1. Because the DNA damage response pathway is continuously activated in NSCLC in response to constant replication stress, and with the knowledge that NPM1 expression minimizes such stress, we hypothesized that NSCLC tumors might require significant expression of NPM1. This hypothesis was validated by analyzing a NSCLC tumor microarray. We then showed that YTR107-mediated targeting of NPM1 is a radiosensitizing event in 6 NSCLC cell lines and in a syngeneic tumor model. We also found that YTR107 is well tolerated in mice, does not produce overt toxicity, and does not potentiate radiation-mediated pulmonary fibrosis. These data suggest that NPM1 represents a potential molecular target for radiosensitization of NSCLC. Citation Format: Konjeti R. Sekhar, Mouadh Benamar, Amudhan Venkateswaran, Narsimha R. Penthala, Peter A. Crooks, Stephen R. Hann, Ling Geng, Ramesh Balusu, Tarek Abbas, Michael L. Freeman. Targeting NPM1 for the radiosensitization of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4905. doi:10.1158/1538-7445.AM2014-4905

Herpes simplex virus 2 ICP34.5 confers neurovirulence by regulating the type I interferon response

The γ34.5 gene of herpes simplex virus (HSV) 2 encodes ICP34.5, which enhances HSV-2 neurovirulence by an unknown mechanism. We found that an HSV-2 γ34.5-null mutant (γ34.5−/−) replicated less robustly than its rescue virus (γ34.5R) in wild-type mouse embryo fibroblasts (MEFs), and in cells primed with IFNβ. Increased eIF2α phosphorylation correlated with γ34.5−/− attenuation. However, γ34.5−/− achieved titers equivalent to γ34.5R in MEFs lacking the type I IFN receptor (IFNα/βR−/−) or lacking protein kinase R. γ34.5−/− also replicated poorly in the vaginal mucosa of wild-type mice, caused little genital inflammation, and spread to the nervous system at lower levels compared to γ34.5R. In IFNα/βR−/− mice, however, γ34.5−/− regained the capacity to replicate and cause disease equivalent to γ34.5R after intravaginal infection or direct inoculation into the central nervous system. Thus, the capacity of HSV-2 ICP34.5 to interdict the type I IFN response in vivo largely determines its neurovirulence.

Contrasting the incorporation of glycerol into lipids caused by the presence of two isoforms of diacylglycerol kinase (605.20)

The mammalian isoforms of diacylglycerol kinase (DGK) include the epsilon isoform (DGKε) and the delta isoform (DGKδ). DGKε has specificity for substrates with an arachidonoyl group, while DGKδ shows no acyl chain specificity for the substrate, but it promotes fatty acid synthesis resulting in an increased fraction of lipids with shorter and less unsaturated acyl chains. We have compared the effects of knocking out each of these isoforms individually using SV40 transformed mouse embryo fibroblasts (MEFs). We compared wild‐type (WT) MEFs with those from knock‐out (KO) mice. We find that DGKδ‐KO MEFs have lower incorporation of 3H‐glycerol into lipids compared with their WT counterparts. This is consistent with our recent demonstration (Biochemistry (2013) 52, 7766) that the depletion of this DGK isoform results in less fatty acid synthesis. This change is not caused by a decreased expression of glycerol kinase in the KO cells. In contrast, the DGKε‐KO MEFs show greater incorporation of 3H‐glycerol into lipids compared with their WT counterparts, with no change in the relative amounts of various lipids between the DGKε‐KO and WT MEFs. This result is explained by our observation that glycerol kinase is more highly expressed in the DGKε‐KO cells than in their WT counterparts.

P seudomonas aeruginosa homoserine lactone triggers apoptosis and B ak/ B ax‐independent release of mitochondrial cytochrome C in fibroblasts

Pseudomonas aeruginosa use N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12-triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild-type (WT) and Bax(-/-) /Bak(-/-) (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψmito ), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax-dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψmito and release of cytochrome C and cell death. Re-expression of Bax or Bak in DKO MEF did not alter the WT-like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax-independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψmito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.