Targeted elimination of senescent Ras-transformed cells by suppression of MEK/ERK pathway

MEK/ERK inhibitor PD0325901 induces AMPK-dependent autophagy thus allowing ERas cells to develop resistance to MEK/ERK inhibitor and to restore their viability

Here, we have used transformed rat embryo fibroblasts expressing E1Aad5 and cHa-Ras oncogenes (ERas cells) as a model to study a role of MEK/ERK pathway in regulation of autophagy, which is involved in the maintenance of viability and implementation of senescence program. Senescence was induced by treatment with HDAC inhibitor sodium butyrate (NaBut, 4 mM). MEK1,2 inhibitor PD0325901 (PD, 1 µM) was used for long-term inhibition of MEK/ERK pathway. The treatment of ERas cells with PD0325901 leads to a complete cessation of ERK1,2 phosphorylation that persists for 2-120 h as evidenced by Western-blot analysis (Fig. 1A).

According to cell growth assay and clonogenic survival data, PD0325901 treatment decreases proliferative activity of ERas cells, albeit the cell growth does not arrest to the full extent (Fig. 1B). The decrease of proliferation is most likely caused by inhibition of ERK1,2 phosphorylation involved in regulation of cell cycle progression [37]. Flow cytometry analysis reveals more than 2-fold decrease of cells in S-phase with simultaneous accumulation of cells in G1-phase (Fig. 1D). ERas cells decrease their viability after 24 h of PD0325901 treatment and then restore it as shown by MTT assay and this recovery is not associated with ERK1,2 phosphorylation (Fig. 1A). Cell proliferation is reactivated after providing the cells with fresh medium without inhibitor after 120h of treatment (Fig. 1C, E).

We further analyzed the role of autophagy in the development of resistance to MEK inhibition as well as in the restoration of viability and proliferation in long-term PD0325901 treated cells. It is well known that autophagy can be activated either by mTOR down regulation or AMPK activation [18–21]. We wondered how the autophagy could be affected upon MEK/ERK suppression by PD0325901. Although Ras-ERK pathway positively regulates mTORC1 by suppressing TSC2-RHEB [17], PD treatment did not lead to mTORC1 inhibition in control cells as shown by 4E-BP1 and S6 protein phosphorylation analysis (Fig. 2A). The level of Ulk1 Ser757 (the mTORC1 target) phosphorylation also did not decrease (Fig. 2A). Therefore, it appears more likely that mTORC1-independent autophagy is activated upon PD0325901 treatment. Then we assayed whether AMPK is activated in ERas cells treated with MEK inhibitor. Upon PD0325901 treatment, the level of AMPK phosphorylation increases more than 2-fold at 2 h and 24 h of treatment (Fig. 2A). The level of pUlk1-Ser555, a pAMPK target responsible for the initiation of autophagy [20,21], also increases throughout 2-72 h of treatment. The dynamics of pAMPK and pUlk1-Ser555 accumulation coincides with the activation of autophagy markers during PD0325901 treatment (LC3-I to LC3-II conversion, p62/SQSTM1 accumulation (Fig. 2B). Thus, PD0325901 induces the AMPK-dependent autophagy in ERas cells, despite the high level of mTORC1 activity, which is a potential negative regulator of autophagy. Given that a key regulator of cellular senescence, mTORC1, is active in PD0325901 treated cells, while cell cycle is blocked, we aimed to analyze the levels of senescence markers. The obtained results show that in ERas cells treated with PD0325901 for 72 h the total protein level and the cell size are slightly decreased (Fig. 3B). In addition, they do not demonstrate senescence-associated β-galactosidase (SA-β-gal) activity (Fig. 3A). Thus, despite the persisting block of the cell cycle and especially the high activity of mTORC1, the suppression of MEK/ERK activates autophagy, which abolishes the development of senescence program. The prolonged suppression of MEK/ERK activity was shown to reduce p38 MAP kinase phosphorylation [38]. As p38 MAPK is involved into regulation of replicative and premature senescence [39], we assessed the levels of p38 phosphorylation in control and senescent cells upon MEK/ERK suppression. According to Western blot data, the level of phosphorylated p38 decreases in both control and senescent cells upon PD0325901 treatment, while senescent cells with active MEK/ERK pathway have a higher level of p38 MAPK phosphorylation (Supplementary Fig. 1). Thus, a decrease of p38 activity in PD-treated ERas cells might contribute to downregulation of senescence markers. Recently, it was shown that inhibition of p38 phosphorylation can promote autophagy [40].

We then assessed autophagic flux by immunofluorescent staining and Western-blot analysis. Immunofluorescent and Western blot data show that PD0325901 treatment activates autophagy as follows from LC3-I to LC3-II conversion and colocalization of LC3 with a lysosomal marker LAMP1 at 24-72 h time interval (Fig. 2B, C). Another marker of autophagy – an autophagy cargo receptor p62/SQSTM1 protein, which degrades after fusion of autophagosomes with lysosomes, is accumulated to 72 h of PD0325901 treatment, but then disappears at 120 h correlating well with Western-blot data of LC3 I-II conversion (Fig. 2B, C). We infer that autophagy flux increases in a period of 24-72 h and then returns to a control level at 120 h. This results correlate with the restoration of cell viability and proliferative activity as assessed by a clonogenic assay and analysis of cell growth of PD0325901-treated cells after removal of inhibitor (Fig. 1C, E). Suppression of autophagy with Bafilomycin A1 decreases the viability of control ERas cells that indicates the ERas-transformed cells require autophagy to maintain their viability. Co-treatment of cells with PD0325901 and Bafilomycin A1 results in a significant decrease of cellular viability compared to cells treated with PD alone, where cellular viability restores 72 h post treatment, thereby suggesting a cytoprotective role of autophagy activation in control ERas cells (Fig. 2D). To clarify whether the development of PD0325901 resistance is linked to AMPK-mediated autophagy, we used an ATP-competitive inhibitor of AMP-kinase activity Compound C together in combination with PD0325901. We found that exposure of PD-treated cells to Compound C decreased cell viability compared to PD0325901 alone (Fig. 2E). Thus, these data support a conclusion that AMPK is involved in autophagic response of ERas cells to MEK/ERK suppression.

Given that mitochondria ensure the viability of Ras-expressing cells and autophagy is required to maintain the pool of functional mitochondria [6,7,10], we analyzed the integrity of mitochondria using transmission electron microscopy (TEM) and immunofluorescent staining with Mitotracker Red (potential-dependent) and Mitotracker Green (potential-independent). TEM data demonstrate that mitochondria have severe ultrastructural alterations in ERas cells treated with PD0325901 for 24 h. They are vacuolated and have disrupted cristae (Fig. 4A). The damaged mitochondria can be observed in numerous autophagosome-like vacuoles (Fig. 4A, B), indicating that they are degraded presumably by mitophagy. After 72 h of PD0325901 treatment the ultrastructure of mitochondria does not differ from control cells, and the damaged mitochondria are no longer detected (Fig. 4C). In addition, both Mitotracker Red and Mitotracker Green fluorescent signals are active in ERas cells treated with PD0325901 for 72 h. Mitotracker Red/Mitotracker Green intensity ratio is not decreased, indicating that the mitochondria retain their membrane potential (Fig. 5A, B). Mitochondria damage is associated with the increased levels of reactive oxygen species (ROS) at 2 h and 24 h of MEK/ERK suppression in senescent cells, as follows from an assay with DCF-DA, which is oxidized by ROS into 2’, 7’–dichlorofluorescein (DCF). DCF is a highly fluorescent compound which can be detected by fluorescence spectroscopy with maximum excitation and emission spectra of 495 nm and 529 nm respectively. However, ROS levels in cells with suppressed MEK/ERK do not exceed those in control cells as damaged mitochondria are no longer present (Fig. 5C). We infer that the effects caused in ERas cells by PD0325901 treatment are at least due to mitochondria damage. However, after the activation of AMPK-mediated autophagy in long-term PD0325901 treated cells the viability of cell population restores. Thus, ERas cells respond to MEK/ERK inhibition by activating AMPK-mediated autophagy, which promotes recovery of mitochondrial integrity and cellular survival.

Inhibition of MEK/ERK pathway in ERas cells induced to senescence with NaBut impairs autophagy and cellular survival

When cell cycle is blocked, the high level mTORC1 favors the protein synthesis and cell hypertrophy, maintains energy homeostasis, senescence-associated secretory phenotype (SASP) and viability [23,25,41]. However, in control Ras-transformed cells elevated activity of mTORC1 provides viability and supports high proliferation rate. Therefore, we asked how MEK/ERK inhibition by PD0325901 affects the viability of cells induced to senescence with NaBut and whether PD0325901 is able to induce AMPK-dependent autophagy in the senescent cells.

First, we analyzed how PD0325901 treatment affects viability of NaBut-treated senescent ERas cells. Growth curves and clonogenic survival data show that MEK-ERK inhibition in cells induced to senescence leads to almost 10-fold suppression of viability as compared with control ERas cells. Similar data follows from the MTT viability assay (Fig. 1E). NaBut induces an irreversible cell cycle arrest and suppression of proliferation in ERas cells resulting in senescence [34,42,43], however the arrested cells remain viable as confirmed by MTT assay (Fig. 2E). The flow cytometry data reveal a sub-diploid peak upon NaBut and PD0325901 co-treatment implying the apoptotic cell death (Fig. 2D).

Next, we investigated whether PD0325901 activates AMPK-regulated autophagy in cells induced to senescence by NaBut. Senescent cells treated with PD0325901 accumulate phosphorylated AMPK at 2 h and 24 h of treatment followed by accumulation of phosphorylated autophagy-responsible target pUlk1-Ser555 (Atg1) (Fig. 6A). However, the fusion of autophagosomes with lysosomes is completely blocked as judged by the lack of colocalization of the LC3 and LAMP1 (Fig. 6B, 7B). Both PD0325901- and NaBut-treated cells demonstrate elevated lysosomal β-galactosidase levels (pH 4.0), a marker of the increased lysosomal activity, that localizes predominantly around the nuclei (Fig. 6D). However senescent cells with suppressed MEK/ERK signaling also demonstrate perinuclear lysosomal β-galactosidase (pH 4.0) localization, but intensity of staining is weaker indicating the lower lysosomal activity. Thus, we suggest that in senescent cells exposed to MEK/ERK inhibitor the activity of lysosomes and autophagosome-lysosome fusion are disrupted. It was previously shown that in senescent cells lysosomes predominantly co-localize with mTOR [44]. Our data indicate that NaBut-treated cells (72 h) demonstrate colocalization of mTOR and LAMP1 in perinuclear regions, while upon MEK/ERK suppression LAMP1 does not colocalize with mTOR (Fig.7A). Besides, senescent cells with suppressed MEK/ERK activity have a weaker LAMP1 signals that are not detected at the periphery of the cytoplasm, where LC3 is mostly localized. These cells also do not demonstrate LAMP1 and LC3 colocalization, as can be seen by immunofluorescent staining data (Figs. 6B and 7B). Low level of LC3-II revealed at 2 h of PD treatment as assessed by Western blotting then disappears concomitantly with a decrease of the overall cytoplasmic level of LC3 (Fig. 6B, C). Analysis of p62/SQSTM1 immunofluorescence staining shows neither accumulation nor its colocalization with LC3 (Fig. 6B, C). Together, this data show that senescent cells with suppressed MEK/ERK pathway are characterized by defective autophagy that fails to support viability of senescent cells. To clarify whether AMPK activation is cytotoxic for these cells, we used AICAR, a compound that is converted to monophosphorylated nucleotide (ZMP) within the cell, thus mimicking a decrease of ATP and hence activating ATP synthesis through AMPK activity. Treatment of senescent ERas cells with AICAR resulted in a significant decrease of cellular viability, while in control cells AICAR increased viability after 72 h of treatment according to MTT assay (Fig. 6E). These data indicate that, while in control cells AMPK activity favors viability, in senescent cells even early steps of AMPK activation are cytotoxic.

We have shown that inhibition of MEK/ERK signaling by PD0325901 initially leads to mitochondrial damage, but then cells can restore the mitochondrial function and proliferative potential. We asked whether the damaged mitochondria also degrade in senescent ERas cells treated with PD0325901. TEM data shows that senescent ERas cells treated for 24 h with PD0325901 have the mitochondria with disordered cristae and electron dense or concentric membrane structures inside the organelles as well as with partial to complete loss of the cristae. The mitochondria devoid of cristae are present as the rounded electron-empty structures with double membranes (Fig. 4D, E). Although the mitochondria are severely disordered, they are not localized within the autophagosomes in contrast to the control ERas cell treated with PD alone (Fig. 4B). Thus, the electron microscopy and immunofluorescence data indicate the absence of autophagosome-lysosome fusion and mitophagy activation in senescent cells. Therefore, senescent cells are not capable of removing damaged mitochondria. Furthermore, cells co-treated with NaBut and PD0325901 for 24 h have the enlarged Golgi complexes with the numerous stacks of flattened cisternae surrounded by multiple vesicles. Considering the well-developed nucleoli, all together indicates that the ongoing active protein synthesis associates with a high level of mTORC1 activity. Senescent ERas cells exposed to PD0325901 for 72 h demonstrate the abundant of damaged mitochondria with swollen cristae and hyperdense matrix (Fig. 4E). Accordingly, senescent cells with suppressed MEK/ERK show the increased levels of ROS caused by the mitochondria damage. ROS levels remain high up to 72 h of treatment, unlike control cells treated with PD0325901. Analysis of the main markers of senescence in NaBut and PD0325901 co-treated cells shows a high level of senescence-associated SA-β-gal activity (pH 6.0) and increased mTORC1 activity in addition to cell cycle arrest and cell hypertrophy. However, these parameters are slightly lower than in NaBut-treated senescent cells (Fig. 3A, B). Despite high mTORC1 activity, the Golgi complexes are numerous, but the nucleoli demonstrate weak development at 72 h of NaBut and PD0325901 co-treatment (Fig. 4E). ERas cells treated with NaBut alone for 24 h and 72 h show numerous stacks of Golgi cisternae and lysosome-like structures (Fig. 4). Mitochondria retain their usual structure in senescent cells (Fig. 4F, G). Interestingly, NaBut-treated cells have increased ROS level (Fig. 5C) that can be explained by senescence activation rather than the mitochondrial damage [45,46].

The tumor cells are characterized by distinctive features such as high proliferative rate and glycolysis as a predominant source of ATP production [47]. Our data demonstrate that the suppression of MEK/ERK pathway in senescent cells does not abolish mTORC1 activity, specific for NaBut-treated cells. It is well known that mTORC1 is involved in up-regulation of glycolysis [48–50]. When mitochondria are damaged, the cells can activate glycolytic activity to maintain viability, thereby surviving the unfavorable conditions until the mitochondria will be restored [51]. Therefore, we checked whether glycolysis is indeed affected in senescence cells treated with PD0325901 by measuring the lactate level in culture medium. The results indicate that suppression of MEK/ERK in senescent cells reduces the production of lactate, while senescent cells show a much higher level of lactate production (Fig. 3C). Similarly, control cells treated with PD0325901 alone for 72 h have lower levels of lactate compared to control cells. We suggest that PD decreases the lactate levels due to the involvement of MEK/ERK pathway in regulation of several glycolytic genes [52–54]. Accordingly, senescent cells with suppressed MEK/ERK pathway are characterized by the lowest levels of lactate at 72 h of treatment, thus confirming that glycolysis in this case is suppressed. We believe that inhibition of glycolysis together with the disturbance of mitochondrial function contributes to cell death of PD0325901-treated senescent cells.

A direct evidence of the activated apoptotic program in senescent ERas cells with repressed MEK/ERK pathway follows from analysis of the nucleosomal DNA fragmentation. As shown by DNA fragmentation assay in 2% agarose gel, inhibition of MEK/ERK in cells undergoing NaBut-induced senescence results in the nucleosomal DNA fragmentation after 24 h and 72 h of treatment (Fig. 8A). According to a clonogenic assay, PD0325901 alone also induces apoptotic cell death at 72 h of treatment, while about 50% of cells in population remain viable (Fig. 1C). The obtained results are in accordance with a 2-fold activation of caspase-3 in NaBut and PD0325901 co-treated cells as compared to control ones, and a 1,5-fold increase in case of PD0325901 treatment alone (Fig. 8B). Interestingly, an increased activity of caspase-8 but not of caspase-9 can be observed in NaBut and PD0325901 co-treated cells.

Thus, inhibition of MEK-ERK pathway in NaBut-treated senescent cells interferes with the autophagy programs which serve to support a stringent balance of catabolic and anabolic processes in senescent cells provided by mTORC1. Due to a defective autophagy, the cells are incapable of maintaining senescence program and viability. Unlike the control cells exposed to PD0325901 alone, the senescent cells are unable to eliminate the damaged mitochondria thus giving rise to accumulation of the defected mitochondria and resulting in apoptotic cell death.

NaBut and PD0325901 co-treatment causes redistribution of Ras from the plasma membrane to the cytosol and its co-localization with LC3

Oncogenic Ras provides both pro-autophagic and anti-apoptotic functions within the cell [55,56]. It is shown that autophagy provides survival of Ras-expressing tumor cells by maintenance of the mitochondrial function [6,7,10]. We showed that the inhibition of MEK/ERK pathway in senescent ERas cells leads to accumulation of damaged mitochondria due to defects of autophagy. We wondered whether the oncogenic Ras and E1A content is a subject of change due to autophagy during PD0325901 treatment of ERas senescent cells. We wanted to know whether the expression and localization of anti-apoptotic Ras oncoprotein, which is also a key regulator of autophagy and cell viability, can change in ERas cells treated with PD0325901, NaBut or both inhibitors together. Western blot data show that unlike treatment with PD0325901 or NaBut alone, inhibition of MEK/ERK in senescent cells causes a decrease in the Ras content (Fig. 9A). Similarly, the expression of complementing E1A oncoprotein did not decrease in PD0325901 treated cells. However, overall amount of E1A decreased in NaBut-treated ERas cells with increasing time and then significantly downregulated with development of senescence.

The most significant changes occur in the colocalization of the Ras protein. While in untreated control, in PD0325901-treated and in senescent ERas cells the Ras protein is localized under plasma membrane (Fig. 9B), no membrane-bound Ras has been detected in senescent cells upon 72 h of PD0325901 treatment. Immunofluorescence staining using antibodies against Ras and LC3 indicates that Ras colocalizes with LC3 in the cytoplasm of cells co-treated with NaBut and PD0325901 for 24 h. Thus, Ras re-localizes from the membranes to the cytoplasmic structures in senescent cells upon PD0325901 treatment, where it most possibly fails to function as a regulator of signal transduction and autophagy. Thus, in senescent cells PD0325901 does not only inhibit the MER/ERK pathway, but also promotes the oncogenic Ras re-localization, thereby disrupting its function as an autophagy regulator in senescent ERas cells [7,8].

To clarify whether the cell response to MEK/ERK inhibition by PD0325901 is specific for the ERas-engineered cell line or inhibition of MEK/ERK signaling would successfully eliminate also other Ras-expressing senescent tumor cells, we used Ki-Ras mutated A549 human lung adenocarcinoma cells. Inhibition of MEK/ERK pathway in A549 cells simultaneously with induction of senescence by NaBut leads to a decrease of cell viability at 72-120 h of treatment (Fig. 10B). Сells undergo non-apoptotic death according to nucleosomal DNA fragmentation analysis (Fig. 10C). According to immunofluorescent staining data, A549 cells show colocalization of LAMP1 and LC3 at 72 h upon MEK/ERK suppression, whereas no LAMP1 signal has been detected 120 h after treatment (Fig. 10D). Our data indicate that MEK/ERK suppression in control A549 cell results in the induction of autophagy that lasts for 72 h. A pronounced LAMP1 staining can be detected in senescent A549 cells nearby the nuclei at 120 h post treatment. In addition, LAMP1 colocalizes with LC3 upon 120 h treatment. However, senescent A549 cells with suppressed MEK/ERK pathway show very weak LAMP1 signal at 120 h of treatment, and no LAMP1-LC3 colocalization can be detected in these cells (Fig. 10D). These data allow us to conclude that in senescent A549 cells lysosomal activity and autophagy are disrupted by PD0325901 treatment. Also, A549 cells induced to senescence by NaBut and treated with PD0325901 demonstrate Ras co-localization with LC3 in the cytoplasm, implying that this phenomenon may be common for various Ras-expressing cells (Fig. 10E).

Cell culture and treatment

The work has been performed using rat embryonic fibroblasts, co-transformed with E1A region of Ad5 DNA and human cHa-Ras oncogene (Q12 and G61 mutated), and human non-small lung adenocarcinoma A549 cells. Cells were cultured in DMEM with 10% FBS (HyClone), penicillin and streptomycin in 5% CO2 at 37 °C. Histone deacetylase inhibitor sodium butyrate (NaBut, 4 mM, Sigma) was used to induce senescence. PD0325901 (PD, 1 µM, Sigma) was used to inhibit MEK1,2 activity and ERK1,2 phosphorylation. Compound C (2 µM, Calbiochem, CAS 866405-64-3) and AICAR (Calbiochem, CAS 2627-69-2) were used to determine the role of AMPK in cellular response to NaBut and PD0325901. Bafilomycin A1 (Calbiochem, 196000-10UG, 10 nM) was used to inhibit autophagy in senescent or PD0325901 treated cells.

Cell proliferation analysis

Cells were seeded at the initial density of 3x10⁴ cells per 35 mm cell culture dish (Costar, USA) in 3 repeats 24 h before the treatment. Cells were treated with NaBut and PD0325901 or left untreated. The medium was replaced by a fresh one supplemented with 10% FBS and inhibitors every second day. The number of cells was daily counted in the cell counting chambers throughout 120 h. The growth curves were plot based on the data obtained in 3 independent experiments.

Flow cytometry

Cells were seeded at initial density 15x10⁴ per 60 mm cell culture dish (Costar, USA) and treated with inhibitors for indicated time. Cells were harvested in phosphate-buffered solution, permeabilized with 0,01% saponin and incubated with 100 µg/ml RNAse A and 40µg/ml propidium iodide for 15 min at 37°C. Cell cycle distribution was analyzed using Odam (Brucker, France) flow cytometer.

Cell viability assay

For clonogenic survival assay, cells were seeded at initial density 3x10⁴ cells per 35 mm cell culture dish and treated with inhibitors for 72 and 120 h. At the indicated time points, cells were trypsinized and plated at the density of 200 cells per 35 mm cell culture dish in fresh, inhibitor-free medium. After 7 days of cultivation, colonies were stained with Crystal Violet counted. For regrowth assay, cells were treated with inhibitors for 120 h, then medium was changed for inhibitor-free one and cells were left to grow. After 120 h, dishes were stained with Crystal Violet. For MTT assay, cells were seeded at 12-well plate at initial density 2x10⁴ cells per well and treated with inhibitors for indicated time. Then medium was replaced with 0,5 mg/ml MTT reagent solution and cells were incubated for 1 h at 37°C. After 1 h, MTT solution was replaced with DMSO to dissolve formazan, and cells were incubated for 30 min. Amount of formazan was measured at 572 nm wavelength, using DMSO as a blank solution. The resulting optical densities were normalized to optical density of untreated control cells, measured in 24 h after seeding.

Senescence-associated β-galactosidase detection

SA-β-Gal expression was analyzed as previously described [83]. Images were acquired using LSM5 Pascal microscope (Carl Zeiss Microscopy). Colorimetric analysis was performed at 595 nm wavelength and normalized to cell number.

Lysosomal activity

Lysosomal β-galactosidase was analyzed as previously described [83], using acetate/phosphate buffer adjusted to pH 4.0. Images were acquired using LSM 5 Pascal microscope (Carl Zeiss Microscopy).

Protein content and cell size

Cells were treated with inhibitors for 72 h, then 1x10⁵ cells were collected and lysed in RIPA buffer. Protein concentration was measured using Bradford method. To evaluate cell size, cells were treated with inhibitors for 72 h, then harvested and subjected to forward light scattering analysis using Odam (Brucker, France) flow cytometer.

Analysis of lactate level in culture medium

Cells were grown in 12-well plates in triplicates and treated with inhibitors. At the indicated time points medium was collected, centrifuged using Amicon filters (Sigma-Aldrich, Z740170-8EA), and lactate was measured using a BioVision kit (BioVision, #K607-100) as described by manufacturer.

ROS levels

Cell were treated with inhibitors for the indicated time points and then incubated with 10 μM 2′-7′-dichlorodihydrofluorescene diacetate (DCF-DA, Invitrogen D399) for 30 min. DCF fluorescence was measured using FLUOstar Omega (BMG Labtech) microplate reader and normalized to cell number.

DNA fragmentation assay

Cells were lysed in a buffer containing 10 mM Tris-HCl (pH 8,0), 0,1M EDTA (pH 8,0) and 0,5% SDS for 20 min at 4°C. Then samples were centrifuged and NaCl (0,15 M) and RNAse A (100 µg/ml) were added to supernatant. The probes were incubated for 1 h at 37° С. Then samples were incubated with 0,5% SDS and 200 µg/ml Proteinase K and incubated for 2 h at 37° С. DNA was deproteinized by a mixture of water-saturated phenol pH 8.0/chloroform. DNA was reprecipitated, washed with 70% of ethanol, and dried. For electrophoresis, DNA was dissolved in 1 mM TE buffer pH 8.0 and subjected to electrophoresis in 2% agarose gel (Sigma). DNA bands were stained with ethidium bromide. DNA isolated from ERas cells starved for 24 h in 0.5% FCS undergoing apoptosis [84] was used as a positive control.

Western-blotting

Cells were lysed in RIPA buffer (1% IGEPAL, 0,5% Sodium deoxycholate; 0,1% SDS; 50 mM TRIS-HCl pH 8,0; 150 mM sodium chloride; 5 mM EDTA pH 8,0; 60 mM sodium fluoride) supplemented with the Roche Protease inhibitor cocktail. 50 µg of protein were separated by SDS-PAGE electrophoresis in 15%, 12% or 10% gels and transferred to PVDF membranes (0,2 µM, EMD Millipore). Membranes were blocked with 5% fat-free milk (Sigma) for 1 h following the incubation with primary antibodies in TBS solution containing 3% BSA and 0,1% Tween-20 overnight at 4°C. The membranes were washed and incubated with secondary antibodies conjugated with horseradish peroxidase. Blots were developed by enhanced chemiluminescence (ECL, ThermoFisher Scientific). GelPro3 Software (Media Cybernetics) was used to perform densitometry.

Immunofluorescence

Cells were seeded at coverslips at initial density 5x10⁴ cells per 35 mm cell culture dish and treated with inhibitors at the indicated time points. Cells were fixed, permeabilized with 0,2% TRITON X100, blocked in 3% BSA in TBS-T, and incubated in primary antibodies overnight at +4°C following the incubation with secondary antibodies for how long and what temperature. Coverslips were mounted with DAPI-containing ProLong Gold mounting medium (Molecular Probes, P36931). Cells were visualized using Leica TSC SP5 microscope (Leica Microsystems).

Caspase activity assay

Cells were lysed in Lysis buffer (50 mM HEPES-HCl, pH 7,4; 0,5% IGEPAL; 0,1% CHAPS; 5 mM DTT) and incubated for 30 min at 4°C. The protein concentration was measured and 50 µg of protein was incubated for 1h at 37°C in the dark with with Reaction buffer (20 mM HEPES-HCL, pH 7,4; 0,1% CHAPS; 5 mM DTT) containing fluorogenic substrates: Ac-DEVD-AMC (EMD Millipore) for caspase-3, Ac-VETD-AMC (Sigma) for caspase-8 and Ac- LEHD-AFC (EMD Millipore) for caspase-9. Serum-starved ERas cells were used as a positive control [84]. Fluorescence was measured at following wavelengths: Ex/Em=380/460 nm for caspase 3 substrate, 435/538 nm for caspase 8 substrate, 400/505 nm for caspase 9 substrate.

Mitotracker Red and Mitotracker Green staining for detection of mitochondrial integrity

Cells were seeded on coverslips and treated with the inhibitors for indicated time. Then cells were stained with Mitotracker Red and Mitotracker Green (Invitrogen, M7513 and M7514, respectively) as described by manufacturer. The intensity of fluorescence was visualized using Leica TCS SP5 microscope (Leica Microsystems) at following wavelengths: Ex/Em=577/599 nm for Mitotracker Red, 490/516 nm for Mitotracker Green. Images were analyzed using ImageJ software.

Transmission electron microscopy

For transmission electron microscopy, cells were seeded on coverslips and treated with inhibitors, fixed for 1 h at 4°C in 2,5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3), containing 0,15 M sucrose, postfixed for 1 h in 1% osmium tetroxide in the cacodylate buffer, dehydrated and embedded in a mixture of Epon and Araldit. Ultrathin sections were cut using a diamond knife on a LKB ultratome, collected on fine mesh copper grids, and stained with uranyl acetate and lead citrate for the examination with Zeiss Libra 120 electron microscope operated at 80 kV.

Statistical analysis

Data are presented as means of three independent experiments; statistical significance was evaluated by Student’s t test, where P<0,05 was considered statistically significant.

Antibodies

The following primary antibodies were used: pan-LC3 (MBL, #PM036); p62/SQSTM1 (BD Transduction, #610077); LAMP1 (Santa-Cruz, sc-17768); pan-Ras (Oncogene Science, #OP40); E1A (Santa-Cruz, sc-58658); phospho-Ulk1 Ser757 (Cell Signaling, #6888S); phospho-Ulk1 Ser555 (EMD Millipore, ABC124); phospho-AMPK T172 (Cell Signaling, #2535S); phospho-4E-BP1 Thr37/46 (Cell Signaling, #2855S); phospho-S6 Ser235/236 (Cell Signaling, #2211S); phospho-42/44 MAPK Thr202/Tyr204 (Cell Signaling, #4377S); phospho-Akt Ser473 (Cell Signaling, #4060S); phospho-p38 MAPK Thr180/Tyr182 (Cell Signalling, #9211); mTOR (Cell Signaling, #2983S). The following secondary antibodies were used: Goat-anti-Rabbit IgG (H+L) Alexa Flour 488 (Invitrogen, A11088); Rabbit-anti-Mouse IgG (H+L) Alexa Fluor 568 (Invitrogen, A11031); Goat-anti-Rabbit IgG HRP Conjugated (Sigma-Aldrich, A0545); Rabbit-anti-Mouse IgG HRP Conjugated (Sigma-Aldrich, A9044).