Signaling pathways in which the candidate genes are involved.

<p>An extensive literature search revealed that the candidate genes are cross-linked to 3 signaling pathways: NF-κB, Akt and MAPK, which all play a role in cancer. NF-κB signaling pathway has a crucial role in regulating immune responses, whereas Akt signaling has been shown to inhibit the growth of GBM cells and GBM stem-like cells that may also be impaired by MAPK signaling disruption. Because of the RT-qPCR results, <i>CTSK</i>'s role has been examined and it was found via cross linking to other candidate genes obtained via osteopontin (<i>OPN</i>) gene functions.</p

RT-qPCR analysis of expression of selected proteases and protease inhibitors in U87-MG and U373 GBM cells, NHA cells and GBM tissues and non-malignant brain <i>(in vivo)</i>.

<p>(A) Upregulated expression of seven genes (<i>TFRC</i>, <i>CTSK</i>, <i>GFPT2</i>, <i>ERAP2</i>, <i>GPR56</i>, <i>CD74</i>, <i>PI3</i>) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the <i>CTSK</i> gene using GBM tissues and cell lines with reference genes <i>TBP</i> and <i>HPRT1</i> in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.</p

Scheme of cellular processes and activities involving overexpressed protease and protease inhibitor genes in GBM.

<p>The overexpressed protease and inhibitor genes in GBM tissues and cells were queried by the Biomine search engine which identified processes and activities ascribed with KEGG and GO identifiers (in circles) in which selected genes (in bold caption) are involved.</p

Immunohistochemstry and immunocytochemstry and Western blot analysis of cathepsin K in GBM cells and tissues.

<p>ICC staining of CatK in U87-MG (A) and U373 (B, D) GBM cells. At high magnification (1000×), a granular pattern of the staining was observed in U373 cells (D), which corresponds to perinuclear endo-lysosomal-like localization of CatK. Strong IHC staining of CatK in GBM tissue (C), and weak staining in control brain tissue (non-malignant brain) (E, F). Mouse jaw with bone tissue (B) with osteoclasts at bone edges (O) was used as positive control for CatK staining (G). Magnifications: A, B, G - 400×; D - 1000×; C, E, F - 200×. (H) Western blot of GBM cells (lanes 7–10) and their conditioned media (CM; lanes 3–6), GBM tissue (lanes 17–25) and non-malignant brain tissue (lanes 11–16) showing positivity for pro-CatK (39 kDa). The active form of the enzyme (27 kDa) was detected only in a small amount in one passage of U87-MG cells (lane 8). At 30 kDa, an intermediate form of CatK was observed (lane 21). Recombinant proform (lane 1) and active CatK (lane 2) were used as positive control. As loading control β-actin was used. Legend: 1 – recombinant pro-CatK, 2 – recombinant active CatK, 3 – U87p38 CM, 4 – U87p39 CM, 5 – U373p41 CM, 6 – U373p42 CM, 7 – U87p45, 8 – U87p48, 9 – U373p45, 10 – U373p48, 11–16 – different non-malignant brain samples, 17–25 – different GBM tissue samples. <b>Please note</b> that Western blotting image does not allow for direct quantitative comparison of CatK expression in control and tumor samples due to variable protein amounts loaded. (I and J) Additional western blot experiments using cell lines U87-MG (I) and U373 (J) and different protease inhibitors. No active form of cathepsin K was observed in any of the conditions tested but in all cases pro-cathepsin K was present. Legend: 1 – without any inhibitor, 2 – 5 µM E-64, 3 – 5 µM CA074, 4 – 5 µM CLIK148, 5 – 5 µM pepstatin A, 6 – 1 mM PMSF, 7 – 20 mM EDTA, 8 – combination of all inhibitors, 9 – control: recombinant pro-CatK, 10 – control: recombinant active CatK.</p

Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells

<div><p>Some epidemiological studies indicate that the use of mobile phones causes cancer in humans (in particular glioblastomas). It is known that DNA damage plays a key role in malignant transformation; therefore, we investigated the impact of the UMTS signal which is widely used in mobile telecommunications, on DNA stability in ten different human cell lines (six brain derived cell lines, lymphocytes, fibroblasts, liver and buccal tissue derived cells) under conditions relevant for users (SAR 0.25 to 1.00 W/kg). We found no evidence for induction of damage in single cell gel electrophoresis assays when the cells were cultivated with serum. However, clear positive effects were seen in a p53 proficient glioblastoma line (U87) when the cells were grown under serum free conditions, while no effects were found in p53 deficient glioblastoma cells (U251). Further experiments showed that the damage disappears rapidly in U87 and that exposure induced nucleotide excision repair (NER) and does not cause double strand breaks (DSBs). The observation of NER induction is supported by results of a proteome analysis indicating that several proteins involved in NER are up-regulated after exposure to UMTS; additionally, we found limited evidence for the activation of the γ-interferon pathway. The present findings show that the signal causes transient genetic instability in glioma derived cells and activates cellular defense systems.</p></div

Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells - Fig 4

<p><b>A-D Measurement of the NER and BER activity in glioblastoma cells after exposure to different RF intensities (Specific Absorption Rate 0.25 W/kg—1 W/kg) for 16 hrs.</b> Bars indicate means ± SEM of results obtained in the three independent experiments. (Six cultures were prepared per experimental point, three were treated with RF and three were sham exposed. From each culture, one slide was made and 50 cells were evaluated per slide). Left bars show results of control experiments, which were performed in parallel (for details see also Collins and Dusinska [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193677#pone.0193677.ref048" target="_blank">48</a>]). Striped bars show results obtained with isolated nuclei which were exposed to buffer only, hatched bars show results with nuclei which were treated either with UV (for determination of NER) or with Ro-19-8022 (determination of BER). Subsequently, the nuclei of the cells were incubated with T4 endonuclease V (for NER measurements) or FPG (for BER measurements). Other bars show results of experiments in which cells were treated either with UV (determination NER) or with Ro-19-8022 (determination BER) and subsequently with cytosols of sham exposed (white bars) or RF treated (grey bars) glioblastoma cells. Stars indicate significance (p < 0.05). Statistical comparisons were performed by linear contrasts after analysis of variance.</p

Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells - Fig 5

<p><b>A-C Results of proteomic analysis of RF exposed glioblastoma cell line U87.</b> The cells were exposed to RF for 16 hrs (Specific Absorption Rate 1.0 W/kg). The distribution of up- and down-regulated proteins is shown in a volcano plot (Fig 5A). For each identified protein, the fold-change on a logarithmic scale to the basis of 2 (ln₂Δt-test) and the corresponding p-value (-log p-value) are indicated. Label-free quantification intensities of proteins related to NER activity are shown in Fig 5B and of proteins related to the γ-interferon pathway in Fig 5C. White bars represent sham exposed cells, and grey bars the exposed cells. Stars indicate statistical significance (p < 0.05).</p

Impact of RF on γH2AX phosphorylation in human glioblastoma cell lines¹.

<p>Impact of RF on γH2AX phosphorylation in human glioblastoma cell lines<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193677#t001fn001" target="_blank">¹</a>.</p

Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells - Fig 1

<p><b>A and B Impact of RF-exposure on% tail DNA in different human derived cell lines.</b> The cells were exposed to RF (Specific Absorption Rate 1.0 W/kg) for 16 hrs. Subsequently, some of the cultures were treated with H₂O₂ (30 μM) for 10 min as a positive control (Fig 1B). % Tail DNA was monitored in SCGE experiments under standard conditions as described in materials and methods. Bars represent means ± SEM of results obtained in three independent experiments. (Six cultures were prepared per experimental point, three were treated with RF and three were sham exposed. From each culture, one slide was made and 50 cells were evaluated per slide). White bars represent sham exposed cells (controls), grey bars exposed cells. Statistical comparisons were performed by linear contrast after analysis of variance.</p

Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells - Fig 2

<p><b>A-D Impact of the cultivation conditions and RF-exposure on the cell cycle.</b> The cells were grown in presence and absence of serum and exposed to different RF intensities (Specific Absorption Rates 0.25, 0.5 and 1.0 W/kg) over a period of 16 hrs before they were analyzed by FACS. Cells in G0/G1, S and G2/M phase were sorted on the basis of their DNA contents. 10 000 cells were analyzed per experimental point and all experiments were performed in triplicate.</p