scholarly journals HPVE6-USP46 Mediated Cdt2 Stabilization Reduces Set8 Mediated H4K20-Methylation to Induce Gene Expression Changes

Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 30
Author(s):  
Shashi Kiran ◽  
Briana Wilson ◽  
Shekhar Saha ◽  
Julia Ann Graff ◽  
Anindya Dutta
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
High Risk ◽  
Cell Lines ◽  
Deletion Mapping ◽  
Helix Loop Helix ◽  
Induced Tumors ◽  
Transformed Cell Lines ◽  
High Risk Hpv

E6 from high-risk strains of HPV is well known to transform cells by deregulating p53. We reported that in HPV transformed cell-lines E6 from high-risk HPV can recruit the USP46 deubiquitinase to substrates such as Cdt2 and stabilize the latter, and that USP46 is important for growth of HPV induced tumors in xenografts. Here we show that in cervical cancer biopsies the stabilization of Cdt2 in the HPV-induced cancers leads to the decrease of a CRL4-Cdt2 substrate, the histone H4K20 mono-methyltransferase Set8, and decrease in H4K20me1 or H4K20me3 that can be detected by immunohistochemistry. In HPV-transformed cancer cell lines in vitro, knockdown of E6 decreases Cdt2 and increases Set8. Co-knockdown of Set8 shows that some of the gene expression changes produced by E6 knockdown is due to the increase of Set8. EGFR and EGFR regulated genes were identified in this set of genes. Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20. Deletion mapping of Cdt2 shows that there are three discrete, but redundant, parts of the substrate that are essential for stabilization by E6: USP46. The helix–loop–helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2. The identification of 50 amino acid stretches in the 731 amino acid Cdt2 protein as being important for the stabilization by E6 underlines the specificity of the process. In summary, E6 activates the deubiquitinase activity of USP46, stabilizes Cdt2 utilizing multiple sites on Cdt2, and leads to degradation of Set8 and changes in gene-expression in HPV-transformed cells.

scholarly journals ATRA Upregulates Cell Surface CD1D on Myeloma Cells and Sensitizes Them to iNKT Cell-Mediated Lysis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2102-2102
Author(s):  
Xin Li ◽  
Tarun K. Garg ◽  
Sarah K. Johnson ◽  
Susann Szmania ◽  
Justin Stivers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Retinoic Acid ◽  
High Risk ◽  
Cell Surface ◽  
Cell Lines ◽  
Target Genes ◽  
Inkt Cell ◽  
Inkt Cells

Abstract Invariant natural killer T (iNKT) cells recognize lipid antigens in the context of CD1D and have been shown to effectively kill CD1D-expressing multiple myeloma (MM) cells. It has been reported that iNKT cells in MM patients are deficient and dysfunctional and that CD1D is variably expressed among MM patients and is completely lost in MM cell lines. Recent clinical study suggests that targeting iNKT cells may prevent progression of asymptomatic MM (AMM) to active disease (Richter at al. Blood 2013). All-Trans Retinoic Acid (ATRA) has been used for the treatment of MM but its modes of action have not been completely elucidated. The aims of this study were to shed light on the mechanism of action of ATRA and to identify ATRA target genes in MM. ATRA at clinically relevant concentrations (1-2 μM, 4 days) had minimal growth inhibitory effect on the majority of MM cell lines (10±13% and 14±16%, respectively; n=10). Gene expression profile (GEP) of MM cell lines and primary cases (n=13) treated with ATRA (1 μM, 2 days) revealed upregulation of CD1D (381±204 vs. 5692±1675 mean±SEM GEP signal in CONT and ATRA groups, respectively; p<0.006). Flow cytometry analysis for cell surface CD1D in 21 MM cases treated in vitro with ATRA revealed significant CD1D induction in 11 cases, intermediate induction in 2 cases and no induction in 8 cases. Clinically, MM cells from patients who received ATRA as part of their salvage therapy also had induced CD1D gene expression compared to levels prior to ATRA therapy. Analysis of abnormal plasma cells based on a single 8-color markers flow cytometry showed higher CD1D coefficient of variation (CV) in AMM cases molecularly classified as high-risk (n=5) than low-risk cases (n=9, p<0.003), while mean florescence intensity (MFI) was insignificantly lower in high-risk AMM cases. In vitro ATRA treatment of BM cells from high-risk AMM patient resulted in increased CD1D MFI by 2 folds and reduced CV in abnormal plasma cell restricted population. To test the functional consequences of CD1D upregulation in MM cells, iNKT cells were purified from healthy donors’ peripheral blood mononuclear cells using anti-Vα24Jα18 immunomagnetic beads and expanded in vitro via stimulation with irradiated autologous α-GalCer-loaded mature dendritic cells (DCs) in the presence of IL-2. ATRA treated (1 μM, 2 days) compared to vehicle treated MM cells (n=4) were more sensitive to iNKT cell-mediated lysis in 5 hour 51chromium-release assays or bioluminescence viability assays (e.g. 21% vs. 75% lysis of H929 cells, p<0.0001, effector: target ratio 2.5:1). ATRA-induced MM cell lysis by iNKT cells was markedly blocked by CD1D neutralizing antibody (1-40 μg/ml) indicating that the enhanced cell lysis was mediated through CD1D. Further GEP analysis identified RARRES3 (retinoic acid receptor responder protein 3) as an additional top ATRA target gene in MM cells. RARRES3 gene expression was commonly but variably induced by ATRA whereas MM cells expressing high baseline RARRES3 were resistant to ATRA-induced CD1D gene and protein expression. Expression levels of CD1D and RARRES3 were inversely correlated in MM cells from newly diagnosed patients (n=449, r=-0.4, p<0.0001). Among molecularly classified groups, the MF subtype, known to be associated with poor survival, had the lowest CD1D/highest RARRES3 expression, whereas the favorable CD2 subtype had a reversed pattern. We conclude that CD1D and RARRES3 are ATRA target genes in MM cells and that stimulation of cell surface CD1D expression on MM cells enhances sensitivity to iNKT cell-mediated lysis. Disclosures van Rhee: Senesco: PI Other.

High-Risk HPV E6 Oncoproteins Assemble into Large Oligomers that Allow Localization of Endogenous Species in Prototypic HPV-Transformed Cell Lines†

Biochemistry ◽  
2007 ◽  
Vol 46 (2) ◽  
pp. 341-349 ◽  
Author(s):  
María M. García-Alai ◽  
Karina I. Dantur ◽  
Clara Smal ◽  
Lía Pietrasanta ◽  
Gonzalo de Prat-Gay
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Transformed Cell ◽  
Hpv E6 ◽  
Transformed Cell Lines ◽  
High Risk Hpv

High-risk HPV-positive human cancer cell lines show different sensitivity to cisplatin-induced apoptosis correlated with the p21Waf1/Cip1 level

1996 ◽  
Vol 108 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Kohsei Funaoka ◽  
Masanobu Shindoh ◽  
Toshiharu Yamashita ◽  
Kei Fujinaga ◽  
Akira Amemiya ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Cancer Cell ◽  
Human Cancer ◽  
Cancer Cell Lines ◽  
Human Cancer Cell ◽  
Human Cancer Cell Lines ◽  
Induced Apoptosis ◽  
High Risk Hpv

Pre-clinical evaluation of PV-10 for in vitro anti-tumor activity in refractory and high-risk adult solid tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e14544-e14544
Author(s):  
Son Tran ◽  
Satbir Thakur ◽  
Mohit Jain ◽  
Chunfen Zhang ◽  
Aru Narendran
Keyword(s):  
High Risk ◽  
Tumor Cell ◽  
Solid Tumors ◽  
Cell Lines ◽  
Therapeutic Agent ◽  
Intratumoral Injection ◽  
Tumor Cell Lines ◽  
Clinical Testing ◽  
Anti Tumor Activity

e14544 Background: PV-10 (10% rose bengal disodium; 4,5,6,7-tetrachloro-2’,4’,5’,7’-tetraiodofluorescein) is a novel therapeutic agent previously shown to have potent anti-tumor activity following intratumoral injection in melanoma and refractory neuroblastoma, and currently is undergoing clinical testing as a single-agent for refractory metastatic neuroendocrine cancer (NCT02693067) and in combination with checkpoint inhibitors for metastatic melanoma (NCT02557321) and metastatic uveal melanoma (NCT00986661). Given the established clinical efficacy of PV-10 in adult melanoma and hepatic cancers via intratumoral injection, there is a need to evaluate the therapeutic potential of PV-10 in high-risk and refractory adult solid tumors via systemic administration. Our study aims to identify the clinical potential of systemically-delivered PV-10 by first generating prerequisite in vitro data for adult malignancies. Methods: Cytotoxicity assays were performed using the Alamar Blue assay to study the effects of PV-10 in vitro 96-hours post-treatment against a panel of adult solid tumor cell lines derived from breast (MCF-7, T-47D, MDA-MB-231), colorectal (HCT-116, LoVo, T-84), head and neck (CAL-27, Detroit-562, FaDu, UM-SCC-1), and testicular (NCC-IT, NTERA-2, TCAM-2) tissues. Light microscopy and Western blotting were used to investigate apoptosis induction and target modulation in tumor cells after PV-10 treatment. Results: In vitro results from our study demonstrate that PV-10 is cytotoxic at pharmacologically relevant concentrations across the indicated cell lines. Specifically, tumor cell lines originating from testicular tissues were highly sensitive to PV-10 treatment (Mean ± SD IC50: 37.5 ± 16.4 µM; n = 3) compared to breast (117.5 ± 71.0 µM; n = 3), colorectal (64.79 µM; n = 3), and head and neck (106.6 ± 29.2 µM; n = 4) cell lines. Western blot analyses showed dose- and time-dependent activation of pro-apoptotic protein markers in caspase-3 and PARP cleavage, indicating drug-induced apoptosis. Conclusions: This study provides the first pre-clinical results of PV-10 as a novel systemically-delivered therapeutic agent for a range of high-risk and refractory adult solid tumors. Data obtained from our in vitro experiments using a broad repertoire of cell lines that represent diverse molecular and phenotypic subtypes of solid tumors in adults can serve as prerequisite pre-clinical data to establish clinical testing in these populations.

Rearrangement and diversification of immunoglobulin light-chain genes in lymphoid cells transformed by reticuloendotheliosis virus

1989 ◽  
Vol 9 (11) ◽  
pp. 4970-4976
Author(s):  
J Y Zhang ◽  
W Bargmann ◽  
H R Bose
Keyword(s):  
Cell Lines ◽  
Light Chain ◽  
Lymphoid Cells ◽  
Chain Gene ◽  
Gene Rearrangements ◽  
B Cell Differentiation ◽  
Immunoglobulin Light Chain ◽  
Light Chain Gene ◽  
Transformed Cell Lines

Avian lymphoid cells transformed by reticuloendotheliosis virus (REV-T) serve as a model to analyze the mechanism by which B-cell differentiation and antibody diversification occur in birds. Immunoglobulin light-chain gene rearrangements, diversification, and expression were analyzed in 72 independently derived REV-T-transformed cell lines. Lymphoid cells transformed as the result of expression of the v-rel oncogene were divided into two distinct groups based on light-chain gene rearrangements. The status of the light-chain gene loci in these REV-T-transformed cell lines was determined in part by the ages of the chickens whose spleen cells were transformed. In embryonic spleen cell lines transformed by the v-rel oncogene, rearrangements were not detected, even after prolonged culture in vitro, indicating that these cells are arrested in B-cell differentiation. REV-T transformants derived from spleens obtained from chickens 2 weeks old or older, however, had at least one light-chain allele rearranged. All of the cell lines analyzed which exhibited rearranged light-chain genes contained light-chain transcripts, and most of the REV-T-transformed cells which displayed light-chain rearrangements expressed immunoglobulin protein. REV-T, therefore, transforms B-lymphoid cells at phenotypically different stages of development. Many REV-T-transformed cells undergo immunoglobulin chain gene rearrangements during prolonged propagation in vitro. Most of the cell lines which rearrange their light-chain alleles also undergo diversification during cultivation in vitro. Light-chain diversification occurs during or after the rearrangement event.

scholarly journals Impact of extracellular matrix properties on neuroblastoma genomic heterogeneity

2020 ◽  
Author(s):  
Amparo López-Carrasco ◽  
Susana Martín-Vañó ◽  
Rebeca Burgos-Panadero ◽  
Ezequiel Monferrer ◽  
Ana P Berbegall ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
High Risk ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Chromosome 9 ◽  
Future Research ◽  
Knock Out ◽  
Specific Distribution

Abstract Background Increased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible. Methods We have applied high density SNPa and NGS techniques to in vivo and in vitro models (orthotropic xenograft vitronectin knock-out mice and 3D bioprinted hydrogels with different stiffness) using two representative neuroblastoma cell lines (the MYCN amplified SK-N-BE(2) and the ALK mutated SH-SY5Y), to discern how tumor genomics patterns and clonal heterogeneity of both cell lines are affected. Results We describe a remarkable subclonal selection of some genomic aberrations in SK-N-BE(2) cells grown in knock-out vitronectin xenograft mice that also emerged when cultured for long times in stiff hydrogels. Specially, we detected an enlarged subclonal cell population with chromosome 9 aberrations in both models. Similar abnormalities were found in human high-risk neuroblastoma with MYCN amplification. Genomics of the SH-SY5Y cell line remained stable when cultured in both models. Conclusions Focus on heterogeneous intratumor segmental chromosome aberrations and mutations, as a mirror image of tumor microenvironment, is a vital area of future research.

scholarly journals Gene Transfer into the Lung by Nanoparticle Dextran-Spermine/Plasmid DNA Complexes

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Syahril Abdullah ◽  
Wai Yeng Wendy-Yeo ◽  
Hossein Hosseinkhani ◽  
Mohsen Hosseinkhani ◽  
Ehab Masrawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Cell Lines ◽  
Plasmid Dna ◽  
Mixing Ratio ◽  
Systemic Delivery ◽  
Clear Trend ◽  
Assay Time

A novel cationic polymer, dextran-spermine (D-SPM), has been found to mediate gene expression in a wide variety of cell lines andin vivothrough systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in cell lines and in the lungs of BALB/c mice via instillation delivery.In vitrostudies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.

scholarly journals Murine Gammaherpesvirus (MHV-68) Transforms Cultured Cells in vitro

Intervirology ◽  
2015 ◽  
Vol 58 (2) ◽  
pp. 69-72 ◽  
Author(s):  
Veronika Mrázová ◽  
Tatiana Betáková ◽  
Marcela Kúdelová ◽  
Miroslava Šupolíková ◽  
Veronika Lachová ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cultured Cells ◽  
Virus Antigen ◽  
Immunofluorescence Assay ◽  
Dermal Fibroblasts ◽  
Murine Gammaherpesvirus ◽  
Transformed Cell ◽  
Transformed Cell Lines ◽  
Nih 3T3

Human dermal fibroblasts and mouse NIH/3T3 cells acquired the transformed phenotype (‘criss-cross' pattern of growth) after infection with ultraviolet-irradiated murine gammaherpesvirus (MuHV-4 strain 68; MHV-68). These cells with changed phenotype could be serially cultured for 5-6 passages (35-40 days), and then they entered into crisis and most of them died. In a small number of cultures, however, foci of newly transformed cells appeared from which two stable cell lines were derived. After 6-9 cell culture passages of the MHV-68 transformed cell lines, MHV-68 DNA and virus antigen could be detected by PCR and immunofluorescence assay along with the disappearance of actin bundles, indicating that both transformed cell lines might be oncogenic.

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