Single-cell cloning of human T-cell lines reveals clonal variation in cell death responses to chemotherapeutics

Hanlon, Kathleen; Thompson, Alex; Pantano, Lorena; Hutchinson, John N.; Al-Obeidi, Arshed; Wang, Shu; Bliss-Moreau, Meghan; Helble, Jennifer D.; Alexe, Gabriela; Stegmaier, Kimberly; Bauer, Daniel E.; Croker, Ben A.

doi:10.1016/j.cancergen.2019.06.003

Cited by 6 publications

(4 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, the fraction of free NADH was lower in quiescent T cells from the blood of healthy donors than in T cells activated for 24 to 72h with tetrameric antibodies (anti-CD2/CD3/CD28) 12 and the authors of the study reported that the lifetime of NADH was consistently the most important biomarker for differentiating quiescent and activated T cells 12 . Our results showing heterogeneity in the fb NADH both in Jurkat cells, which are known to present clonal heterogeneity 51 and in primary activated CD4 + T cells are in accordance with this published study. The decreased fb NADH we observed in activated T cells is also consistent with this published study although the timing was different.…”

Section: Such a Regulation Is Exemplified By A Study Reporting That I...supporting

confidence: 93%

Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes

Paillon

Ung

Dogniaux

et al. 2023

Preprint

View full text Add to dashboard Cite

T cell activation induces a metabolic switch generating energy required for proliferation, survival, and fueling their functions. Thus, it is essential to monitor metabolism associated to subcellular functional and structural changes. We used non-invasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NADH co-enzyme during T lymphocyte activation. 2P-FLIM measurements of the protein-bound and free NADH ratios provides a readout of the redox state (NAD+/ NADH) of the cells, and thus of their OXPHOS and glycolysis rates. Using this method, we followed the dynamics of fraction of bound NADH (fb NADH) in live single cells. Comparing fb NADH between resting and activated T cells, we show that T cell activation induces a rapid switch toward glycolysis. The switch takes only 10 minutes and remains stable for at least one hour. Three-dimensional (3D) analysis revealed that the intracellular distribution of fb NADH is symmetrically distributed in resting cells, whereas increases at the contact zone in activated cells. Finally, we show that fb NADH negatively correlates with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study shows that 2P-FLIM measurement of fb NADH is well suited to follow a fast metabolic switch in 3D, in single T lymphocytes with subcellular resolution.

show abstract

Section: Such a Regulation Is Exemplified By A Study Reporting That I...supporting

confidence: 93%

Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes

Paillon

Ung

Dogniaux

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Oxidative Medicine and Cellular Longevity after colony formation was visible to the naked eye. Then, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for counting [15].…”

Section: Follow-up For Prognosismentioning

confidence: 99%

LINC00958 Inhibits Autophagy of Bladder Cancer Cells via Sponge Adsorption of miR-625-5p to Promote Tumor Angiogenesis and Oxidative Stress

Xiao

Wang

Cheng

et al. 2022

Oxidative Medicine and Cellular Longevity

View full text Add to dashboard Cite

Objective. This study further explored LINC00958’s role in promoting tumor angiogenesis (AG) and oxidative stress (OS) development by inhibiting BC cell autophagy through sponge adsorption of miR-625-5p. Methods. BC patients and healthy controls who visited our hospital between June 2017 and February 2019 were selected as the research group (RG) and the control group (CG), respectively, with a total of 133 study subjects. Peripheral blood LINC00958 and miR-625-5p in both cohorts of participants were detected. Additionally, human bladder transitional cell carcinoma cells (T24 and J82) and human normal urothelial cells (SV-HUC-1) were purchased. Alterations in cell biological behavior were observed after transfecting miR-625-5p-mimics, miR-625-5p-inhibition, and miR-625-5p-NC sequences into these cells, respectively. Besides, ELISA was performed to quantify inflammatory factors (IFs), AG indicators, and OS indexes in cells. Subsequently, a double luciferase reporter (DLR) assay was performed to verify the targeting relationship between LINC00958 and miR-625-5p. Finally, BALB/c-nu nude mice were purchased, and T24 cells transfected with silenced LINC00958 and miR-625-5p expression sequences were used to establish subcutaneous tumors to observe tumor growth and pathological changes. Results. RG exhibited higher LINC00958 and lower miR-625-5p than CG. LINC00958 and miR-625-5p were strongly linked to myometrial invasion (MI), lymph node metastasis (LNM), distant metastasis (DM), and histology in BC patients, and the increase of LINC00958 and the decrease of miR-625-5p predicted an increased risk of prognostic death in such patients. After miR-625-5p inhibition, the capacity of BC cells to proliferate, invade, and migrate enhanced and the AG, inflammatory response, and OS injury increased, while the apoptosis rate and autophagy ability decreased. The DLR assay revealed inhibited LINC00958WT fluorescence activity by miR-625-5p-mimics, while the biological behavior of BC cells cotransfected with sh-LINC00958 and miR-625-5p-inhibition had no difference with the functions of sh-control and miR-625-5p-NC cotransfected cells. Finally, the nude mouse tumorigenesis experiment showed that the tumor mass, volume, and histopathological features of the sh-LINC00958 group were decreased compared with the sh-control group, while those of the miR-625-5p-inhibition group were increased versus miR-625-5p-NC. Conclusions. In BC, LINC00958 is highly expressed while miR-625-5p is underexpressed. LINC00958 can inhibit cell autophagy to enhance cell activity; promote OS, inflammation, and AG; and regulate tumor immunity by targeting miR-625-5p, thus participating in the development of BC.

show abstract

“…1A). To avoid clonal variations [ 61 ], we mixed individual CASP8-/- knock-out clones—Clones K5, 7 and 8 for HeLa and Clones 7, 11 and 22 for SiHa, to form a mixed knock-out population (henceforth KO) and determined different biological traits of the resulting cells. We first determined the 2D cell-proliferation of the WT vs. KO cells over 120 h and observed that the KO HeLa cells proliferated at a significantly slower rate than the WT cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

The non-apoptotic function of Caspase-8 in negatively regulating the CDK9-mediated Ser2 phosphorylation of RNA polymerase II in cervical cancer

Mandal

Raab²,

Rödel³

et al. 2022

Cell. Mol. Life Sci.

View full text Add to dashboard Cite

Cervical cancer is the fourth most frequently diagnosed and fatal gynecological cancer. 15–61% of all cases metastasize and develop chemoresistance, reducing the 5-year survival of cervical cancer patients to as low as 17%. Therefore, unraveling the mechanisms contributing to metastasis is critical in developing better-targeted therapies against it. Here, we have identified a novel mechanism where nuclear Caspase-8 directly interacts with and inhibits the activity of CDK9, thereby modulating RNAPII-mediated global transcription, including those of cell-migration- and cell-invasion-associated genes. Crucially, low Caspase-8 expression in cervical cancer patients leads to poor prognosis, higher CDK9 phosphorylation at Thr186, and increased RNAPII activity in cervical cancer cell lines and patient biopsies. Caspase-8 knock-out cells were also more resistant to the small-molecule CDK9 inhibitor BAY1251152 in both 2D- and 3D-culture conditions. Combining BAY1251152 with Cisplatin synergistically overcame chemoresistance of Caspase-8-deficient cervical cancer cells. Therefore, Caspase-8 expression could be a marker in chemoresistant cervical tumors, suggesting CDK9 inhibitor treatment for their sensitization to Cisplatin-based chemotherapy.

show abstract

Single-cell cloning of human T-cell lines reveals clonal variation in cell death responses to chemotherapeutics

Cited by 6 publications

References 44 publications

Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes

Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes

LINC00958 Inhibits Autophagy of Bladder Cancer Cells via Sponge Adsorption of miR-625-5p to Promote Tumor Angiogenesis and Oxidative Stress

The non-apoptotic function of Caspase-8 in negatively regulating the CDK9-mediated Ser2 phosphorylation of RNA polymerase II in cervical cancer

Contact Info

Product

Resources

About