magnetic-resonance-guided-stereotaxy-for-infusions-to-the-pig-brain

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide ...

Houston Methodist Research Institute

Ionizing radiation (IR) induces highly cytotoxic double-strand breaks (DSBs) and also clustered oxidized bases in mammalian genomes. Base excision repair (BER) of bi-stranded oxidized bases could generate additional DSBs as repair intermediates in the vicinity of direct DSBs, leading to loss of DNA fragments. This could be avoided if DSB repair via DNA-PK-mediated nonhomologous end joining (NHEJ) precedes BER initiated by NEIL1 and other DNA glycosylases (DGs). Here we show that DNA-PK subunit Ku inhibits DGs via direct interaction. The scaffold attachment factor (SAF)-A, (also called hnRNP-U), phosphorylated at Ser59 by DNA-PK early after IR treatment, is linked to transient release of chromatin-bound NEIL1, thus preventing BER. SAF-A is subsequently dephosphorylated. Ku inhibition of DGs in vitro is relieved by unphosphorylated SAF-A, but not by the phosphomimetic Asp59 mutant. We thus propose that SAF-A, in concert with Ku, temporally regulates base damage repair in irradiated cell genome.

Scaffold attachment factor A (SAF-A) and Ku temporally regulate repair of radiation-induced clustered genome lesions.

Etiological role of viral proteins E6 and E7 of high-risk HPV in cervical carcinogenesis is well established. However, their contribution in chemoresistance and epithelial-mesenchymal transition (EMT) that leads to advanced metastatic lesions and chemoresistance is poorly defined. In the present study, contribution of viral oncoproteins in acquisition of EMT character during onset of chemoresistance was assessed. A chemoresistant cell line (SiHaCR) was developed from an established HPV16-positive cervical cancer cell line, SiHa, by escalating selection pressure of 5-fluorouracil (5-FU). Expression of Survivin, ABCG2, Snail, Slug, Twist, and Vimentin was examined in SiHa and SiHaCR cells by reverse transcriptase-PCR (RT-PCR) and immunoblotting assays. Mesenchymal phenotype in SiHaCR cells was confirmed by assessment of migration and invasion potentials. SiHaCR cells displayed elevated level of functional and molecular markers associated with chemoresistance (Survivin, ABCG2) and EMT (Snail, Slug, Twist, Vimentin) and reduced E-cadherin. SiHaCR also showed increased levels of HPV16 E6 and E7 transcripts. Specific silencing of HPV16 E6, but not E7 using corresponding siRNA, demonstrated a differential involvement of HPV oncogenes in manifestation of EMT. HPV16 E6 silencing resulted in reduction of Slug and Twist expression. However, the expression of Snail and Vimentin was only marginally affected. In contrast, there was an increase in the expression of E-cadherin. A reduced migration and invasion capabilities were observed only in E6-silenced SiHaCR cells, which further confirmed functional contribution of HPV16 E6 in manifestation of EMT. Taken together, our study demonstrated an active involvement of HPV16 E6 in regulation of EMT, which promotes chemoresistance in cervical cancer.

Human papillomavirus oncoproteins differentially modulate epithelial-mesenchymal transition in 5-FU-resistant cervical cancer cells.

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex.

Genomic fidelity in the humans is continuously challenged by genotoxic reactive oxygen species (ROS) generated both endogenously during metabolic processes, and by exogenous agents. Mispairing of most ROS-induced oxidized base lesions during DNA replication induces mutations. Although bulky base adducts induced by ultraviolet light and other environmental mutagens block replicative DNA polymerases, most oxidized base lesions do not block DNA synthesis. In 8-oxo-G:A mispairs generated by the incorporation of A opposite unrepaired 8-oxo-G, A is removed by MutYH (MYH) for post-replicative repair, and other oxidized base lesions must be repaired prior to replication in order to prevent mutation fixation. Our earlier studies documented S phase-specific overexpression of endonuclease VIII-like 1 (NEIL1) DNA glycosylase (DG), one of five oxidized base excision repair (BER)-initiating enzymes in mammalian cells, and its high affinity for replication fork-mimicking single-stranded (ss)DNA substrates. We recently provided experimental evidence for the role of NEIL1 in replicating-strand repair, and proposed the "cowcatcher" model of pre-replicative BER, where NEIL1's nonproductive binding to the lesion base in ssDNA template blocks DNA chain elongation, causing fork regression. Repair of the lesion in the then re-annealed duplex is carried out by NEIL1 in association with the DNA replication proteins. In this commentary, we highlight the critical role of pre-replicative BER in preventing mutagenesis, and discuss the distinction between pre-replicative vs. post-replicative BER.

Pre-Replicative Repair of Oxidized Bases Maintains Fidelity in Mammalian Genomes: The Cowcatcher Role of NEIL1 DNA Glycosylase.

Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson's disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (∼2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.

Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson's Disease.

Posttranslational modifications of DNA repair proteins have been linked to their function. However, it is not clear if posttranslational acetylation affects subcellular localization of these enzymes. Here, we show that the human DNA glycosylase NEIL1, which is involved in repair of both endo- and exogenously generated oxidized bases via the base excision repair (BER) pathway, is acetylated by histone acetyltransferase p300. Acetylation occurs predominantly at Lys residues 296, 297 and 298 located in NEIL1's disordered C-terminal domain. NEIL1 mutant having the substitution of Lys 296-298 with neutral Ala loses nuclear localization, whereas Lys > Arg substitution (in 3KR mutant) at the same sites does not affect NEIL1's nuclear localization or chromatin binding, presumably due to retention of the positive charge. Although non-acetylated NEIL1 can bind to chromatin, acetylated NEIL1 is exclusively chromatin-bound. NEIL1 acetylation while dispensable for its glycosylase activity enhances it due to increased product release. The acetylation-defective 3KR mutant forms less stable complexes with various chromatin proteins, including histone chaperones and BER/single-strand break repair partners, than the wild-type (WT) NEIL1. We also showed that the repair complex with WT NEIL1 has significantly higher BER activity than the 3KR mutant complex. This is consistent with reduced resistance of non-acetylable mutant NEIL1 expressing cells to oxidative stress relative to cells expressing the acetylable WT enzyme. We thus conclude that the major role of acetylable Lys residues in NEIL1 is to stabilize the formation of chromatin-bound repair complexes which protect cells from oxidative stress.

Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress.

Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in  mutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects.

Localization and differential expression of STAT3 and survivin in cancer cells are often related to distinct cellular functions. The involvement of survivin and STAT3 in gastric cancer has been reported in separate studies but without clear understanding of their kinetics in cancer progression.

Differentially localized survivin and STAT3 as markers of gastric cancer progression: Association with Helicobacter pylori.

It has been suggested that particle size plays an important role in determining the genotoxicity of gold nanoparticles (GNPs). The purpose of this study was to compare the potential radio-sensitization effects of two different sized GNPs (3.9 and 37.4 nm) fabricated and examined in vitro in Lewis lung carcinoma (LLC) as a model of non-small cell lung cancer through use of comet and clonogenic assays. After treatment with 2Gy X-ray irradiation, both particle sizes demonstrated increased DNA damage when compared to treatment with particles only and radiation alone. This radio-sensitization was further translated into a reduction in cell survival demonstrated by clonogenicity. This work indicates that GNPs of both sizes induce DNA damage in LLC cells at the tested concentrations, whereas the 37.4 nm particle size treatment group demonstrated greater significance in vitro. The presented data aids in the evaluation of the radiobiological response of Lewis lung carcinoma cells treated with gold nanoparticles.

Gold Nanoparticles Radio-Sensitize and Reduce Cell Survival in Lewis Lung Carcinoma.

New strategies that improve median survivals of only ~15-20 months for glioblastoma (GBM) with the current standard of care (SOC) which is concurrent temozolomide (TMZ) and radiation (XRT) treatment are urgently needed. Inhibition of polo-like kinase 1 (PLK1), a multifunctional cell cycle regulator, overexpressed in GBM has shown therapeutic promise but has never been tested in the context of SOC. Therefore, we examined the mechanistic and therapeutic impact of PLK1 specific inhibitor (volasertib) alone and in combination with TMZ and/or XRT on GBM cells. We quantified the effects of volasertib alone and in combination with TMZ and/or XRT on GBM cell cytotoxicity/apoptosis, mitochondrial membrane potential (MtMP), reactive oxygen species (ROS), cell cycle, stemness, DNA damage, DNA repair genes, cellular signaling and in-vivo tumor growth. Volasertib alone and in combination with TMZ and/or XRT promoted apoptotic cell death, altered MtMP, increased ROS and G2/M cell cycle arrest. Combined volasertib and TMZ treatment reduced side population (SP) indicating activity against GBM stem-like cells. Volasertib combinatorial treatment also significantly increased DNA damage and reduced cell survival by inhibition of DNA repair gene expression and modulation of ERK/MAPK, AMPK and glucocorticoid receptor signaling. Finally, as observed in-vitro, combined volasertib and TMZ treatment resulted in synergistic inhibition of tumor growth in-vivo. Together these results identify new mechanisms of action for volasertib that provide a strong rationale for further investigation of PLK1 inhibition as an adjunct to current GBM SOC therapy.

Arvind Pandey

Department of Neurosurgery

Houston Methodist Research Institute

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

Arvind Pandey

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurosurgery

Houston Methodist Research Institute

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

Department of Neurosurgery