Konjeti R. Sekhar

Inhibition of mammalian target of rapamycin or apoptotic pathway induces autophagy and radiosensitizes PTEN null prostate cancer cells

The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis. The mTOR inhibitor rapamycin and its derivatives are cytotoxic to a number of cell lines. Recently, mTOR inhibition has also been shown to radiosensitize endothelial and breast cancer cells in vitro. Because radiation is an important modality in the treatment of prostate cancer, we tested the ability of the mTOR inhibitor RAD001 (everolimus) to enhance the cytotoxic effects of radiation on two prostate cancer cell lines, PC-3 and DU145. We found that both cell lines became more vulnerable to irradiation after treatment with RAD001, with the PTEN-deficient PC-3 cell line showing the greater sensitivity. This increased susceptibility to radiation is associated with induction of autophagy. Furthermore, we show that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA in these cell lines enhances radiation-induced mortality and induces autophagy. Together, these data highlight the emerging importance of mTOR as a molecular target for therapeutic intervention, and lend support to the idea that nonapoptotic modes of cell death may play a crucial role in improving tumor cell kill.

Novel n-3 Fatty Acid Oxidation Products Activate Nrf2 by Destabilizing the Association between Keap1 and Cullin3

Consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can mitigate the progression of diseases in which oxidative stress represents a common underlying biochemical process. Nrf2-regulated gene expression regulates detoxification of reactive oxygen species. EPA and DHA were subjected to an in vitro free radical oxidation process that models in vivo conditions. Oxidized n-3 fatty acids reacted directly with the negative regulator of Nrf2, Keap1, initiating Keap1 dissociation with Cullin3, thereby inducing Nrf2-directed gene expression. Liquid chromatography-tandem mass spectrometry analyses of oxidized EPA demonstrated the presence of novel cyclopentenone-containing molecules termed J3-isoprostanes in vitro and in vivo and were shown to induce Nrf2-directed gene expression. These experiments provide a biochemical basis for the hypothesis that formation of J-ring compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based cellular defense systems.

Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3.

The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl- N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in vivo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.

Multi-faceted regulation of ?-glutamylcysteine synthetase

Glutathione is an important antioxidant that is involved in numerous cellular activities. γ‐Glutamylcysteine synthetase (γGCS) is a key regulatory enzyme in the synthesis of glutathione. It is a heterodimeric zinc metalloprotein that belongs to a unique class of proteins that gain activity due to formation of a reversible disulfide bond. The two subunits of γGCS exhibit differential and coordinate transcription regulation. In addition, the subunits are regulated at the posttranscriptional and posttranslational levels. These various levels of regulation allow numerous stimuli to induce or inhibit activity. J. Cell. Physiol. 182:163–170, 2000.

Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response.

While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox‐cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose‐dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non‐native disulfides on protein conformation, Ca2+‐ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non‐native intermolecular or glutathione (GHS)‐mixed disulfides. Differential scanning calorimetry profiles and 1‐anilinonaphthalene‐8‐sulfonic acid fluorescence indicated that formation of non‐native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent‐insoluble aggregates. Cells treated with menadione exhibited activation of HSF‐1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non‐native intermolecular and GSH‐mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response. J. Cell. Physiol. 171:143–151, 1997.

INCREASED EXPRESSION OF NUCLEAR FACTOR E2 p45-RELATED FACTOR 2 (NRF2) IN HEAD AND NECK SQUAMOUS CELL CARCINOMAS

Head and neck squamous cell carcinoma (HNSCC) continues to cause significant morbidity and mortality. Overexpression of specific phase II gene products may represent an important biomarker. One regulator of phase II gene expression is the transcription factor nuclear factor E2 p45‐related factor 2 (Nrf2). Nrf2 expression was evaluated in HNSCC, to determine whether it might serve as a biomarker for early detection of disease.

Cysteine-based Regulation of the CUL3 Adaptor Protein Keap1

Nrf2 (NF-E2-related factor 2) is a master transcription factor containing a powerful acidic transcriptional activation domain. Nrf2-dependent gene expression impacts cancer chemoprevention strategies, inflammatory responses, and progression of neurodegenerative diseases. Under basal conditions, association of Nrf2 with the CUL3 adaptor protein Keap1 results in the rapid Nrf2 ubiquitylation and proteasome-dependent degradation. Inhibition of Keap1 function blocks ubiquitylation of Nrf2, allowing newly synthesized Nrf2 to translocate into the nucleus, bind to ARE sites and direct target gene expression. Site-directed mutagenesis experiments coupled with proteomic analysis support a model in which Keap1 contains at least 2 distinct cysteine motifs. The first is located at Cys 151 in the BTB domain. The second is located in the intervening domain and centers around Cys 273 and 288. Adduction or oxidation at Cys151 has been shown to produce a conformational change in Keap1 that results in dissociation of Keap1 from CUL3, thereby inhibiting Nrf2 ubiquitylation. Thus, adduction captures specific chemical information and translates it into biochemical information via changes in structural conformation.

Redox-sensitive interaction between KIAA0132 and Nrf2 mediates indomethacin-induced expression of γ-glutamylcysteine synthetase

Exposure of HepG2 cells to nonsteroidal anti-inflammatory drugs (i.e., indomethacin and ibuprofen; NSAIDs) as well as resveratrol, caused increased expression of the mRNAs coding for the catalytic (Gclc) and modifier (Gclm) subunits of the glutathione synthetic enzyme, gamma-glutamylcysteine synthetase. In addition, indomethacin exposure increased intracellular glutathione content as well as inhibited glutathione depletion and cytotoxicity caused by diethyl maleate. Indomethacin-induced increases in the expression of gamma-glutamylcysteine synthetase mRNA were preceded by increases in steady state levels of intracellular pro-oxidants and glutathione disulfide accumulation. Simultaneous incubation with the thiol antioxidant N-acetylcysteine (NAC) inhibited indomethacin-mediated increases in GCLC mRNA, suggesting that increases in GCLC message were triggered by changes in intracellular oxidation/reduction (redox) reactions. Indirect immunofluorescence using intact cells demonstrated that indomethacin induced the nuclear translocation of Nrf2, a transcription factor believed to regulate GCLC expression. Immunoprecipitation studies showed that indomethacin treatment also inhibited Nrf2 tethering to KIAA0132 (the human homolog of Keap1 accession #D50922), which is believed to be a negative regulator of Nrf2. Consistent with this idea, over-expression of Nrf2 increased GCLC reporter gene expression and over-expression of KIAA0132 inhibited GCLC reporter gene activity as well as inhibited indomethacin-induced increases in the expression of GCLC. Finally, simultaneous treatment with NAC inhibited both indomethacin-induced release of Nrf2 from KIAA0132 and indomethacin-induced nuclear translocation of Nrf2. These results demonstrate that NSAIDs and resveratrol cause increases in the expression of gamma-glutamylcysteine synthetase mRNA and identify these agents as being capable of stimulating glutathione metabolism. These results also support the hypothesis that indomethacin-induced transcriptional activation of GCLC involves the redox-dependent release of KIAA0132 from Nrf2 followed by the nuclear translocation of Nrf2.

Activating Transcription Factor 3 Is a Novel Repressor of the Nuclear Factor Erythroid-Derived 2–Related Factor 2 (Nrf2)–Regulated Stress Pathway

The transcription factor nuclear factor erythroid-derived 2-related factor 2 (Nrf2) regulates induction of an extensive cellular stress response network when complexed with the cAMP-responsive element binding protein (CBP) at antioxidant response elements (ARE) located in the promoter region of target genes. Activating transcription factor 3 (ATF3) can repress Nrf2-mediated signaling in a manner that is not well understood. Here, we show that ATF3-mediated suppression is a consequence of direct ATF3-Nrf2 protein-protein interactions that result in displacement of CBP from the ARE. This work establishes ATF3 as a novel repressor of the Nrf2-directed stress response pathway.

Alteration of transcriptional and post-transcriptional expression of gamma-glutamylcysteine synthetase by diethyl maleate.

Gamma-glutamylcysteine synthetase (gamma-GCS), also known as glutamate-cysteine ligase (EC 6.3.2.2), is the rate-limiting enzyme in the synthesis of glutathione (GSH). The gene GLCLC encodes the catalytic subunit while GLCLR encodes the regulatory subunit. Although it has been shown that GLCLC can respond to a variety of stresses by increased transcription, it is not known whether a similar response occurs for GLCLR. Nor is it known whether post-transcriptional regulation of either gene product is altered during stress. The present investigation was undertaken to explore transcriptional and post-transcriptional regulation of GLCLC and GLCLR gene products when HepG2 cells were challenged with the radiation sensitizer diethyl maleate (DEM). Expression of steady-state GLCLC and GLCLR mRNA was enhanced 5-20-fold after DEM challenge. Nuclear run-off assays were performed on unstressed and stressed cells to determine whether the increased expression of GLCLC and GLCLR mRNA was due to altered transcriptional activity of these genes. The DEM treatment increased the transcription rates of both genes 2-5-fold. In unstressed HepG2 cells, the half-life of GLCLC mRNA transcripts was approximately 4 h. In contrast, the half-life of GLCLR transcripts was approximately 8 h. In cells treated with DEM, the half-lives of all transcripts were increased, indicating that message stabilization contributed to the increased expression of gene products. Finally, a PEST algorithm has identified a PEST (proline, glutamate, serine, threonine) motif within the catalytic subunit of gamma-GCS, suggesting that this subunit might exhibit conditional proteolytic regulation. These results imply that regulation of the products of the GLCLC and GLCLR genes may be altered at multiple levels during exposure to stress.