Resistance of Hypoxic Cells to Ionizing Radiation Is Mediated in Part via Hypoxia-Induced Quiescence

Abstract

: Double strand breaks (DSBs) are highly toxic to a cell, a property that is exploited in radiation therapy. A critical component for the damage induction is cellular oxygen, making hypoxic tumor areas refractory to the efficacy of radiation treatment. During a fractionated radiation regimen, these hypoxic areas can be re-oxygenated. Nonetheless, hypoxia still constitutes a negative prognostic factor for the patient’s outcome. We hypothesized that this might be attributed to specific hypoxia-induced cellular traits that are maintained upon reoxygenation. Here, we show that reoxygenation of hypoxic non-transformed RPE-1 cells fully restored induction of DSBs but the cells remain radioresistant as a consequence of hypoxia-induced quiescence. With the use of the cell cycle indicators (FUCCI), cell cycle-specific radiation sensitivity, the cell cycle phase duration with live cell imaging, and single cell tracing were assessed. We observed that RPE-1 cells experience a longer G1 phase under hypoxia and retain a large fraction of cells that are non-cycling. Expression of HPV oncoprotein E7 prevents hypoxia-induced quiescence and abolishes the radioprotective effect. In line with this, HPV-negative cancer cell lines retain radioresistance, while HPV-positive cancer cell lines are radiosensitized upon reoxygenation. Quiescence induction in hypoxia and its HPV-driven prevention was observed in 3D multicellular spheroids. Collectively, we identify a new hypoxia-dependent radioprotective phenotype due to hypoxia-induced quiescence that accounts for a global decrease in radiosensitivity that can be retained upon reoxygenation and is absent in cells expressing oncoprotein E7.

Indisulam synergizes with palbociclib to induce senescence through inhibition of CDK2 kinase activity

“…In order to determine CDK2 activity following 24h drug treatment, cells were seeded in 96 well plate, treated with indisulam and palbociclib for 24h and then imaged on a spinning disc microscope using Andor 505 Dragonfly system equipped with 20x 0.75 NA objective and Zyla 4.2+, sCMOS camera. CDK2 activity was determined by calculating the nucleo/cytoplasmic ratio of the CDK2 reporter, using a custom macro for ImageJ, as described before [ 65 ]. For real-time analysis of CDK2 activity immediately following drug treatment, cells were seeded in chambered covered slides (LabtekII), and subsequently imaged for 25h.…”

Magnetic-Based Human Tissue 3D Cell Culture: A Systematic Review

“…Magnetic-based models allow us to effectively produce homogeneous 3D aggregates and with desirable compactness [ 1 , 17 ]. Of the twenty-five studies included in this review, nine studies used only magnetic levitation [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ], eleven studies used only the bioprinting method [ 14 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ], and three studies used both methods [ 15 , 36 , 37 ]. Only two studies used the magnetic ring formation method [ 22 , 38 ].…”

“…The 24-well plates were the most used for studies using the levitation method; whereas, the 96-well plates were the most used for the bioprinting method. More precisely, nine studies used the 24-well Bio-Assembler Kit [ 15 , 23 , 24 , 25 , 26 , 30 , 32 , 37 , 39 ], eight studies used the 96-well Bioprinting Kit [ 14 , 27 , 28 , 29 , 31 , 33 , 35 , 36 ], two studies used both 6-well and 24-well Bio-Assembler Kit [ 17 , 19 ], one study used only the 6-well Bio-Assembler Kit [ 34 ], and one study used the 96-well Ring Drive for ring structure formation [ 38 ]. There is an additional study that used tissue culture Petri dishes covered by a top cover with an attached neodymium magnet [ 22 ].…”

Micro-RNAs, the Cornerstones of the Future of Radiobiology in Head and Neck Cancers?

“…Factors involved in HNSCC radioresistance or radiosensitivity are numerous and are generally common to all cancers including traditional variable like cells lesions repair capacity, hypoxia, cell cycle position, and cell growth fraction, but also new “actors” such as hepatocyte growth factor receptor (HGFR) or programmed cell death protein 1 and programmed cell death protein 1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1) [ 98 , 99 , 100 , 101 ]. HNSCC is a malignancy of a special interest for the improvement of therapeutic ratio due to the importance of radiotherapy in both adjuvant treatment and definitive treatment in association with chemotherapy or in particular cases with target therapy.…”