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UV irradiation/cold shock-mediated apoptosis is switched to bubbling cell death at low temperatures.

Chen SJ, Lin PW, Lin HP, Huang SS, Lai FJ, Sheu HM, Hsu LJ, Chang NS - Oncotarget (2015)

Bottom Line: Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death.Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53.Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan, ROC.

ABSTRACT
When COS7 fibroblasts and other cells were exposed to UVC irradiation and cold shock at 4°C for 5 min, rapid upregulation and nuclear accumulation of NOS2, p53, WWOX, and TRAF2 occurred in 10-30 min. By time-lapse microscopy, an enlarging gas bubble containing nitric oxide (NO) was formed in the nucleus in each cell that finally popped out to cause "bubbling death". Bubbling occurred effectively at 4 and 22°C, whereas DNA fragmentation was markedly blocked at 4°C. When temperature was increased to 37°C, bubbling was retarded and DNA fragmentation occurred in 1 hr, suggesting that bubbling death is switched to apoptosis with increasing temperatures. Bubbling occurred prior to nuclear uptake of propidium iodide and DAPI stains. Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death. Unlike apoptosis, there were no caspase activation and flip-over of membrane phosphatidylserine (PS) during bubbling death. Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53. Together, UV/cold shock induces bubbling death at 4°C and the event is switched to apoptosis at 37°C. Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

No MeSH data available.


Related in: MedlinePlus

Suppression of bubbling by NOS2 inhibitor Nω-LAME(A) COS7 cells were pretreated with Nω-LAME (0.125–4 mM) for 30 min, followed by exposure to UV irradiation (480 mJ/cm2) and cold shock (4°C) for 30 to 180 min. (B) Induction of NOS2 expression by UV/cold shock was shown in 30 min. The levels of intracellular NFκB (p65) and α-tubulin remained largely unchanged. A representative data is shown from 2 experiments. (C–D) By immunofluorescence microscopy, UV/cold shock together significantly induced the expression of NOS2 in COS7 cells (n = 10; mean ± standard deviation; Student's t-test).
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Figure 5: Suppression of bubbling by NOS2 inhibitor Nω-LAME(A) COS7 cells were pretreated with Nω-LAME (0.125–4 mM) for 30 min, followed by exposure to UV irradiation (480 mJ/cm2) and cold shock (4°C) for 30 to 180 min. (B) Induction of NOS2 expression by UV/cold shock was shown in 30 min. The levels of intracellular NFκB (p65) and α-tubulin remained largely unchanged. A representative data is shown from 2 experiments. (C–D) By immunofluorescence microscopy, UV/cold shock together significantly induced the expression of NOS2 in COS7 cells (n = 10; mean ± standard deviation; Student's t-test).

Mentions: To determine the underlying mechanisms, Nω-nitro-L-arginine methyl ester hydrochloride (Nω-LAME), an analog of arginine, was shown to suppress the formation of gas bubbles (Figure 5A), suggesting that NO is in the generated gas. When COS7 cells were pretreated with Nω-LAME for 1 hr, and then exposed to UV and chilled at 4°C for indicated times, Nω-LAME blocked bubbling in a time- and a dose-dependent manner (Figure 5A). UV/cold shock rapidly induced the expression of nitric oxide synthase 2 (NOS2) in 30 min, as determined by Western blotting (Figure 5B). The cellular levels of α-tubulin were relatively unchanged (Figure 5B). Similarly, UV/cold shock significantly induced the generation of NOS2 in 5–10 min, as determined by immunofluorescence staining (Figure 5C and 5D). UV irradiation or cold shock alone was less effective (Figure 5C and 5D).


UV irradiation/cold shock-mediated apoptosis is switched to bubbling cell death at low temperatures.

Chen SJ, Lin PW, Lin HP, Huang SS, Lai FJ, Sheu HM, Hsu LJ, Chang NS - Oncotarget (2015)

Suppression of bubbling by NOS2 inhibitor Nω-LAME(A) COS7 cells were pretreated with Nω-LAME (0.125–4 mM) for 30 min, followed by exposure to UV irradiation (480 mJ/cm2) and cold shock (4°C) for 30 to 180 min. (B) Induction of NOS2 expression by UV/cold shock was shown in 30 min. The levels of intracellular NFκB (p65) and α-tubulin remained largely unchanged. A representative data is shown from 2 experiments. (C–D) By immunofluorescence microscopy, UV/cold shock together significantly induced the expression of NOS2 in COS7 cells (n = 10; mean ± standard deviation; Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480731&req=5

Figure 5: Suppression of bubbling by NOS2 inhibitor Nω-LAME(A) COS7 cells were pretreated with Nω-LAME (0.125–4 mM) for 30 min, followed by exposure to UV irradiation (480 mJ/cm2) and cold shock (4°C) for 30 to 180 min. (B) Induction of NOS2 expression by UV/cold shock was shown in 30 min. The levels of intracellular NFκB (p65) and α-tubulin remained largely unchanged. A representative data is shown from 2 experiments. (C–D) By immunofluorescence microscopy, UV/cold shock together significantly induced the expression of NOS2 in COS7 cells (n = 10; mean ± standard deviation; Student's t-test).
Mentions: To determine the underlying mechanisms, Nω-nitro-L-arginine methyl ester hydrochloride (Nω-LAME), an analog of arginine, was shown to suppress the formation of gas bubbles (Figure 5A), suggesting that NO is in the generated gas. When COS7 cells were pretreated with Nω-LAME for 1 hr, and then exposed to UV and chilled at 4°C for indicated times, Nω-LAME blocked bubbling in a time- and a dose-dependent manner (Figure 5A). UV/cold shock rapidly induced the expression of nitric oxide synthase 2 (NOS2) in 30 min, as determined by Western blotting (Figure 5B). The cellular levels of α-tubulin were relatively unchanged (Figure 5B). Similarly, UV/cold shock significantly induced the generation of NOS2 in 5–10 min, as determined by immunofluorescence staining (Figure 5C and 5D). UV irradiation or cold shock alone was less effective (Figure 5C and 5D).

Bottom Line: Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death.Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53.Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan, ROC.

ABSTRACT
When COS7 fibroblasts and other cells were exposed to UVC irradiation and cold shock at 4°C for 5 min, rapid upregulation and nuclear accumulation of NOS2, p53, WWOX, and TRAF2 occurred in 10-30 min. By time-lapse microscopy, an enlarging gas bubble containing nitric oxide (NO) was formed in the nucleus in each cell that finally popped out to cause "bubbling death". Bubbling occurred effectively at 4 and 22°C, whereas DNA fragmentation was markedly blocked at 4°C. When temperature was increased to 37°C, bubbling was retarded and DNA fragmentation occurred in 1 hr, suggesting that bubbling death is switched to apoptosis with increasing temperatures. Bubbling occurred prior to nuclear uptake of propidium iodide and DAPI stains. Arginine analog Nω-LAME inhibited NO synthase NOS2 and significantly suppressed the bubbling death. Unlike apoptosis, there were no caspase activation and flip-over of membrane phosphatidylserine (PS) during bubbling death. Bubbling death was significantly retarded in Wwox knockout MEF cells, as well as in cells overexpressing TRAF2 and dominant-negative p53. Together, UV/cold shock induces bubbling death at 4°C and the event is switched to apoptosis at 37°C. Presumably, proapoptotic WWOX and p53 block the protective TRAF2 to execute the bubbling death.

No MeSH data available.


Related in: MedlinePlus