Co-incubation of cisplatin with isopropanol leads to enhanced apoptosis without an increase in intercellular cisplatin concentration.

<p>(A) Expression levels of cleaved PARP, an apoptotic marker, as measured by western blot for cells incubated with 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone along with cisplatin free controls (B) Levels of intracellular cisplatin were measured using optical emission spectrometry for the three treatments, 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone. The differences between the three treatments are not statistically significant (n = 8 trials). Also shown are predicted levels of cisplatin obtained by assuming that intracellular cisplatin that is directly proportional to the external concentration determines the extent of cell death (as described in Methods). The solid line denotes the predicted mean theoretical value and corresponding dashed lines denote error bounds. (C) Levels of intracellular cisplatin for UMSCC1 and the more resistant cell line Me-180pt treated with 10μM cisplatin. The solid line as described previously denotes predicted levels given the assumptions stated in 4B and dashed lines indicate error bounds.</p

Changes in transition temperature in GPMVs correlate with the cell lines resistance to cisplatin.

<p>(A) GPMVs were isolated from four cell-lines as described in the Methods section. The transition temperature shifts are reported by comparing the transition temperatures of GPMVs probed in the presence of 10 μM cisplatin to untreated GPMVs. (B) Data points in panel A were plotted against a previously reported measure of surviving fraction to cisplatin obtained using clonogenic assays for the same four cell lines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.ref008" target="_blank">8</a>]. Surviving fraction was measured using a clonogenic survival assay. Surviving fractions were measured 72 hours after treatment with 10uM cisplatin. The straight line is drawn to visually distinguish sensitive and resistant celllines. Transition temperature shifts upon incubation with 10 μM cisplatin or exposure to 10 Gy irradiation for GPMVs isolated from ME-180 pt cells (C) and RBL cells (D). In all cases, points represent the average of at least 3 independent measurements and error bounds represent the standard error of the mean. Significance between transition temperature shift measurements were evaluated using t-tests.</p

Tristetraprolin Mediates Radiation-Induced TNF-α Production in Lung Macrophages

<div><p>The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For <i>in vitro</i> studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the <i>in vivo</i> relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from <i>ttp</i> (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP^Ser178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.</p> </div

Schematic model explaining the role of post-translational TTP modifications (phosphorylation and degradation) in radiation-induced TTP inactivation as an upstream regulator involved in increased TNF-α secretion by mouse lung macrophages.

<p>Schematic model explaining the role of post-translational TTP modifications (phosphorylation and degradation) in radiation-induced TTP inactivation as an upstream regulator involved in increased TNF-α secretion by mouse lung macrophages.</p

Radiation-induced TTP degradation and TNF-α secretion is inhibited significantly by the proteasome inhibitor MG132.

<p>(A) MH-S cells were metabolically labeled with ³⁵S-methionine, were either sham-irradiated or irradiated with 4 Gy, and chased with cold methionine for indicated time periods. After the chase period cell lysates were subjected to immunoprecipitation using TTP antibody, immunocomplexes were resolved by SDS-PAGE and autoradiography. (B) TTP protein’s half life in sham-irradiated (-RT) and irradiated (4 Gy) groups were determined by densitometric scanning of the autoradiographs followed by quantitation using Image J1.32j software (NIH, Bethesda, MD). Relative protein levels were determined in comparison to sample isolated immediately after the pulse labeling (0 h chase). (C) MH-S cells were either sham-irradiated or radiated with 4 Gy, and 44 h after radiation 2 µM of MG132 was added. Cell lysates were collected 4 h after MG132 addition and immunoblotted for TTP and TNF-α. GAPDH was used as loading control.</p

Radiation increased the TNF-α transcript and its release by MH-S cells 48 h post–radiation.

<p>(A) MH-S cells were left untreated (0 Gy) or irradiated (4 Gy) and culture supernatants were collected at the indicated time points. Released TNF-α levels were quantified using ELISA kits according to the manufacturer’s instruction. (B) Cells were treated as above, and RNA was isolated and quantified at the indicated time points as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057290#s2" target="_blank">materials and methods</a>. (C) MH-S cells were either sham irradiated or irradiated with 4 Gy. 48 h post-irradiation TNF-α mRNA level, stability and synthesis was determined using BrU pulse-chase labeling experiment as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057290#pone.0057290-Fu1" target="_blank">[28]</a>.</p

p38 kinase controls radiation-induced TTP phosphorylation and TNF-α secretion by MH-S cells.

<p>(A) CHO cells overexpressing TTP were treated with 4 Gy in the presence of either DMSO (vehicle control) or p38 inhibitor (SB203580), or PI3K inhibitor (Wortmannin), or GSK3ß inhibitors (SB415286, SB216763). Cell lysates were prepared 10 min post-radiation and immunoblotted using indicated antibodies. (B) MH-S cells were pretreated with either p38 or GSK3ß inhibitors as above and cell lysates were prepared 10 min post-radiation and immunoblotted with the indicated antibodies. (C) MH-S cells were irradiated with 4 Gy in the presence or absence of a p38 inhibitor (SB203580), and radiation-induced TNF-α secretion was quantified using ELISA. (D) MH-S cells were treated with either control (C) or TTP (T) siRNA. 24 h post-transfection, cells were either left un-irradiated or radiated with 4 Gy. Culture supernatants were collected 48 h post-radiation, and TNF-α levels were quantified. In the inset, the effectiveness of p38 siRNA is shown in cell lysates isolated from C or T siRNA treated cells.</p