Immunoblot analysis of the placental form of glutathione S-transferase in protein extracted from paraffin-embedded human glioma tissue

Gliomas are the most common type of malignant brain tumors. Glioma diagnosis is not very effective, and there are few therapeutic biomarkers. The aims of this study were to detect Pokemon and its regulatory genes and explore the potential mechanism between them in glioma. The Fe3O4 nanoparticles identified using TEM were used to isolate cell and tissue RNA, qRT-PCR was used to detect Pokemon, survivin, and cyclin B1 mRNA expression. Western blotting was used to detect Pokemon, survivin, and cyclin B1 protein expression. Immunofluorescence and immunohistochemistry were used to detect Pokemon expression. CCK-8 assay, EdU staining, and TUNEL staining were used to assess cell viability and apoptosis. Pokémon was over-expressed in human glioma tissue and cells. In U251 cells, Pokemon knockdown significantly decreased survivin and cyclin B1 expression, cell viability, and Pokemon expression and increased apoptosis. Pokemon overexpression had an opposite effect. In addition, over-expressed Pokemon reversed these results. Overall, we found that Pokemon promotes tumorigenesis, and the potential mechanism might be related to the Pokemon-related genes survivin and cyclin B1.

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TAMI-13. IMPORTANCE OF CD38 INHIBITION IN PRE-RECURRENT GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.902 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii215-ii216

Author(s):

Renee Hirte ◽

Ian E Olson ◽

Masum Rahman ◽

Moustafa Mansour ◽

Amanda Munoz Casabella ◽

...

Keyword(s):

Glioma Cell ◽

Large Scale ◽

Progression Free Survival ◽

Standards Of Care ◽

Screening Methods ◽

Human Glioma ◽

Glioma Tissue ◽

Free Survival ◽

Nad Synthesis ◽

Large Scale Screening

Abstract Glioblastoma multiforme (GBM) is the most aggressive CNS neoplasm with a mere 15 month progression-free survival following current standards-of-care. Furthermore, conventional therapies have been effective, in part, by inducing a senescent-like phenotype in at least a proportion of glioma, as well as within the tumor-adjacent, healthy brain cells. Through an N-glyco-capture large-scale screening method, we were able to identify 25 genes exclusively overexpressed on glioma cell surfaces. Of those 25 targets, CD38 was an attractive target due to pre-existing FDA approved therapeutics. Recent studies have shown, however, that senescent cells, such as those induced via chemo- and radiotherapies, secrete a pro-inflammatory array of cytokines, known collectively as senescence associated secretory phenotype (SASP), that are capable of increasing CD38 expression in monocytes. CD38 functions as an ectoenzyme to convert extracellular NAD+ to nicotinamide (required for glioma cell salvage pathway NAD+ synthesis) and ADPR/cADPR (a cellular proliferation signal). To examine the effects of radiation on human glioma tissue, we performed reverse cyclase assays (RCA) on paired primary and recurrent human glioma tissue samples and found an increase in CD38 activity in post-irradiated tissues (recurrent glioma) compared to pre-irradiated (primary glioma). Through proliferation assays, we also found an increase in glioma cell growth following treatment with cADPR compared to untreated. Our results demonstrate that CD38 activity is tumorgenic, and furthermore that conventional chemo- and radiotherapies increase this CD38 activity. This indicates that treating CD38 with previously FDA approved therapeutics may provide hope for increasing progression free survival in our GBM patient population, and moreover emphasize the importance of leveraging novel large scale screening methods for identifying additional opportunities for treatment.

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Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells.

Molecular Biology of the Cell ◽

10.1091/mbc.8.4.555 ◽

1997 ◽

Vol 8 (4) ◽

pp. 555-566 ◽

Cited By ~ 47

Author(s):

C M Niessen ◽

E H Hulsman ◽

E S Rots ◽

P Sánchez-Aparicio ◽

A Sonnenberg

Keyword(s):

Protein A ◽

Immunoblot Analysis ◽

Cytoplasmic Domain ◽

Glutathione S Transferase ◽

Type Iii ◽

Fibronectin Type Iii ◽

Cytoskeletal Networks ◽

Integrin Alpha 6 ◽

Fibronectin Type

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.

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