DNA double-strand breaks and Aurora B mislocalization induced by exposure of early mitotic cells to H 2 O 2 appear to increase chromatin bridges and resultant cytokinesis failure
“…1a). Notably, neither 50 nor 100 μM H 2 O 2 , concentrations that are easily achievable in a pathological setting (e.g., a rat ischemia/reperfusion model 34 ), caused cell death after 24 h, as we reported previously 30 .Fig. 1H 2 O 2 treatment forms a more abnormal nuclear shape in mitotic cells than in asynchronous cells. a Left panel: Asynchronous (upper) or mitotic (lower) HeLa cells were treated with 100 μM H 2 O 2 for 10 h and subjected to immunocytochemical staining for Lamin B1 (green), α-tubulin (red) and DAPI (blue).…”
Section: Resultsmentioning
confidence: 57%
“…Mitotic cell synchronization was performed by thymidine/RO3306 block as described in a previous paper 30 . Briefly, cells (5 × 10 5 ) were seeded into 100-mm culture plates (BD FALCON, 353003), cultured in DMEM/F12 or DMEM containing 10 % FBS for 1 day and subsequently treated with 2 mM thymidine (Sigma-Aldrich, T9250) to arrest at the G1/S transition.…”
Section: Methodsmentioning
confidence: 99%
“…The intracellular level of H 2 O 2 was measured using the fluorescent sensor pHyper-Cyto 32 ; the experimental procedure was described in a previous paper 30 . Briefly, 2 days before the experiment, HeLa cells were transfected with pHyper-Cyto using electroporation, and fluorescence was monitored by using a microscope (a Nikon Eclipse Ti with a 20 × 14 NA Plan Apochromat objective).…”
Many types of cancer cells exhibit abnormal nuclear shapes induced by various molecular changes. However, whether reactive oxygen species (ROS) induce nuclear deformation has not been fully addressed. Here, we show that hydrogen peroxide (H
2
O
2
) treatment induced concentration-dependent alterations in nuclear shape that were abolished by pretreatment with the antioxidant N-acetyl-L-cysteine or by catalase overexpression. Interestingly, treatment with H
2
O
2
induced nuclear shape alterations significantly more frequently in mitotic cells than in asynchronous cells, suggesting that H
2
O
2
mainly affects nuclear envelope disassembly and/or reassembly processes. Because protein phosphatase 2 A (PP2A) activity is reported to be involved in nuclear envelope reassembly during mitosis, we investigated the possible involvement of PP2A. Indeed, H
2
O
2
reduced the activity of PP2A, an effect that was mimicked by the PP1 and PP2A inhibitor okadaic acid. Moreover, overexpression of PP2A but not PP1 or PP4 partially rescued H
2
O
2
-induced alterations in nuclear shape, indicating that the decrease in PP2A activity induced by H
2
O
2
is specifically involved in the observed nuclear shape alterations. We further show that treatment of mitotic cells with H
2
O
2
induced the mislocalization of BAF (barrier-to-autointegration factor), a substrate of PP2A, during telophase. This effect was associated with Lamin A/C mislocalization and was rescued by PP2A overexpression. Collectively, our findings suggest that H
2
O
2
preferentially affects mitotic cells through PP2A inhibition, which induces the subsequent mislocalization of BAF and Lamin A/C during nuclear envelope reassembly, leading to the formation of an abnormal nuclear shape.
“…1a). Notably, neither 50 nor 100 μM H 2 O 2 , concentrations that are easily achievable in a pathological setting (e.g., a rat ischemia/reperfusion model 34 ), caused cell death after 24 h, as we reported previously 30 .Fig. 1H 2 O 2 treatment forms a more abnormal nuclear shape in mitotic cells than in asynchronous cells. a Left panel: Asynchronous (upper) or mitotic (lower) HeLa cells were treated with 100 μM H 2 O 2 for 10 h and subjected to immunocytochemical staining for Lamin B1 (green), α-tubulin (red) and DAPI (blue).…”
Section: Resultsmentioning
confidence: 57%
“…Mitotic cell synchronization was performed by thymidine/RO3306 block as described in a previous paper 30 . Briefly, cells (5 × 10 5 ) were seeded into 100-mm culture plates (BD FALCON, 353003), cultured in DMEM/F12 or DMEM containing 10 % FBS for 1 day and subsequently treated with 2 mM thymidine (Sigma-Aldrich, T9250) to arrest at the G1/S transition.…”
Section: Methodsmentioning
confidence: 99%
“…The intracellular level of H 2 O 2 was measured using the fluorescent sensor pHyper-Cyto 32 ; the experimental procedure was described in a previous paper 30 . Briefly, 2 days before the experiment, HeLa cells were transfected with pHyper-Cyto using electroporation, and fluorescence was monitored by using a microscope (a Nikon Eclipse Ti with a 20 × 14 NA Plan Apochromat objective).…”
Many types of cancer cells exhibit abnormal nuclear shapes induced by various molecular changes. However, whether reactive oxygen species (ROS) induce nuclear deformation has not been fully addressed. Here, we show that hydrogen peroxide (H
2
O
2
) treatment induced concentration-dependent alterations in nuclear shape that were abolished by pretreatment with the antioxidant N-acetyl-L-cysteine or by catalase overexpression. Interestingly, treatment with H
2
O
2
induced nuclear shape alterations significantly more frequently in mitotic cells than in asynchronous cells, suggesting that H
2
O
2
mainly affects nuclear envelope disassembly and/or reassembly processes. Because protein phosphatase 2 A (PP2A) activity is reported to be involved in nuclear envelope reassembly during mitosis, we investigated the possible involvement of PP2A. Indeed, H
2
O
2
reduced the activity of PP2A, an effect that was mimicked by the PP1 and PP2A inhibitor okadaic acid. Moreover, overexpression of PP2A but not PP1 or PP4 partially rescued H
2
O
2
-induced alterations in nuclear shape, indicating that the decrease in PP2A activity induced by H
2
O
2
is specifically involved in the observed nuclear shape alterations. We further show that treatment of mitotic cells with H
2
O
2
induced the mislocalization of BAF (barrier-to-autointegration factor), a substrate of PP2A, during telophase. This effect was associated with Lamin A/C mislocalization and was rescued by PP2A overexpression. Collectively, our findings suggest that H
2
O
2
preferentially affects mitotic cells through PP2A inhibition, which induces the subsequent mislocalization of BAF and Lamin A/C during nuclear envelope reassembly, leading to the formation of an abnormal nuclear shape.
“…Moreover, it is speculated that the entire chromatin structure also plays a regulatory function during cell migration and invasion in 3D extracellular matrix confinements, as it has been shown that chromatin needs to be stretched in addition to the polymeric structural network within the nuclear skeleton. [110][111][112][113] Moreover, the position of the cell's nucleus is not distinct and unchangeable; rather, the nucleus can be moved in relation to other organelles through diverse cytoskeletal restructuring events. 114 In several cell types, the directed motion of cells involves a change in the localization of the centrosomes, as they become positioned between the cell's nucleus and its migration front, the cell's leading edge.…”
Section: Nuclear Jammingmentioning
confidence: 99%
“…In addition, the local chromatin distribution of the nuclear structure plays a prominent role in regulating the transcription efficiency of distinct migration‐related genes. Moreover, it is speculated that the entire chromatin structure also plays a regulatory function during cell migration and invasion in 3D extracellular matrix confinements, as it has been shown that chromatin needs to be stretched in addition to the polymeric structural network within the nuclear skeleton …”
In addition to genetic, morphological and biochemical alterations in cells, a key feature of the malignant progression of cancer is the stroma, including cancer cell motility as well as the emergence of metastases. Our current knowledge with regard to the biophysically driven experimental approaches of cancer progression indicates that mechanical aberrations are major contributors to the malignant progression of cancer. In particular, the mechanical probing of the stroma is of great interest. However, the impact of the tumor stroma on cellular motility, and hence the metastatic cascade leading to the malignant progression of cancer, is controversial as there are two different and opposing effects within the stroma. On the one hand, the stroma can promote and enhance the proliferation, survival and migration of cancer cells through mechanotransduction processes evoked by fiber alignment as a result of increased stroma rigidity. This enables all types of cancer to overcome restrictive biological capabilities. On the other hand, as a result of its structural constraints, the stroma acts as a steric obstacle for cancer cell motility in dense three-dimensional extracellular matrices, when the pore size is smaller than the cell's nucleus. The mechanical properties of the stroma, such as the tissue matrix stiffness and the entire architectural network of the stroma, are the major players in providing the optimal environment for cancer cell migration. Thus, biophysical methods determining the mechanical properties of the stroma, such as magnetic resonance elastography, are critical for the diagnosis and prediction of early cancer stages. Fibrogenesis and cancer are tightly connected, as there is an elevated risk of cancer on cystic fibrosis or, subsequently, cirrhosis. This also applies to the subsequent metastatic process.
Acquired resistance to conventional chemotherapeutic agents limits their effectiveness and can cause cancer treatment to fail. Because enzymes in the aurora kinase family are vital regulators of several mitotic events, we reasoned that targeting these kinases with tozasertib, a pan-aurora kinase inhibitor, would not only cause cytokinesis defects, but also induce cell death in high-grade pediatric and adult glioma cell lines. We found that tozasertib induced cell cycle arrest, increased mitochondrial permeability and reactive oxygen species generation, inhibited cell growth and migration, and promoted cellular senescence and pro-apoptotic activity.However, sustained exposure to tozasertib at clinically relevant concentrations conferred resistance, which led us to examine the mechanistic basis for the emergence of drug resistance. RNA-sequence analysis revealed a significant upregulation of the gene encoding pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), a pyruvate dehydrogenase (PDH) inhibitory kinase that plays a crucial role in the control of metabolic flexibility under various physiological conditions. Upregulation of PDK1, PDK2, PDK3 or PDK4 protein levels is positively correlated with tozasertib-induced resistance through inhibition of PDH activity. Tozasertib-resistant cells exhibited increased mitochondrial mass as measured by 10-N-nonyl-Acridine Orange. Inhibition of PDK with dichloroacetate (DCA) resulted in increased mitochondrial permeability and cell death in tozasertib-resistant
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