Elucidating cellular response to treatment with viral immunotherapies in pediatric high-grade glioma and medulloblastoma.

HSV G207, a double-stranded, DNA virus, and the polio:rhinovirus chimera, PVSRIPO, a single positive-strand RNA virus, are viral immunotherapies being used to treat pediatric malignant brain tumors in clinical trials. The purpose of this work is to elucidate general response patterns and putative biomarkers of response. Multiple pediatric high-grade glioma and medulloblastoma cell lines were treated with various multiplicities of infection of G207 or PVSRIPO. There was a significant inverse correlation between expression of one HSV cellular receptor, CD111, and the lethal dose of 50% of cells (LD50) of cells treated with G207 (r = -0.985, P<0.001) but no correlation between PVSRIPO cellular receptor expression (CD155) and LD50. RNA sequencing of control cells and cells treated for 8 and 24 h revealed that there were few shared differentially expressed (DE) genes between cells treated with PVSRIPO and G207: GCLM, LANCL2, and RBM3 were enriched whilst ADAMTS1 and VEGFA were depleted. Likewise, there were few shared DE genes enriched between medulloblastoma and high-grade glioma cell lines treated with G207: GPSM2, CHECK2, SEPTIN2, EIF4G2, GCLM, GDAP1, LANCL2, and PWP1.  Treatment with G207 and PVSRIPO appear to cause disparate gene enrichment and depletion suggesting disparate molecular mechanisms in malignant pediatric brain tumors.

ABL1 and ABL2 promote medulloblastoma leptomeningeal dissemination.

Background: Medulloblastoma is the most common malignant pediatric brain tumor, and leptomeningeal dissemination (LMD) of medulloblastoma both portends a poorer prognosis at diagnosis and is incurable at recurrence. The biological mechanisms underlying LMD are unclear. The Abelson (ABL) tyrosine kinase family members, ABL1 and ABL2, have been implicated in cancer cell migration, invasion, adhesion, metastasis, and chemotherapy resistance, and are upstream mediators of the oncogene c-MYC in fibroblasts and lung cancer cells. However, their role in medulloblastoma has not yet been explored. The purpose of this work was to elucidate the role of ABL1/2 in medulloblastoma LMD. Methods: ABL1 and ABL2 mRNA expression of patient specimens was analyzed. shRNA knockdowns of ABL1/2 and pharmacologic inhibition of ABL1/2 were used for in vitro and in vivo analyses of medulloblastoma LMD. RNA sequencing of ABL1/2 genetic knockdown versus scrambled control medulloblastoma was completed. Results: ABL1/2 mRNA is highly expressed in human medulloblastoma and pharmacologic inhibition of ABL kinases resulted in cytotoxicity. Knockdown of ABL1/2 resulted in decreased adhesion of medulloblastoma cells to the extracellular matrix protein, vitronectin (P = .0013), and significantly decreased tumor burden in a mouse model of medulloblastoma LMD with improved overall survival (P = .0044). Furthermore, both pharmacologic inhibition of ABL1/2 and ABL1/2 knockdown resulted in decreased expression of c-MYC, identifying a putative signaling pathway, and genes/pathways related to oncogenesis and neurodevelopment were differentially expressed between ABL1/2 knockdown and control medulloblastoma cells. Conclusions: ABL1 and ABL2 have potential roles in medulloblastoma LMD upstream of c-MYC expression.

Structures of maintenance of carboxysome distribution Walker-box McdA and McdB adaptor homologs.

Carboxysomes, protein-coated organelles in cyanobacteria, are important in global carbon fixation. However, these organelles are present at low copy in each cell and hence must be segregated to ensure transmission from one generation to the next. Recent studies revealed that a DNA partition-like ParA-ParB system mediates carboxysome maintenance, called McdA-McdB. Here, we describe the first McdA and McdB homolog structures. McdA is similar to partition ParA Walker-box proteins, but lacks the P-loop signature lysine involved in ATP binding. Strikingly, a McdA-ATP structure shows that a lysine distant from the P-loop and conserved in McdA homologs, enables ATP-dependent nucleotide sandwich dimer formation. Similar to partition ParA proteins this ATP-bound form binds nonspecific-DNA. McdB, which we show directly binds McdA, harbors a unique fold and appears to form higher-order oligomers like partition ParB proteins. Thus, our data reveal a new signature motif that enables McdA dimer formation and indicates that, similar to DNA segregation, carboxysome maintenance systems employ Walker-box proteins as DNA-binding motors while McdB proteins form higher order oligomers, which could function as adaptors to link carboxysomes and provide for stable transport by the McdA proteins.

Influence of POLG on Radiosensitivity of Nasopharyngeal Carcinoma Cells.

BACKGROUND AND OBJECTIVE: There is a high incidence of nasopharyngeal carcinoma (NPC), malignant head and neck tumors, in southern China. Radioresistance is the main cause affecting the efficacy of NPC treatments. The POLG gene particularly plays an important role in radiation-induced damage repair. In this study, the authors established RNAi CNE-1 and CNE-2 knockdown in two NPC cell lines to observe whether this gene affects the radiosensitivity of NPC cells. MATERIALS AND METHODS: Four short hairpin RNA (shRNA) expression plasmids targeting POLG gene were constructed and transfected into the NPC cell lines CNE-1 and CNE-2. Screening was performed to evaluate the stable expression of cloned cells, which were named CNE-1/POLG-shRNA1, CNE-1/POLG-shRNA2, CNE-2/POLG-shRNA1, and CNE-2/POLG-shRNA2. The negative controls CNE-1/Neg-shRNA and CNE-2/Neg-shRNA were additionally used. The MTT method, flow cytometry, clone formation analysis, cell migration, and other experimental methods were employed to verify changes in the radiosensitivity of the NPC cells. RESULTS: Fluorescent quantitative PCR and Western blot confirmed the downregulation of the PLOG gene through diminished PLOG messenger RNA and protein levels. Consequently, the authors report the stable knockdown of the POLG gene in an NPC model. Dose-dependent radiation exposure of POLG inhibited NPC cell growth and increased apoptosis compared with control cells (p < 0.01), as demonstrated through colony formation assay and flow cytometry. Functional assays indicated that knockdown of the POLG in CNE-1 and CNE-2 cells remarkably reduced cell viability and proliferation. Specifically, POLG knockdown led to G1 phase arrest and apoptosis. CONCLUSIONS: Overall, the authors conclude that POLG downregulation alters the radiosensitivity of NPC cells, indicating that the gene is likely involved in conferring the radiation response of the cells. In addition, findings in this study suggest a novel role for POLG as a potential predictive marker for NPC radiotherapy efficiency. POLG gene can be used as a potential clinical target to effectively improve the radiosensitivity of NPC.

Structures of partition protein ParA with nonspecific DNA and ParB effector reveal molecular insights into principles governing Walker-box DNA segregation.

Walker-box partition systems are ubiquitous in nature and mediate the segregation of bacterial and archaeal DNA. Well-studied plasmid Walker-box partition modules require ParA, centromere-DNA, and a centromere-binding protein, ParB. In these systems, ParA-ATP binds nucleoid DNA and uses it as a substratum to deliver ParB-attached cargo DNA, and ParB drives ParA dynamics, allowing ParA progression along the nucleoid. How ParA-ATP binds nonspecific DNA and is regulated by ParB is unclear. Also under debate is whether ParA polymerizes on DNA to mediate segregation. Here we describe structures of key ParA segregation complexes. The ParA-ß,γ-imidoadenosine 5'-triphosphate (AMPPNP)-DNA structure revealed no polymers. Instead, ParA-AMPPNP dimerization creates a multifaceted DNA-binding surface, allowing it to preferentially bind high-density DNA regions (HDRs). DNA-bound ParA-AMPPNP adopts a dimer conformation distinct from the ATP sandwich dimer, optimized for DNA association. Our ParA-AMPPNP-ParB structure reveals that ParB binds at the ParA dimer interface, stabilizing the ATPase-competent ATP sandwich dimer, ultimately driving ParA DNA dissociation. Thus, the data indicate how harnessing a conformationally adaptive dimer can drive large-scale cargo movement without the requirement for polymers and suggest a segregation mechanism by which ParA-ATP dimers equilibrate to HDRs shown to be localized near cell poles of dividing chromosomes, thus mediating equipartition of attached ParB-DNA substrates.

Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.

UNLABELLED: Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans, Candida glabrata, and Aspergillus fumigatus Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo, where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts. IMPORTANCE: FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus, C. albicans, C. glabrata, and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.

Global genetic determinants of mitochondrial DNA copy number.

Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role.

Reduction of decoy receptor 3 enhances TRAIL-mediated apoptosis in pancreatic cancer.

Most human pancreatic cancer cells are resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. However, the mechanisms by which pancreatic cancer cells utilize their extracellular molecules to counteract the proapoptotic signaling mediated by the TNF family are largely unknown. In this study, we demonstrate for the first time that DcR3, a secreted decoy receptor that malignant pancreatic cancer cells express at a high level, acts as an extracellular antiapoptotic molecule by binding to TRAIL and counteracting its death-promoting function. The reduction of DcR3 with siRNA unmasked TRAIL and greatly enhanced TRAIL-induced apoptosis. Gemcitabine, a first-line drug for pancreatic cancer, also reduced the level of DcR3. The addition of DcR3 siRNA further enhanced gemcitabine-induced apoptosis. Notably, our in vivo study demonstrated that the therapeutic effect of gemcitabine could be enhanced via further reduction of DcR3, suggesting that downregulation of DcR3 in tumor cells could tip the balance of pancreatic cells towards apoptosis and potentially serve as a new strategy for pancreatic cancer therapy.

Transition pattern and mechanism of B-lymphocyte precursors in regenerated mouse bone marrow after subtotal body irradiation.

Little is known about the effects of ionizing radiation on the transition and the related signal transduction of progenitor B cells in the bone marrow. Thus, using an NIH Swiss mouse model, we explored the impact of ionizing radiation on the early stage of B-cell development via an examination of the transition of CLP to pro-B to pre-B cells within bone marrow as a function of radiation doses and times. Our results showed that while the total number of bone marrow lymphoid cells at different stages were greatly reduced by subtotal body irradiation (sub-TBI), the surviving cells continued to transition from common lymphoid progenitors to pro-B and then to pre-B in a reproducible temporal pattern. The rearrangement of the immunoglobulin heavy chain increased significantly 1-2 weeks after irradiation, but no change occurred after 3-4 weeks. The rearrangement of the immunoglobulin light chain decreased significantly 1-2 weeks after sub-TBI but increased dramatically after 3-4 weeks. In addition, several key transcription factors and signaling pathways were involved in B-precursor transitions after sub-TBI. The data indicate that week 2 after irradiation is a critical time for the transition from pro-B cells to pre-B cells, reflecting that the functional processes for different B-cell stages are well preserved even after high-dose irradiation.

B1 sequence-based real-time quantitative PCR: a sensitive method for direct measurement of mouse plasma DNA levels after gamma irradiation.

PURPOSE: Current biodosimetric techniques for determining radiation exposure have inherent delays, as well as quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA. METHODS AND MATERIALS: Real-time quantitative polymerase chain reaction (PCR) was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 h from mice receiving 0-10 Gy total- or partial-body irradiation ((137)Cs gamma-ray source at approximately 1.86 Gy/min; homogeneity: +/- 6.5%). RESULTS: The correlation coefficient between DNA levels and the threshold cycle value (C(T)) was 0.996, and the average recoveries of DNA in the assay were 87%. This assay revealed that when BALB/c mice were exposed to 10 Gy total-body irradiation (TBI), plasma DNA levels gradually increased beginning at 3 h after irradiation, peaked at 9 h, and returned to baseline within 48 h. Increased plasma DNA levels were also detected following upper-torso or lower-torso partial-body irradiation; however, TBI approximately doubled those plasma DNA levels at the same radiation dose. This technique therefore reflects total body cell damage. The advantages of this assay are that DNA extraction is not required, the assay is highly sensitive (0.002 ng), and results can be obtained within 2.5 h after collection of plasma samples. CONCLUSIONS: A radiation dose-dependent increase of plasma DNA was observed in the dose range from 2 to 10 Gy, suggesting that plasma DNA may be a useful radiation biomarker and adjunct to existing cell-based assays.

Anti-cancer effect of resveratrol is associated with induction of apoptosis via a mitochondrial pathway alignment.

Resveratrol, a phytoalexin found in the skin of grapes, is believed to have multiple bioactivities including anti-cancer, anti-carcinogenesis and antiinflammatory. The mechanisms by which resveratrol might produce these effects are not well understood. In this study, malignant human pancreatic cancer cells were treated without or with resveratrol in combination with ionizing radiation (IR), and then the mitochondrial function of treated cells was evaluated using several standardized assays. They include the Calcein AM method for mitochondria transition pore; the JC-1 staining method for mitochondria membrane potential; the CM-H2DCFDA method for reactive oxygen species; and the Annexin V/propidium iodide (PI) method for apoptosis/cell death. Our results indicated that (1) pore function was partially intact after resveratrol, but resveratrol probably interfered with the accumulation of intracellular Calcein AM; (2) depolarization of the mitochondria membrane was increased in the resveratrol treated cells, consistent with mitochondrial dysfunction; (3) ROS was slightly increased with resveratrol, a phenomenon that was greatly increased when this agent was combined with IR; and (4) in parallel with the above changes in mitochondrial and drug transport, cells treated with resveratrol showed increased apoptosis as measured by Annexin V/PI staining. In summary, the anti-cancer effect of resveratrol is associated with the damage of mitochondrial function that leads to increased ROS, apoptosis, and possibly intracellular drug accumulation via inhibition of proteins involved in multi-drug resistance (MDR).

Antioxidants reduce consequences of radiation exposure.

Antioxidants have been studied for their capacity to reduce the cytotoxic effects of radiation in normal tissues for at least 50 years. Early research identified sulfur-containing antioxidants as those with the most beneficial therapeutic ratio, even though these compounds have substantial toxicity when given in-vivo. Other antioxidant molecules (small molecules and enzymatic) have been studied for their capacity to prevent radiation toxicity both with regard to reduction of radiation-related cytotoxicity and for reduction of indirect radiation effects including long-term oxidative damage. Finally, categories of radiation protectors that are not primarily antioxidants, including those that act through acceleration of cell proliferation (e.g. growth factors), prevention of apoptosis, other cellular signaling effects (e.g. cytokine signal modifiers), or augmentation of DNA repair, all have direct or indirect effects on cellular redox state and levels of endogenous antioxidants. In this review we discuss what is known about the radioprotective properties of antioxidants, and what those properties tell us about the DNA and other cellular targets of radiation.

Age-related effects of chlorpyrifos on muscarinic receptor-mediated signaling in rat cortex.

Chlorpyrifos (CPF) is a widely used organophosphorus pesticide. Earlier work from our laboratory and others has demonstrated that the sensitivity to CPF exposure changes markedly during maturation. A number of studies suggest that in addition to inhibiting acetylcholinesterase (AChE), CPF oxon may also interact directly with m2 and/or m4 subtypes of muscarinic acetylcholine receptors (mAChRs). In the present study, we investigated the in vivo effects of CPF exposure on phosphoinositide (PI) hydrolysis and cAMP formation, second-messenger systems coupled to m1, m3 and m5 (PI hydrolysis) or m2 and m4 (cAMP formation) mAChRs. Neonatal (7-day), juvenile (21-day) and adult (90-day) rats were treated with either peanut oil s.c. or CPF s.c. at 0.3x or 1x the maximum tolerated dosage (MTD: 45, 127 and 279 mg/kg for 7-day, 21-day and 90-day rats, respectively). Neurochemical end-points including AChE activity, muscarinic receptor ([3H]quinuclidinyl benzilate, and [3H]oxotremorine) binding, PI hydrolysis, and cAMP formation in cortex were evaluated at 4 h, 24 h, or 96 h after treatment. Under these conditions, relatively similar maximal degrees of cholinesterase (ChE) inhibition were noted, but times to peak inhibition varied among these age groups (24 h in neonates and juveniles, 96 h in adults). Total muscarinic receptor (QNB) binding was reduced in all three age groups with 1x MTD exposure, at both 24 h and 96 h in neonates and juveniles, but only at 96 h in adults. Oxotremorine binding was also reduced at 96 h after MTD exposure in all three age groups. Neither basal nor carbachol-stimulated IP accumulation was affected in any age group or at any time point following CPF exposure. In contrast, basal cAMP formation was significantly increased by MTD exposure in all three age groups 4 h after exposure, and at 4 h, 24 h, and 96 h after exposure in juveniles. Forskolin/Mn2+-stimulated cAMP formation was increased in neonates and juveniles at 96 h, and in juveniles also at 24 h, but was significantly decreased in adults at 96 h after MTD exposure. Oxotremorine-mediated inhibition of cAMP formation was significantly greater at 96 h after MTD exposure in all three age groups. These results provide further evidence that the cortical cAMP signaling pathway may be particularly sensitive to CPF exposure in neonatal, juvenile, and adult rats, possibly due to a direct interaction between CPF (or its oxon) and mAChRs or other components of the adenylyl cyclase cascade.