3-methyladenine Pretreatment Research Articles

Repeated radon exposure induced ATM kinase-mediated DNA damage response and protective autophagy in mice and human bronchial epithelial cells.

Radon ( 222 Rn) is a naturally occurring radioactive gas that has been closely linked with the development of lung cancer. In this study, we investigated the radon-induced DNA strand breaks, a critical event in lung carcinogenesis, and the corresponding DNA damage response (DDR) in mice and human bronchial epithelial (BEAS-2B) cells. Biomarkers of DNA double-strand breaks (DSBs), DNA repair response to DSBs, ataxia-telangiectasia mutated (ATM) kinase, autophagy, and a cell apoptosis signaling pathway as well as cell-cycle arrest and the rate of apoptosis were determined in mouse lung and BEAS-2B cells after radon exposure. Repeated radon exposure induced DSBs indicated by the increasing expressions of γ-Histone 2AX (H2AX) protein and H2AX gene in a time and dose-dependent manner. Additionally, a panel of ATM-dependent repair cascades [i.e. non-homologous DNA end joining (NHEJ), cell-cycle arrest and the p38 mitogen activated protein kinase (p38MAPK)/Bax apoptosis signaling pathway] as well as the autophagy process were activated. Inhibition of autophagy by 3-methyladenine pre-treatment partially reversed the expression of NHEJ-related genes induced by radon exposure in BEAS-2B cells. The findings demonstrated that long-term exposure to radon gas induced DNA lesions in the form of DSBs and a series of ATM-dependent DDR pathways. Activation of the ATM-mediated autophagy may provide a protective and pro-survival effect on radon-induced DSBs.

Silica nanoparticles induce ferroptosis of HUVECs by triggering NCOA4-mediated ferritinophagy

Silica nanoparticles (SiNPs) have been widely used in electronics, chemistry, and biomedicine. Human exposure to SiNPs and possible health effects have attracted much attention. The potential cardiovascular toxicity of SiNPs and their related mechanisms are still unclear. Therefore, in this study, we investigated the toxic effects of SiNPs on human umbilical vein endothelial cells (HUVECs). We found that SiNPs could induce HUVECs ferroptosis. The results showed that the level of intracellular divalent iron and lipid peroxidation increased, and mitochondrial cristae decreased. In addition, the pretreatment of the iron chelator deferoxamine mesylate (DFO) could alleviate the ferroptosis of cells. Interestingly, pretreatment of 3-methyladenine (3-MA), an autophagy/PI3K inhibitor could partially inhibit autophagy and reduce ferroptosis, which indicated that autophagy played an important role in cell ferroptosis. Additionally, after knocking down nuclear receptor coactivator 4 (NCOA4), Ferritin Heavy Chain 1 (FTH1) expression was up-regulated, and the levels of divalent iron and lipid peroxidation decreased, which suggested that NCOA4 mediated the ferroptosis of HUVECs induced by SiNPs. In conclusion, this study shows that SiNPs can induce cardiovascular toxicity in which there is ferroptosis. NCOA4-mediated ferritinophagy and resultant ferroptosis by SiNPs may play an important role. This study provides a new theoretical strategy for the treatment and prevention of cardiovascular diseases in the future.

Curcumin Protects Human Dermal Fibroblasts Exposed to Hydrogen Peroxide by Regulating Autophagy Level and Reactive Oxygen Species Generation.

Curcumin is getting more and more attention in wound healing and scar prevention because of its wide range of pharmacological effects, such as anti-inflammation, antioxidant, and anti-fibrosis. The activity of fibroblasts suffering from oxidative stress is reduced, affecting wound repair. In this study, we investigated whether curcumin treatment (10 μM, 24 hours) had protective effects on human dermal fibroblasts (HDFs) exposed to hydrogen peroxide (H2O2, 300 μM, 12 hours). We found that curcumin alleviated H2O2-induced accumulation of reactive oxygen species (ROS, the fold change relative to the untreated control was 1.75 [SD ± 0.21] vs 5.23 [SD ± 0.51], P < .001) and improved the expression and activities of antioxidant enzymes superoxide dismutase 1 (66.61 U [SD ± 7.47] vs 46.39 U [SD ± 6.82]/106 cells, P < .05) and catalase (9.77 U [SD ± 1.82] vs 4.61 U [SD ± 0.94]/106 cells, P < .01), accompanied with increased cell proliferation and migration but decreased senescence. In addition, we found that curcumin reduced the inhibition of autophagy by H2O2, as manifested in the increased autophagic vacuoles (P < .05) and higher expression of autophagy-related proteins including phosphoinositide-3-kinase class III (P < .001), light chain 3 form II (P < .001), and Beclin1 (P < .01). However, intracellular redox status deteriorated again and curcumin's protection effects were partially canceled after autophagy was inhibited by 3-methyladenine pretreatment. These data suggest that rescue of HDFs from oxidative damage by curcumin may related to the regulation of autophagy levels and ROS generation.

Intermittent hypoxia-induced autophagy via AMPK/mTOR signaling pathway attenuates endothelial apoptosis and dysfunction in vitro.

The aim of this study was to examine whether or notintermittent hypoxia (IH) upregulated autophagy and the contributions of autophagy to endothelial apoptosis and dysfunction in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated under normoxia and IH conditions. After 3-, 6-, 12-, and 24-h exposure, the autophagic vacuoles and autophagosomes were observed by transmission electron microscopy and monodansylcadaverine staining. The protein levels of autophagy-related biomarkers and AMPK/mTOR pathway were measured by Western blot. The apoptosis-related proteins and the percentage of apoptotic cells were evaluated by Western blot and flow cytometry, respectively, while the levels of endothelial function biomarkers were assessed by ELISA. IH induced autophagy, as determined by the increased numbers of the autophagic vacuoles, autophagosomes, and by the elevated levels of Beclin-1 protein, the LC3II/LC3I ratio, and p62 degradation. IH-induced autophagic flux peaked at 12-h duration and weakened at 24 h. IH increased the ratio of p-AMPK/AMPK and decreased the ratio of p-mTOR/mTOR, while compound C restored the alteration. A significant decrease in the Bcl-2 level and the Bcl-2/Bax ratio and a significant increase in the protein expression levels of Bax and cleaved caspase 3 and in the percentage of apoptosis were observed under IH exposure. Moreover, the NO level was reduced, while the ET-1 and VEGF levels were raised under IH condition. These alterations were suppressed by the pretreatment of 3-methyladenine. IH upregulates autophagy through AMPK/mTOR pathway in HUVECs in vitro, which might be protective against endothelial apoptosis and dysfunction caused by IH.

Rapamycin and 3-Methyladenine Influence the Apoptosis, Senescence, and Adipogenesis of Human Adipose-Derived Stem Cells by Promoting and Inhibiting Autophagy: An In Vitro and In Vivo Study

We aimed to clarify the changes in apoptosis, proliferation, senescence, and adipogenesis after promoting and inhibiting autophagy in adipose-derived stem cells (ADSCs) by rapamycin and 3-methyladenine in vitro and in vivo. After rapamycin and 3-methyladenine pretreatment, ADSC autophagy was detected by immunofluorescence for LC3, RT-PCR for ATG genes, and western blotting (WB) for the LC3 II/I and p62 proteins. TUNEL staining, PCR of BAX, and WB of Caspase-3 were preformed to assess ADSC apoptosis. The adipogenesis of ADSCs was evaluated by Oil red O staining and PCR of PPAR-γ. CCK8 assays were conducted to detect proliferation. Senescence was tested by Sa-β-gal staining and PCR of the P16/ 19/21 genes. Moreover, the mass and volume retention rate were determined, and perilipin and CD31 staining were performed in vivo. Rapamycin and 3-methyladenine pretreatment increased and decreased autophagy of ADSCs, respectively, under normal and oxygen-glucose deprivation conditions. Apoptosis and senescence of ADSCs were decreased, and adipogenesis was increased along with the upregulation of autophagy. However, the proliferation of ADSCs was inhibited after either rapamycin or 3-methyladenine pretreatment. In vivo, the volume and mass retention rate and the angiogenesis of the grafts were also improved after rapamycin pretreatment. Rapamycin pretreatment reduced apoptosis, delayed senescence, and promoted adipogenesis of ADSCs. These effects were inhibited by 3-methyladenine, indicating that the changes may be mediated by autophagy. Moreover, the survival rate and angiogenesis of the grafts were increased after upregulation of ADSC autophagy in vivo, which may help improve the efficiency of clinical fat transplantation. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

RSL3 induced autophagic death in glioma cells via causing glycolysis dysfunction

RSL3 is a type of small molecular compound which can inactivate glutathione peroxidase 4 (GPX4) and induce ferroptosis, but its role in glioma cell death remains unclear. In this study, we found RSL3 inhibited the viabilities of glioma cells and induced glioma cell death in a dose-dependent manner. In vitro studies revealed that RSL3-induced cell death was accompanied with the changes of autophagy-associated protein levels and was alleviated by pretreatment of 3-Methyladenine, bafilomycin A1 and knockdown of ATG5 with siRNA. The ATP and pyruvate content as well as the protein levels of HKII, PFKP, PKM2 were decreased in cells treated by RSL3, indicating that RSL3 induced glycolysis dysfunction in glioma cells. Moreover, supplement of exterior sodium pyruvate, which was a final product of glycolysis, not only inhibited the changes of autophagy-associated protein levels caused by RSL3, but also prevented RSL3-induced cell death. In vivo data suggested that the inhibitory effect of RSL3 on the growth of glioma cells was associated with glycolysis dysfunction and autophagy activation. Taken together, RSL3 induced autophagic cell death in glioma cells via causing glycolysis dysfunction.

Adipose derived mesenchymal stem cells alleviated osteoarthritis and chondrocyte apoptosis through autophagy inducing.

We aim to explore the effect of adipose derived mesenchymal stem cells (ADMSCs) on a knee osteoarthritis rat model and analyze how ADMSCs affect chondrocyte apoptosis. A surgically induced rat knee osteoarthritis (OA) model was constructed. ADMSCs were engrafted into the right knee cavity. Hematoxylin and eosin (H&E), Masson, and Safranin O were used to compare the histopathology of synovial membrane and cartilage. Immunohistochemical (IHC) was used to measure MMP-13, Collagen 2 (Col-2), Caspase-3 (Cas-3), PARP, p62, LC3b, DDR-2, FGFR-1, Wnt, P-AKT/AKT, p-CAMKII/CAMKII, and p-Smad1/Smad1 expression in the articular cartilage. qPCR and Western blot analysis were used to detect mRNA and protein levels of markers in chondrocytes. TUNEL and Annexin-V were used to assess apoptosis. Histological analysis showed that ADMSCs alleviated the deterioration of cartilage and osteoarthritis. ADMSCs coculture increase the expression of Col2 and Sox-9, while down regulated MMP-13 in IL-1β stimulated chondrocytes. ADMSCs decreased proinflammatory cytokines IL-1β, IL-6, and TNF-α. ADMSCs enhanced the viability of IL-1β stimulated chondrocytes. ADMSC attenuated chondrocyte apoptosis. The pretreatment of 3-methyladenine (3-MA) reversed the reduction of Caspase-3 caused by ADMSCs, showing that the antiapoptotic effect was associated with autophagy inducing. ADMSCs significantly reduced the expression of FGFR-1, DDR-2, and Wnt in IL-1β stimulated chondrocytes. ADMSCs reduced the ratio of p-Smad1/Smad1 and p-CAMK II/CAMKII, and increased the ratio of p-AKT/AKT. ADMSCs treatment alleviate osteoarthritis in rat OA models. AMDSCs reduced the secretion of proinflammatory cytokines and protected against apoptosis through autophagy inducing. ADMSCs' function could be related to multiple signaling pathway.

CHOP favors endoplasmic reticulum stress-induced apoptosis in hepatocellular carcinoma cells via inhibition of autophagy.

C/EBP-homologous protein (CHOP) is an important component of the endoplasmic reticulum (ER) stress response. We demonstrated the induction of ER stress in response to tunicamycin stimulation, as evidenced by increased expression of chaperone proteins Grp78, Grp94, and enhanced eukaryotic initiation factor 2 subunit 1 (eIF2α) phosphorylation in hepatocellular carcinoma cells. Tunicamycin-induced ER stress resulted in apoptosis and autophagy simultaneously. While inhibition of autophagy mediated by 3-methyladenine pretreatment or direct knockdown of LC3B promoted cell apoptosis, activation of autophagy with rapamycin decreased tunicamycin- induced apoptosis in HCC cells. Furthermore, CHOP was shown to be significantly upregulated upon treatment with tunicamycin in HCC cells. Specific knockdown of CHOP not only enhanced tunicamycin-induced autophagy, but also significantly attenuated ER stress-induced apoptosis in HCC cells. Accordingly, simultaneous inhibition of autophagy in HCC cells with CHOP-knockdown could partially resensitize ER stress-induced apoptosis. Taken together, our data indicate that CHOP may favor ER stress-induced apoptosis in HCC cells via inhibition of autophagy in vitro.

Autophagy mediates free fatty acid effects on MDA-MB-231 cell proliferation, migration and invasion

Epidemiological and animal studies indicate an association between high levels of dietary fat intake and an increased risk of breast cancer. The multifaceted role of autophagy in cancer has been revealed in previous years. However, the mechanism of this role remains unknown. In the present study, the two most common free fatty acids, palmitate acid (PA) and oleic acid (OA), were used to determine the effect on human breast cancer MDA-MB-231 cells, and the possible role of autophagy was investigated by detecting light chain 3 (LC3)-II/I. Bafliomycin A1 was used to detect autophagy flux. High palmitate acid condition-induced MDA-MB-231 cell death and invasion were mitigated by 3-methyladenine pretreatment or transfection with shRNA against autophagy protein 5. By contrast, high oleic acid condition induced MDA-MB-231 cell proliferation, migration and invasion were mitigated using rapamycin. The present results suggest that autophagy has an important role in the effects of PA and OA on breast cancer growth and metastasis in vitro.

Autophagy Induced by Areca Nut Extract Contributes to Decreasing Cisplatin Toxicity in Oral Squamous Cell Carcinoma Cells: Roles of Reactive Oxygen Species/AMPK Signaling.

Chewing areca nut is closely associated with oral squamous cell carcinoma (OSCC). The current study aimed to investigate potential associations between areca nut extract (ANE) and cisplatin toxicity in OSCC cells. OSCC cells (Cal-27 and Scc-9) viability and apoptosis were analyzed after treatment with ANE and/or cisplatin. The expressions of proteins associated with autophagy and the AMP-activated protein kinase (AMPK) signaling network were evaluated. We revealed that advanced OSCC patients with areca nut chewing habits presented higher LC3 expression and poorer prognosis. Reactive oxygen species (ROS)-mediated autophagy was induced after pro-longed treatment of ANE (six days, 3 μg). Cisplatin toxicity (IC50, 48 h) was decreased in OSCC cells after ANE treatment (six days, 3 μg). Cisplatin toxicity could be enhanced by reversed autophagy by pretreatment of 3-methyladenine (3-MA), N-acetyl-l-cysteine (NAC), or Compound C. Cleaved-Poly-(ADP-ribose) polymerase (cl-PARP) and cleaved-caspase 3 (cl-caspase 3) were downregulated in ANE-treated OSCC cells in the presence of cisplatin, which was also reversed by NAC and Compound C. Collectively, ANE could decrease cisplatin toxicity of OSCC by inducing autophagy, which involves the ROS and AMPK/mTOR signaling pathway.

Neuroprotective effects of autophagy inhibition on hippocampal glutamate receptor subunits after hypoxia-ischemia-induced brain damage in newborn rats.

Autophagy has been suggested to participate in the pathology of hypoxic-ischemic brain damage (HIBD). However, its regulatory role in HIBD remains unclear and was thus examined here using a rat model. To induce HIBD, the left common carotid artery was ligated in neonatal rats, and the rats were subjected to hypoxia for 2 hours. Some of these rats were intraperitoneally pretreated with the autophagy inhibitor 3-methyladenine (10 mM in 10 μL) or the autophagy stimulator rapamycin (1 g/kg) 1 hour before artery ligation. Our findings demonstrated that hypoxia-ischemia-induced hippocampal injury in neonatal rats was accompanied by increased expression levels of the autophagy-related proteins light chain 3 and Beclin-1 as well as of the AMPA receptor subunit GluR1, but by reduced expression of GluR2. Pretreatment with the autophagy inhibitor 3-methyladenine blocked hypoxia-ischemia-induced hippocampal injury, whereas pretreatment with the autophagy stimulator rapamycin significantly augmented hippocampal injury. Additionally, 3-methyladenine pretreatment blocked the hypoxia-ischemia-induced upregulation of GluR1 and downregulation of GluR2 in the hippocampus. By contrast, rapamycin further elevated hippocampal GluR1 levels and exacerbated decreased GluR2 expression levels in neonates with HIBD. Our results indicate that autophagy inhibition favors the prevention of HIBD in neonatal rats, at least in part, through normalizing GluR1 and GluR2 expression.

Canonical autophagy does not contribute to cellular radioresistance

Background(Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. Materials and methodsChanges in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. ResultsIR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. ConclusionOur data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation.

The synergistic effect of combination temozolomide and chloroquine treatment is dependent on autophagy formation and p53 status in glioma cells

Temozolomide (TMZ) is an alkylating agent used for the treatment of glioblastoma. The late autophagy inhibitor chloroquine (CQ) inhibits glioblastoma tumors in a p53-independent and p53-dependent manner. We addressed a possible beneficial effect of combination treatment with TMZ and CQ by examining the molecular and cellular mechanism of co-treatment. Combination treatment of U87 cell (wild type p53) with TMZ and CQ synergistically reduced cell proliferation and enhanced apoptosis, with increased sub-G1 hypodiploid cells and caspase activation. This effect was abolished by a pan-caspase inhibitor, Z-VAD-FMK. TMZ induced autophagy, and the addition of CQ further increased autophagic vacuoles. Inhibition of early stages of autophagy by Beclin 1 knockdown and 3-methyladenine pretreatment prevented the enhanced effect of the combination treatment. The combination treatment also upregulated p53 and phospho-p53 levels, whereas p53 knockdown or overexpression of mutant p53 abolished the combination effect. In contrast, combination therapy had no enhanced effect on U373 cell (mutant p53) proliferation and apoptosis within 3 d, although TMZ induced autophagy and co-treatment with CQ increased autophagic vacuole accumulation. However, long term combination treatment for 9–10 d effectively decreased clonal and cellular growth with increased G2–M arrest. This effect was also abolished by Beclin 1 knockdown. Our data support the beneficial effect of combination treatment with TMZ and CQ in glioma via differential autophagy-associated mechanisms, depending on p53 status.

Tetrahydrocurcumin, a major metabolite of curcumin, induced autophagic cell death through coordinative modulation of PI3K/Akt‐mTOR and MAPK signaling pathways in human leukemia HL‐60 cells

Autophagy (type II programmed cell death) is crucial for maintaining cellular homeostasis. Several autophagy-deficient or knockout studies indicate that autophagy is a tumor suppressor. Tetrahydrocurcumin (THC), a major metabolite of curcumin, has been demonstrated with anti-colon carcinogenesis and antioxidation in vivo. In the present study, we found that treatment with THC induced autophagic cell death in human HL-60 promyelocytic leukemia cells by increasing autophage marker acidic vascular organelle (AVO) formation. Flow cytometry also confirmed that THC treatment did not increase sub-G1 cell population whereas curcumin did with strong apoptosis-inducing activity. At the molecular levels, the results from Western blot analysis showed that THC significantly down-regulated phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase signalings including decreasing the phosphorylation of mammalian target of rapamycin, glycogen synthase kinase 3β and p70 ribosomal protein S6 kinase. Further molecular analysis exhibited that the pretreatment of 3-methyladenine (an autophagy inhibitor) also significantly reduced acidic vascular organelle production in THC-treated cells. Taken together, these results demonstrated the anticancer efficacy of THC by inducing autophagy as well as provided a potential application for the prevention of human leukemia.

Abstract C59: The effects of combined treatment of erlotinib and cisplatin in resistant lung cancer and the role of autophagy in response to chemotherapy

Abstract Introduction: Non-small cell lung cancer (NSCLC) accounts for about 80% of all cases of lung cancer. The treatment of choice for NSCLC is complete surgical resection. However, in advance stages with metastasis and/or aggressive local infiltrate chemotherapy becomes the only option. At high therapeutic doses, chemotherapy can cause significant side effects, and the development of resistance may arise if initial treatment doses of therapy become ineffective. In order to prevent serious side effects and resistance, low-dose combination regimens that offer comparable results to current high-dose regimens are needed. The combination of cisplatin and erlotinib has been shown to inhibit tumor growth more effectively than a single drug in NSCLC cells. However, the mechanism underlying this finding remains unclear. Autophagy has been suggested to play a critical role in cancer and chemotherapy, but there is ongoing controversy among its role (promotion vs. prevention of cancer). In this study, we examined whether the combination of low-dose cisplatin and erlotinib is able to induce higher levels of cancer cell death than each drug alone in both sensitive and erlotinib-resistant lung adenocarcinoma. In addition, we looked into the role of autophagy in cancer cells and its association with chemotherapy. Methods: Alamar blue assay was used to construct dose response curves in which IC 50 values of cisplatin and erlotinib were determined. Erlotinib-resistant PC9 (PC9/ER) cells were developed by culturing PC9 (EGFR overexpression adenocarcinoma) cells in media containing an erlotinib dose 1000 times the IC50 value over a two week period; PC9/ER cells were verified to be 26-fold more resistant than PC9 sensitive cancer cells. Baseline levels of autophagy of normal human bronchial epithelial (NHBE) cells, PC9, and PC9/ER were examined by Western blot with antibodies against LC3I/II. The IC 25 of each drug was chosen as the low-dose treatments (3 uM cisplatin and 10 nM erlotinib). Cells were treated accordingly: control, cisplatin, erlotinib, and combination. Cell death and autophagy levels were measured. Next, cells were pretreated in one day in advance with rapamycin and cell death and changes in autophagy were assessed. Results: At baseline, there were higher levels of LC3II in PC9/ER compared to PC9 and NHBE. Combined treatment of cisplatin and erlotinib was able to induce significantly more cell death than individual drugs at the same dose (p&amp;lt;0.001). In fact, combination treatment provided synergistic killing effects in PC9 and PC9/ER cells. In PC9 cells, erlotinib alone had 79.9% survival, cisplatin 76.3%, and combination 45.0%. In PC9/ER, erlotinib alone had 96.6% survival, cisplatin 89.3%, and combination 61.1%. Levels of autophagy decreased in a similar pattern to killing-effects in both PC9 and PC9/ER cells. In addition, a significant increase in cell survival of about 20% was observed when pretreated with rapamycin (p&amp;lt;0.001). NHBE demonstrated no significant killing and no changes in autophagy levels. Conclusion: Our data is consistent for autophagy's role in promoting cancer cell survival, work as a protective mechanism for cancer-resistant cells, and a critical mechanism in chemotherapy-induced cancer cell death. The combination of erlotinib and cisplatin offers synergistic killing properties in vitro and may be a potential regimen against resistant lung cancer. In addition, the combination treatment does not induce significant cytotoxic effects and death in NHBE cells indicating its safe dosing. We plan to further characterize autophagy with 3-methyladenine pretreatment, changes in apoptosis, and invasiveness of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C59.