Green Fluorescent Protein-light Chain Research Articles

RETRACTED: Allicin promotes autophagy and ferroptosis in esophageal squamous cell carcinoma by activating AMPK/mTOR signaling

The antitumor effects of allicin have been demonstrated in various cancers. However, whether allicin improves esophageal squamous cell carcinoma (ESCC) has not yet been explored. The present study aimed to explore the function and underlying mechanism of action of allicin in ESCC treatment. Our data showed that allicin significantly suppressed ESCC cell proliferation in a dose- and time-dependent manner. A green fluorescent protein-light chain 3 (LC3) transfection assay showed that autophagosomes were elevated in ESCC cells treated with allicin compared with control ESCC cells and that 3-methyladenine (an autophagy inhibitor) reversed allicin-induced LC3 puncta. Furthermore, allicin significantly elevated the ratio of LC3II/LC3I but decreased p62 expression in ESCC cells. Allicin also increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation but decreased that of the mechanistic target of rapamycin kinase (mTOR), which then induced the elevation of autophagy-related 5 and autophagy-related 7 proteins in ESCC cells. Furthermore, allicin treatment increased the expression of nuclear receptor coactivator 4 (a selective cargo receptor) but suppressed the expression of ferritin heavy chain 1 (the major intracellular iron-storage protein) in ESCC cells and elevated malondialdehyde and Fe2+ production levels. In vivo assays showed that allicin significantly decreased tumor weight and volume. In summary, allicin may induce cell death in ESCC cells by activating AMPK/mTOR-mediated autophagy and ferroptosis. Therefore, allicin may have excellent potential for use in the treatment of ESCC.

Dynorphin/KOR inhibits neuronal autophagy by activating mTOR signaling pathway to prevent acute seizure epilepsy.

In previous studies, we found that dynorphin exerts antiepileptic effect by activating the kappa opioid receptor (KOR). However, the role of neuronal autophagy in dynorphin/KOR-mediated antiepileptic is still unclear. This study aimed to investigate the molecular mechanism of dynorphin's antiepileptic effect by inhibiting autophagy and reducing neuronal apoptosis. Here, a pilocarpine-induced rat model of epilepsy was established and hippocampal neurons were treated with Mg2+ -free exposed for epileptiform activity induction. The real-time polymerase chain reaction and Western blot analysis were used to evaluate messengerRNA and protein expression. The TdT-mediated dUTP-biotin nick end labeling staining and flow cytometry were used to analyze cell apoptosis in vivo and in vitro. Neuron cells viability was detected by Cell Counting Kit-8 assay. Immunofluorescent staining and green fluorescent protein-light chain 3 immunofluorescence were used to measure autophagy in vivo and in vitro. Results showed that overexpression ofprodynorphinalleviated neuronal apoptosis, activated the mammalian target of rapamycin (mTOR) signaling pathway, and inhibited neuronal autophagy in epileptic rats. Dynorphin inhibited Mg2+ -free-induced seizure-like neuron apoptosis, partially reversing the effect of Mg2+ -free on the mTOR signaling pathway and seizure-like neuron autophagy. Further, using rapamycin, we found that dynorphin inhibited Mg2+ -free-induced seizure-like neuron autophagy and apoptosis by activating the mTOR signaling pathway. In conclusion, dynorphin inhibits autophagy by activating the mTOR signaling pathway and has a protective effect on epilepsy acute seizure and epilepsy-induced brain injury.

Autophagy induction and antiproliferative effect of a novel curcumin derivative MOMI-1 on the human lung cancer cells A549.

To date, there are some chemically synthesized curcumin derivatives which were produced and identified to evade the disadvantages of physiochemical stability and solubility of curcumin. Here, one novel curcumin derivative, (2-(3-{(1E)-{(E)-3-(4-hydroxy-3-methoxybenzylidene)-2-oxocyclohexylidene)methyl)-1H-indol-1-yl)acetic acid}, (abbreviated as MOMI-1) was first used to detect the antiproliferation activity with MTT assays in different cancer cells including A549 lung cancer cells, MCF-7, and HEPG2 cell lines, and exhibited its wide inhibition spectrum. Next, we found that MOMI-1 could induce autophagic genesis of A549 cells by acridine orange or monodansylcadaverine (MDC) staining and green fluorescent protein-light chain 3 (GFP-LC3) recombinant plasmid transfection analysis, respectively. Western blot analysis confirmed the LC3-I/II conversion, beclin-1 increase and p62 reduction of A549 cells after exposure of MOMI-1, which suggested the typical autophagy induction. The following cell cycle test showed that MOMI-1 could block A549 cells in G0/G1 phase. Furthermore, wounding healing experiment and transwell assays demonstrated that MOMI-1 also possessed the antimigration ability of A549 cells. Our current results confirmed that MOMI-1 could inhibit the proliferation and induce autophagy of A549 cells, which provide a new potential chemical candidate of antigrowth of A549 lung cancer cells. Future work needs to focus on the mechanism of autophagy pathway of A549 cells.

LINC00641 regulates autophagy and intervertebral disc degeneration by acting as a competitive endogenous RNA of miR-153-3p under nutrition deprivation stress.

Emerging evidence supports the involvement of autophagy in the pathogenesis of intervertebral disc degeneration (IDD). MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) play fundamental roles in various cellular processes, including autophagy. However, it remains largely unknown as to how autophagy is regulated by miRNAs and lncRNAs in IDD. Biological functions of miR-153-3p and long intergenic nonprotein coding RNA 641 (LINC00641) were investigated. Luciferase reporter assays was done to validate miR-153-3p targets. To induce nutritional stress, nucleus pulposus (NP) cells were cultured in the normal nutritional condition and the low nutritional condition. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to analyze miR-153-3p and LINC00641 in response to nutrient deprivation. Autophagic activity was assessed by transmission electron microscopy, western blot analysis and green fluorescent protein-light chain 3 puncta. Pull-down assay and RNA fluorescent in situ hybridization were performed to validate LINC00641 target and the location. MiR-153-3p is downregulated in NP tissues from IDD patients. Further, LINC00641 can affect collagen II and matrix metalloproteinase-3 expressions. Upregulation of LINC00641 and downregulation of miR-153-3p are detected in NP cells under nutritional stress. LINC00641 can regulate autophagic cell death by targeting miR-153-3p and autophagy-related gene 5 (ATG5). MiR-153-3p inhibits autophagy and IDD by targeting ATG5. More important, LINC00641 targets miR-153-3p, and thus affects ATG5 expression, autophagic cell death and IDD. These findings uncover a novel regulatory pathway that is composed of LINC00641, miR-153-3p, and ATG5 in IDD. This mechanism may stimulate to a more understanding of IDD pathogenesis and provide new sights for the treatment of this disorder.

Autophagy activation contributes to glutathione transferase Mu 1-mediated chemoresistance in hepatocellular carcinoma.

Glutathione transferase Mu 1 (GSTM1) induces cancer drug resistance by hydrolyzing cancer chemotherapeutics or activating the anti-apoptosis pathway. However, the chemoresistance-inducing mechanism of GSTM1 in hepatocellular carcinoma (HCC) remains unknown. In the present study, the expression of GSTM1 was examined in three HCC cell lines. Oxaliplatin and sorafenib were selected as chemotherapeutic agents. Small interfering RNA was used to decrease GSTM1 expression. Cell death was measured using MTT and annexin V/propidium iodide assays. Activation of autophagy was evaluated by green fluorescent protein-light chain 3 redistribution and analysis of autophagy-related 5 expression in MHCC97-H and Huh-7 cells. A stepwise increase in GSTM1 expression with increasing metastatic potential of HCC cell lines was revealed. Cell death induced by oxaliplatin and sorafenib was significantly increased following GSTM1-knockdown in MHCC97-H and Huh-7 cells. Activation of autophagy was significantly inhibited by silencing GSTM1 expression. The results of the present study suggest that GSTM1 may protect HCC cells against the effect of oxaliplatin treatment through activating autophagy. The present study provides a novel perspective on HCC drug-resistance.

Quantitative analysis of autophagy in green fluorescent protein-light chain 3 transgenic murine keratinocytes

Objective To explore a high-throughput method for quantitative analysis of autophago-somes. Methods Green fluorescent protein-light chain 3 (GFP-LC3) transgenic murine keratinocytes were randomly divided into 4 groups: control group receiving no treatment, starvation group subjected to starved culture, 20 J/cm2 ultraviolet A (UVA) group treated with 20 J/cm2 UVA radiation, and 40 J/cm2 UVA group treated with 40 J/cm2 UVA radiation. After 6-hour treatment, the cells were fixed, and images were acquired by confocal laser scanning microscopy. A macro was created by the ImageJ software to automatically quantify the GFP-LC3 puncta in the cells and the number of cells. Then, the level of autophagy was compared among different groups. Results By using the macro created by the ImageJ software, autophago-somes in the keratinocytes were successfully identified and quantified. Less than 0.6 second was needed for analyzing an image of 4.2 mega pixels in a test computer. The average number of autophagosomes in keratinocytes was significantly higher in the starvation group, 20-J/cm2 UVA group and 40-J/cm2 UVA group than in the control group whether with the treatment with pepstatin A (F = 20.05, P <0.05) or not (F = 5.01, P < 0.05) . This method could successfully differentiate the autophagy levels among the starvation group, UVA irradiation groups and control group. Conclusion A new high-throughput method, which can rapidly and accurately quantify GFP-LC3 puncta in cells, is established successfully to quantificationally detect autophagy. Key words: Autophagy; Keratinocytes; Green fluorescent proteins; Microscopy, confocal; Image processing, computer-assisted

CD147 modulates autophagy through the PI3K/Akt/mTOR pathway in human prostate cancer PC-3 cells

The multifunctional glycoprotein cluster of differentiation (CD)147 is highly expressed on the cell surface of the majority of cancer cells, and promotes tumor invasion, metastasis and growth. However, the role of CD147 in autophagy has not yet been explored in prostrate cancer cells. In the present study, prostate cancer PC-3 cells were cultured under starvation conditions, and the expression level of CD147 gradually increased. Therefore, RNA interference was used to inhibit CD147 expression, in order to investigate the biological role of this glycoprotein in autophagy progression. Autophagic activity was monitored by the changes in green fluorescent protein-light chain 3 (GFP-LC3) location and the expression of the autophagy-associated protein LC3-II. It was found that downregulation of CD147 significantly promoted GFP-LC3 puncta formation and the expression of LC3-II. Furthermore, the levels of phosphorylated serine/threonine protein kinase B (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR) were significantly decreased, and the level of LC3-II was inversely associated with levels of p-Akt and p-mTOR in cells with downregulated expression of CD147. The results of a trypan blue exclusion assay revealed that starvation-induced cell death was increased in PC-3/shCD147 cells compared with control PC-3/Scramble cells (37.7±6.4 vs. 21.7±5.5%). Together, these results indicate that CD147 may be important in the inhibition of autophagy via the PI3K/Akt/mTOR pathway, which prevents cell death from unrestrained autophagy.

Inhibition of autophagy augments apoptosis in human oral squamous cell carcinoma under nutrient depletion.

There has been little research conducted regarding autophagy in oral squamous cell carcinoma (OSCC). Given the prevalence of oral cancers which are OSCC and the severe side effects of current treatments, there is a pressing need to develop effective alternative therapies. In this study, we have endeavored to explore the biological characteristics of oral squamous cell carcinoma cell line KB cells, in particular with regard to the role played by autophagy in their survival. Autophagy was activated by nutrient depletion via culturing cells in Earle's balanced salts (EBSS) and was measured via indices relating to Beclin 1, microtubule-associated protein light chain 3 (MAPLC3, LC3), p62, and Green fluorescent protein-light chain 3 plasmid transfection (GFP-LC3). Cell death and apoptosis induced by nutrient depletion was measured using both MTT assay and flow cytometry (FCM). Compared to initial levels at 0 h, Beclin 1 density in EBSS-treated cells was found to have increased at 6, 12, and 18 h in a time-dependent manner and was found to have subsequently declined at 24 and 48 h. p62 levels, LC3-II/LC3-I ratio, and GFP-LC3 levels increased at 6, 12, 18, 24, and 48 h in a time-dependent manner. 3-methyladenine (3-MA) was found to inhibit autophagy and the expression of Beclin 1 and significantly enhanced nutrient depletion-induced apoptosis and death. We concluded that nutrient depletion enhances OSCC cell autophagy in time-course patterns and that the inhibition of autophagy augments apoptosis in OSCC cells. We also deduced that Beclin 1 takes part in the development and progression of autophagy, potentially playing an important role in the crosstalk between apoptosis and autophagy in OSCC cells. These findings suggest that nutrient depletion may be an effective way to explore autophagy and that autophagy inhibitors should be investigated as a potential novel agent for the adjuvant treatment of human OSCC.

Onjisaponin B Derived from Radix Polygalae Enhances Autophagy and Accelerates the Degradation of Mutant α-Synuclein and Huntingtin in PC-12 Cells

Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae—i.e., polygalacic acid, senegenin and onjisaponin B—onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level.

Inhibition of autophagy augments chemotherapy in human salivary adenoid cystic carcinoma

Although cisplatin (DDP)-based adjuvant chemotherapy is widely used in the treatment of salivary adenoid cystic carcinoma (SACC), SACCs have developed resistance to cisplatin, resulting in chemotherapy failure. Autophagy serves as a critical adaptive response, which was increased in tumor cells in chemotherapy. However, the function of autophagy is not clear in SACC. In this study, apoptosis induced by DDP in SACC high metastatic cell line (ACC-M) was revealed using MTT assay, flow cytometry, and caspase-3 immunoblotting. The autophagy activation induced by DDP treatment was measured by transmission electron microscopy, green fluorescent protein-light chain 3 plasmid transfection LC3 immunoblotting and p62 immunoblotting. 3-methyladenine (3-MA) or small interference RNA targeting beclin 1 (beclin 1 siRNA) inhibited autophagy and significantly enhanced DDP-induced apoptosis. ACC-M xenografts in nude mice further verified the synergistic effect of DDP and 3-MA. In conclusion, autophagy activation was caused to protect cancer cells from DDP-induced apoptosis and autophagy inhibition could be a promising strategy for adjuvant chemotherapy in SACC.

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7

BackgroundMetal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses. MethodsIn this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~30nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD50 doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM. ResultsThis study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis. ConclusionsA combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells. General significanceCuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.

Diarylquinoline compounds induce autophagy-associated cell death by inhibiting the Akt pathway and increasing reactive oxygen species in human nasopharyngeal carcinoma cells

Diarylquinoline compounds are newly synthesized derivatives of the new anti-tuberculosis drug, TMC207. In this study, nine diarylquinoline compounds were screened for cytotoxic activity against human tumor cells, and their mechanisms of action were investigated. Among the nine compounds, STM-57 [N-((6-bromo-2-methoxyquinolin-3-yl)(phenyl)methl)-N-(3,4-dichlorophenyl)-3-(4 -methylpiperazin-1-yl)propanamide] showed potent cytotoxic activity. STM-57 induced caspase-independent cell death in the human nasopharyngeal carcinoma cell line, CNE-2. Further investigation showed that STM-57 induced autophagy, as determined with the increased expression of green fluorescent protein-light chain3 (GFP-LC3) and increased LC3-II levels. STM-57 inhibited the phosphorylation of Akt and the mammalian target of rapamycin (mTOR) in CNE-2 cells. The intracellular calcium concentration and reactive oxygen species levels were increased in CNE-2 cells following treatment with STM-57, whereas the mitochondrial transmembrane potential (ΔΨm) and ATP concentrations were decreased.

Proteasome inhibitor interacts synergistically with autophagy inhibitor to suppress proliferation and induce apoptosis in hepatocellular carcinoma

The ubiquitin-proteasome system and autophagy-lysosome system are 2 major protein degradation pathways in eukaryotic cells, which are tightly linked to cancer. Proteasome inhibitors have been approved in clinical use against hematologic malignancies, but their application in solid tumors is uncertain. Moreover, the role of autophagy after proteasome inhibition is controversial. Two proteasome inhibitors, 2 autophagy inhibitors, and 3 hepatocellular carcinoma (HCC) cell lines were investigated in the current study. In vitro, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis was evaluated by flow cytometry analysis of annexin-V/propidium iodide staining, and autophagy was evaluated by green fluorescent protein-light chain 3 (GFP-LC3) redistribution and LC3 Western blot analysis. In vivo, Ki-67 staining was used to detect cell proliferation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was used to detect apoptosis, and electron microscopy and p62 immunohistochemical staining were used to detect autophagy. Proteasome inhibitors suppressed proliferation, induced apoptosis, and activated autophagy in HCC cell lines in vitro, and autophagy exerted a protective role after proteasome inhibition. In vivo, anticancer effects of bortezomib on the MHCC-97H orthotopic model (human HCC cells) were different from the effects observed on the Huh-7 subcutaneous model (human HCC cells). The autophagy inhibitor chloroquine interacted synergistically with bortezomib to suppress proliferation and induce apoptosis in both tumor models. The current results indicated that simultaneous targeting of the proteasome and autophagy pathways may represent a promising method for HCC treatment.

Proteasome activity and autophagosome content in liver are reciprocally regulated by ethanol treatment

The proteasome and autophagy are two major intracellular protein degradation pathways and the regulation of each by ethanol metabolism affects cellular integrity. Using acute and chronic ethanol feeding to mice in vivo, and precision-cut rat liver slices (PCLS) ex vivo, we examined whether ethanol treatment altered these proteolytic pathways. In acute studies, we gave C57Bl/6 mice either ethanol or phosphate-buffered saline (PBS) by gastric intubation and sacrificed them 12h later. PCLS were exposed to 0 or 50mM ethanol for 12 and 24h with or without 4-methylpyrazole (4MP). In chronic studies we pair-fed control and ethanol liquid diets for 4–6weeks to transgenic mice, expressing the green fluorescent protein (GFP) fused to the autophagic marker, microtubule associated protein-1 light chain 3 (LC3). Acute ethanol administration elevated autophagosomes (AVs), as judged by a 1.5-fold increase in LC3II content over PBS-gavaged control mice. Hepatic proteasome activity was unaffected by this treatment. Compared with controls, ethanol exposure for 12 and 24h to PCLS inhibited proteasome activity by 1.5- to 3-fold and simultaneously enhanced AVs by 2- to 5-fold. The decrease in proteasome activity and the rise in AVs both depended on ethanol oxidation as its inhibition by 4-methylpyrazole (4MP) blocked both proteasome inhibition and AV induction. Hepatocytes from mice chronically consuming ethanol exhibited a 1.6-fold decline in proteasome activity, and a 4-fold rise in GFP–LC3 puncta compared with pair-fed control mice. When we exposed hepatocytes from these animals to MG262, a proteasome inhibitor, LC3II puncta per cell further increased 2- to 5-fold over untreated cells. Conclusion: Our findings demonstrate that ethanol metabolism generates oxidants, the levels of which differentially influence the activities of the proteasome and autophagy.

Mitochondrial Extrusion through the Cytoplasmic Vacuoles during Cell Death

Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.

Human Methyl Purine DNA Glycosylase and DNA Polymerase β Expression Collectively Predict Sensitivity to Temozolomide

Overexpression of N-methylpurine DNA glycosylase (MPG) has been suggested as a possible gene therapy approach to sensitize tumor cells to the cell-killing effects of temozolomide, an imidazotetrazine-class chemotherapeutic alkylating agent. In the present study, we show that both elevated MPG expression and short hairpin RNA-mediated loss of DNA polymerase beta (Pol beta) expression in human breast cancer cells increases cellular sensitivity to temozolomide. Resistance to temozolomide is restored by complementation of either wild-type human Pol beta or human Pol beta with an inactivating mutation specific to the polymerase active site yet functional for 5'-deoxyribose-phosphate (5'dRP) lyase activity. These genetic and cellular studies uniquely demonstrate that overexpression of MPG causes an imbalance in base excision repair (BER), leading to an accumulation of cytotoxic 5'dRP lesions, and that the 5'dRP lyase activity of Pol beta is required to restore resistance to temozolomide. These results imply that Pol beta-dependent 5'dRP lyase activity is the rate-limiting step in BER in these cells and suggests that BER is a tightly balanced pathway for the repair of alkylated bases such as N7-methylguanine and N3-methyladenine. Furthermore, we find that 5'dRP-mediated cell death is independent of caspase-3 activation and does not induce the formation of autophagosomes, as measured by green fluorescent protein-light chain 3 localization. The experiments presented herein suggest that it will be important to investigate whether an active BER pathway could be partially responsible for the temozolomide-mediated resistance seen in some tumors and that balanced BER protein expression and overall BER capacity may help predict sensitivity to temozolomide.