5-ethynyl-2--deoxyuridine and Brain-Neoplasms

Breast cancer cells (BCCs) can remain dormant at the metastatic site, which when revoked leads to formation of metastasis several years after the treatment of primary tumor. Particularly, awakening of dormant BCCs in the brain results in breast cancer brain metastasis (BCBrM) which marks the most advanced stage of the disease with a median survival period of ~4-16 months. However, our understanding of dormancy associated with BCBrM remains obscure, in part, due to the lack of relevant in vitro platforms to model dormancy associated with BCBrM. To address this need, we developed an in vitro hyaluronic acid (HA) hydrogel platform to model dormancy in brain metastatic BCCs via exploiting the bio-physical cues provided by HA hydrogels while bracketing the normal brain and metastatic brain malignancy relevant stiffness range. In this system, we observed that MDA-MB-231Br and BT474Br3 brain metastatic BCCs exhibited a dormant phenotype when cultured on soft (0.4 kPa) HA hydrogel compared to stiff (4.5 kPa) HA hydrogel as characterized by significantly lower EdU and Ki67 positivity. Further, we demonstrated the nuclear localization of p21 and p27 (markers associated with dormancy) in dormant MDA-MB-231Br cells contrary to their cytoplasmic localization in the proliferative population. We also demonstrated that the stiffness-based dormancy in MDA-MB-231Br cells was reversible and was, in part, mediated by focal adhesion kinases and the initial cell seeding density. Finally, RNA sequencing confirmed the dormant phenotype in MDA-MB-231Br cells. This platform could further our understanding of dormancy in BCBrM and could be adapted for anti-metastatic drug screening. STATEMENT OF SIGNIFICANCE: Our understanding of dormancy associated with BCBrM remains obscure, in part, due to the lack of relevant in vitro platforms to model dormancy associated with BCBrM. Herein, we present a HA hydrogel-based platform to model dormancy in brain metastatic BCCs while recapitulating key aspects of brain microenvironment. We demonstrated that the biophysical cues provided the HA hydrogel mediates dormancy in brain metastatic BCCs by assessing both proliferation and cell cycle arrest markers. We also established the role of focal adhesion kinases and initial cell seeding density in the stiffness-mediated dormancy in brain metastatic BCCs. Further, RNA-seq. confirmed the dormant phenotype in brain metastatic BCCs. This platform could be utilized to further our understanding of microenv

Topics: Biomarkers; Brain Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cytoplasm; Deoxyuridine; DNA; Focal Adhesion Kinase 1; Gene Expression Regulation, Neoplastic; Humans; Hyaluronic Acid; Hydrogels; Ki-67 Antigen; Phenotype

Mounting literatures have revealed the crucial effects of long noncoding RNA (lncRNA) in various cancers, including glioma. HNF1A-AS1, a novel lncRNA, is reported to modulate tumorigenesis and development of multiple cancers. However, the tumorigenic function of lncRNA HNF1A-AS1 in glioma remains largely unknown. quantitative reverse transcription and polymerase chain reaction and western blot assays were applied to evaluate the expression of relevant mRNAs and proteins. 5-Ethynyl-2'- deoxyuridine, terminal deoxynucleotidyl transferase dUTP nick-end labeling, flow cytometry, and transwell assays were conducted for examining the influence of HNF1A-AS1 on glioma cell functions. The relationship among RNAs was investigated by mechanical experiments. The results demonstrated that HNF1A-AS1 was predominantly highly expressed in glioma cell lines compared with nontumor glial epithelial cell, which was associated with the stimulation of transcription factor myelocytomatosis oncogene. Knockdown of HNF1A-AS1 remarkably inhibited glioma cells proliferation, migration, and invasion, while accelerating cell apoptosis in vitro. Mechanically, HNF1A-AS1 served as a miR-32-5p sponge. Moreover, SOX4 was discovered as a target of miR-32-5p. Inhibited miR-32-5p or upregulated SOX4 could markedly counteract the inhibitory effects of silencing HNF1A-AS1 on glioma malignant biological behaviors. HNF1A-AS1 exerted oncogenic property in glioma progression via upregulating miR-32-5p-mediated SOX4 expression, suggesting potential novel therapeutic target for future glioma treatment.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Deoxyuridine; Disease Progression; DNA Nucleotidylexotransferase; Flow Cytometry; Gene Silencing; Glioma; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, Nude; MicroRNAs; Neoplasm Proteins; Proto-Oncogene Proteins c-myc; RNA, Long Noncoding; SOXC Transcription Factors; Transcription Factors

Glioblastomas (GBMs) are a heterogeneous group of primary brain tumors. These tumors are resistant to therapeutic interventions and invariably recur after surgical resection. The multifunctional protein transglutaminase 2 (TG2) has been shown to promote cell survival in a number of different tumors. There is also evidence that TG2 may be a pro-survival factor in GBMs. However, the roles that TG2 plays in facilitating GBM survival and proliferation have not yet been clearly delineated .. The functions of TG2 are often cell- and context-specific. Therefore, in this study we examined the ability of TG2 to facilitate GBM proliferation using colony formation assays and 5-ethynyl-2'-deoxyuridine (EdU) incorporation in several different GBM cell lines as well as neurospheres derived from patient tumors representing the 3 major subtypes of GBM tumors (mesenchymal, proneural, and classical) and maintained in the absence of serum. TG2 knockdown or selective TG2 inhibitors were used to modulate TG2 expression and activity.. We show that TG2 plays differential roles in the proliferative process depending on the cell type. In most, but not all, GBM models TG2 plays a crucial role in the proliferative process, and some but not all TG2 inhibitors were highly effective at reducing proliferation in a large subset of the GBM models.. Our results show that TG2 plays an important-but notoriously context-specific-role in GBM cell biology. Nonetheless, as future studies unravel the genetic "fingerprints" that make TG2 inhibitors effective, this information could be exploited to develop TG2 inhibitors into personalized GBM therapies.

Topics: Apoptosis; Brain Neoplasms; Cell Proliferation; Cell Survival; Deoxyuridine; Gene Expression Regulation, Neoplastic; Glioblastoma; GTP-Binding Proteins; Humans; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases

The characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints.. We focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis.. 5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression.. BrdU "immune-labelling", the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA.. The technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation.

Topics: Animals; Brain; Brain Neoplasms; Cells, Cultured; Click Chemistry; Deoxyuridine; Embryonic Stem Cells; Female; Gene Expression Profiling; Glioma; Humans; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Neural Stem Cells; Neurogenesis; Neurons; Olfactory Mucosa; RNA; Single-Cell Analysis