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Background: Ramucirumab plus erlotinib (RAM + ERL) demonstrated superior progression-free survival (PFS) over placebo + ERL (PBO + ERL) in the phase III RELAY study of patients with epidermal growth factor receptor (EGFR)-mutated metastatic non-small-cell lung cancer (EGFR+ mNSCLC; NCT02411448). Next-generation sequencing (NGS) was used to identify clinically relevant alterations in circulating tumor DNA (ctDNA) and explore their impact on treatment outcomes., Patients and Methods: Eligible patients with EGFR+ mNSCLC were randomized 1 : 1 to ERL (150 mg/day) plus RAM (10 mg/kg)/PBO every 2 weeks. Liquid biopsies were to be prospectively collected at baseline, cycle 4 (C4), and postdiscontinuation follow-up. EGFR and co-occurring/treatment-emergent (TE) genomic alterations in ctDNA were analyzed using Guardant360 NGS platform., Results: In those with valid baseline samples, detectable activating EGFR alterations in ctDNA (aEGFR+) were associated with shorter PFS [aEGFR+: 12.7 months (n = 255) versus aEGFR-: 22.0 months (n = 131); hazard ratio (HR) = 1.87, 95% confidence interval (CI) 1.42-2.51]. Irrespective of detectable/undetectable baseline aEGFR, RAM + ERL was associated with longer PFS versus PBO + ERL [aEGFR+: median PFS (mPFS) = 15.2 versus 11.1 months, HR = 0.63, 95% CI 0.46-0.85; aEGFR-: mPFS = 22.1 versus 19.2 months, HR = 0.80, 95% CI 0.49-1.30]. Baseline alterations co-occurring with aEGFR were identified in 69 genes, most commonly TP53 (43%), EGFR (other than aEGFR; 25%), and PIK3CA (10%). PFS was longer in RAM + ERL, irrespective of baseline co-occurring alterations. Clearance of baseline aEGFR by C4 was associated with longer PFS (mPFS = 14.1 versus 7.0 months, HR = 0.481, 95% CI 0.33-0.71). RAM + ERL improved PFS outcomes, irrespective of aEGFR mutation clearance. TE gene alterations were most commonly in EGFR [T790M (29%), other (19%)] and TP53 (16%)., Conclusions: Baseline aEGFR alterations in ctDNA were associated with shorter mPFS. RAM + ERL was associated with improved PFS outcomes, irrespective of detectable/undetectable aEGFR, co-occurring baseline alterations, or aEGFR+ clearance by C4. aEGFR+ clearance by C4 was associated with improved PFS outcomes. Monitoring co-occurring alterations and aEGFR+ clearance may provide insights into mechanisms of EGFR tyrosine kinase inhibitor resistance and the patients who may benefit from intensified treatment schedules., Competing Interests: Disclosure EBG declares grants from ABL-Bio, AstraZeneca, Bristol-Myers Squibb, Daiichi-Sankyo, Dynavax Technologies, EMD Serono, Eli Lilly and Company, Genentech, Iovance Biotherapeutics, Merck, Mirati Therapeutics, Neon, and Novartis; consulting fees from AbbVie, ABL-Bio, AstraZeneca, Boehringer-Ingelheim, Bristol-Myers Squibb, Dracen Pharmaceuticals, EMD Serono, Eisai, Eli Lilly and Company, Gilead, GlaxoSmithKline, Ipsen, Merck, Natera, Novartis, Personalis, Regeneron, Sanofi, Shionogi, and Xilio; payment for expert testimony from UCLA relating to motif neoepitopes for cancer immunotherapy; leadership/fiduciary role with LUNGevity and Jonsson Comprehensive Cancer Center at UCLA. JVH declares advisory board/committee membership with Genentech, Mirati Therapeutics, Eli Lilly and Company, Janssen Pharmaceuticals, Boehringer-Ingelheim Pharmaceuticals, DAVA Oncology, Regeneron, Takeda Pharmaceuticals, BerGenBio, Jazz Pharmaceuticals, Curio Science, Immunocore, and Novartis; research support from AstraZeneca, Boehringer-Ingelheim, Spectrum, and Takeda; royalties and licensing fees from Spectrum. MR declares consulting fees from Amgen, AstraZeneca, Boehringer-Ingelheim, BeiGene, BMS, GSK, Mirati, Merck, MSD, Eli Lilly and Company, Novartis, Pfizer, Roche, Regeneron, and Sanofi; honoraria from Amgen, AstraZeneca, Boehringer-Ingelheim, BeiGene, BMS, GSK, Mirati, Merck, MSD, Eli Lilly and Company, Novartis, Pfizer, Roche, Regeneron, and Sanofi; support for meetings/travel from Amgen, AstraZeneca, Boehringer-Ingelheim, BeiGene, BMS, GSK, Mirati, Merck, MSD, Eli Lilly and Company, Novartis, Pfizer, Roche, Regeneron, and Sanofi; board participation for Daiichi and Sanofi. KN declares grants from Nippon Boehringer-Ingelheim, West Japan Oncology Group, Thoracic Oncology Research Group, North East Japan Study Group, Clinical Research Support Center Kyushu, Nichirei Biosciences Inc., Eli Lilly and Company, Hitachi, Sysmex, and Otsuka Pharmaceutical; consulting fees from SymBio Pharmaceuticals, Solasia Pharma, Eli Lilly and Company, and Otsuka Pharmaceutical; honoraria from Chugai, Pfizer, Eli Lilly and Company, MSD, Novartis Pharma, AstraZeneca, Amgen, Merck Biopharma, Roche Diagnostics, Yakult Honsha, Guardant Health, Takeda Pharmaceuticals, Boehringer-Ingelheim Japan, FUJIREBIO, Bristol-Myers Squibb, Merck Biopharma, Janssen Pharmaceutical Daiichi-Sankyo, and Ono Pharmaceutical. MN declares honoraria from Ono Pharmaceuticals, Chugai Pharmaceutical, Taiho Pharmaceutical, Bristol-Myers Squibb, Daiichi-Sankyo, Eli Lilly and Company, AstraZeneca, MSD, AbbVie, Takeda, Pfizer, Boehringer-Ingelheim, Novartis, Nippon Kayaku, Merck, and Janssen. SN declares grants from Eli Lilly and Company; consulting fees from BMS, Eli Lilly and Company, Takeda, Roche, Pfizer, AstraZeneca, BI, MSD, AbbVie, PharmaMar, and BeiGene; support for meetings and/or travel from Eli Lilly and Company, Thermo Fisher, and Takeda; participation on advisory boards for Guardant Health, GSK, Novocure, Amgen, AstraZeneca, Janssen, and Eli Lilly and Company; and declares leadership/fiduciary roles as President of WALCE (Women against Lung Cancer in Europe) Onlus. LPA declares grants from MSD, AstraZeneca, Pfizer, and BMS; consulting fees from Eli Lilly and Company, MSD, Roche, PharmaMar, Merck, AstraZeneca, Novartis, Servier, Amgen, Pfizer, Sanofi, Bayer, BMS, Mirati, GSK, Janssen, Takeda, and Daiichi-Sankyo; honoraria from AstraZeneca, Janssen, Merck, and Mirati; member of the board of directors of Altum Sequencing and Genomica; principal investigator for studies sponsored by Alkermes, Amgen, AstraZeneca, Bristol-Myers Squibb, Daiichi-Sankyo, IO Biotech, Janssen-Cilag, Eli Lilly and Company, MSD, Novartis, Pfizer, PharmaMar, Roche, Sanofi, Takeda, and Tesaro. SP declares consulting fees from Amgen, AstraZeneca, Bayer, BeiGene, Blueprint, BMS, Boehringer-Ingelheim, Daiichi-Sankyo, Guardant Health, Incyte, Janssen, Eli Lilly and Company, Merck Serono, MSD, Novartis, Roche, Takeda, Pfizer, Seattle Genetics, Turning Point Therapeutics, and EQRx; honoraria from AstraZeneca, Bayer, Guardant Health, Janssen, Merck Serono, Roche, Takeda, and Pfizer; payment for expert testimony from Roche and Merck Serono; support for attending meetings and/or travel from Janssen and Roche; unpaid leadership/fiduciary roles in British Thoracic Oncology Group, ALK Positive UK, Lung Cancer Europe, Ruth Strauss Foundation, Mesothelioma Applied Research Foundation, and ETOP-IBCSG Partners Foundation Board. SPA declares no conflicts of interest. HG was an employee and stockholder of Eli Lilly and Company during the time of study analysis and manuscript preparation; contributions to the manuscript were made as part of the roles at Eli Lilly and Company. BDB and CVG are full-time employees and minor shareholders of Eli Lilly and Company. KN declares grants to institution from AstraZeneca, MSD, Ono Pharmaceutical Co. Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis, Pfizer Japan Inc., Bristol-Myers Squibb, Eli Lilly and Company, Chugai Pharmaceutical, Daiichi-Sankyo, Merck, Paraxel International Corp., PRA Health Sciences, EPS Corporation, Kissei Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co. Ltd., PPD-SNBL K.K., SymBio Pharmaceuticals Limited, IQVIA Services Japan K.K., Syneos Health Clinical K.K., Nippon Kayaku Co. Ltd., EP-CRSU Co. Ltd., Mebix, Janssen Pharmaceutical K.K., AbbVie Inc., Bayer Yakuhin Ltd., Eisai, Mochida Pharmaceutical Co. Ltd. Covance Japan Inc., Japan Clinical Research Operations, Takeda Pharmaceutical Co. Ltd., GlaxoSmithKline, Sanofi, Sysmex Corporation, Medical Research Support, Otsuka Pharmaceutical Co. Ltd., SRL Inc., Pfizer R&D Japan G.K., and Amgen Inc.; consulting fees from Eli Lilly and Company, KYORIN Pharmaceutical Co. Ltd., Ono Pharmaceutical Co. Ltd., and Pfizer Japan Inc.; honoraria from Ono Pharmaceutical Co. Ltd., Amgen Inc. Nippon Kayaku Co. Ltd., AstraZeneca K.K., Chugai Pharmaceutical Co. Ltd., Eli Lilly and Company, MSD, Pfizer Japan Inc., Nippon Boehringer-Ingelheim Co. Ltd., Taiho Pharmaceutical Co. Ltd., Bayer Yakuhin, CMIC ShiftZero K.K., Life Technologies Japan Ltd., Neo Communication, Roche Diagnostics K.K., AbbVie Inc, Merck Biopharma Co. Ltd., Kyowa Kirin Co. Ltd., Takeda Pharmaceutical Co. Ltd., 3H Clinical Trial Inc., Care Net Inc., Medical Review Co. Ltd., Medical Mobile Communications Co. Ltd., Yodosha Co. Ltd., Nikkei Business Publications Inc., Japan Clinical Research Operations, CMIC Co. Ltd., Novartis, TAIYO Pharma Co. Ltd., KYORIN Pharmaceutical Co. Ltd., and Bristol-Myers Squibb K.K.; patents planned, issued, or pending with Daiichi-Sankyo. Data sharing Lilly provides access to all individual participant data collected during the trial, after anonymization, with the exception of pharmacokinetic or genetic data. Data are available to request 6 months after the indication studied has been approved in the United States and European Union and after primary publication acceptance, whichever is later. No expiration date of data requests is currently set once data are made available. Access is provided after a proposal has been approved by an independent review committee identified for this purpose and after receipt of a signed data sharing agreement. Data and documents, including the study protocol, statistical analysis plan, clinical study report, blank or annotated case report forms, will be provided in a secure data sharing environment. For details on submitting a request, see the instructions provided at www.vivli.org., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-μm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted β-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies., Methods: Genetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A "click" chemistry labeling method is developed for the detection of O-GlcNAcylated tau., Results: Substantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity., Conclusion: The present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer's disease and other neurodegenerative tauopathies.

Apoptosis plays a crucial role in brain development by ensuring that only appropriately growing, migrating, and synapse-forming neurons and their associated glial cells survive. This process involves an intimate relationship between cell-cell interactions and developmental cues and is further impacted by environmental stress during neurogenesis and disease. Oligodendrocytes (OLs), the major myelin-forming cells in the central nervous system, largely form after this wave of neurogenesis but also show a selective vulnerability to cell death stimuli depending on their stage of development. This can affect not only embryonic and early postnatal brain formation but also the response to demyelinating pathologies. In the present review, we discuss the stage-specific sensitivity of OL lineage cells to damage-induced death and how this might impact myelin survival and regeneration during injury or disease.

The genome sequence of the Salmonella enterica serovar Anatum-specific, serotype-converting bacteriophage epsilon15 has been completed. The nonredundant genome contains 39,671 bp and 51 putative genes. It most closely resembles the genome of phiV10, an Escherichia coli O157:H7-specific temperate phage, with which it shares 36 related genes. More distant relatives include the Burkholderia cepacia-specific phage, BcepC6B (8 similar genes), the Bordetella bronchiseptica-specific phage, BPP-1 (8 similar genes) and the Photobacterium profundum prophage, P Pphipr1 (6 similar genes). epsilon15 gene identifications based on homologies with known gene families include the terminase small and large subunits, integrase, endolysin, two holins, two DNA methylase enzymes (one adenine-specific and one cytosine-specific) and a RecT-like enzyme. Genes identified experimentally include those coding for the serotype conversion proteins, the tail fiber, the major capsid protein and the major repressor. epsilon15's attP site and the Salmonella attB site with which it interacts during lysogenization have also been determined.

The role of the proteasome in neuronal apoptosis is poorly understood since both anti- and pro-apoptotic effects result from proteasome inhibition. We studied the effects of proteasome inhibition in cultured rat cerebellar granule neurons. Acute exposure to proteasome inhibitors MG-132 and lactacystin blocked caspase activation induced by removal of depolarizing medium. However, chronic treatment with MG-132 activated caspases in neurons maintained in depolarizing potassium. The biphasic effect of MG-132 was hypothesized to be due to differential degradation of anti- and pro-apoptotic proteins. Accordingly, acute exposure to MG-132 inhibited the hyperphosphorylation, loss of DNA binding, ubiquitination, and degradation of the pro-survival transcription factor MEF2D induced by removal of depolarizing medium. In contrast, chronic exposure to MG-132 increased the expression and phosphorylation of c-Jun, elevated levels of the pro-apoptotic protein Bim, and triggered neuronal apoptosis, even in the presence of depolarizing medium. Thus, proteasome inhibition exerts an acute pro-survival action by stabilizing MEF2 transcription factors. However, chronic proteasome inhibition causes a build-up of phosphorylated c-Jun and Bim, which eventually overwhelms the effects of MEF2 and triggers apoptosis.

Primary cerebellar granule neurons (CGNs) require depolarizing extracellular potassium for their survival. Removal of depolarizing potassium triggers CGN apoptosis that requires induction of Bim, a BH3-only Bcl-2 family member. Bim is classically thought to promote apoptosis by neutralizing pro-survival Bcl-2 proteins. To determine if this is the principal function of Bim in CGNs, we contrasted Bim-mediated apoptosis to neuronal death induced by HA14-1, a BH3-domain mimetic that antagonizes Bcl-2 and Bcl-x(L). HA14-1 elicited CGN apoptosis characterized by caspase 3 and 9 activation, cytochrome c release, conformational activation of Bax, and mitochondrial depolarization. HA14-1 provoked CGN apoptosis in the absence of Bim induction and negative regulators of Bim transcription did not prevent HA14-1-induced cell death. However, the antioxidant glutathione and its precursor, N-acetyl-l-cysteine, suppressed HA14-1-induced apoptosis. Similarly, apoptosis induced by either a structurally distinct Bcl-2/Bcl-x(L) inhibitor (compound 6) or Bcl-2 antisense oligonucleotides was diminished by glutathione. In contrast, antioxidants had no effect on CGN apoptosis provoked by either removal of depolarizing potassium or overexpression of a GFP-Bim fusion protein, two models of Bim-dependent death. These data show that antagonism of Bcl-2/Bcl-x(L) function elicits oxidative stress-dependent CGN apoptosis that is mechanistically distinct from Bim-mediated cell death. These results further indicate that, although Bcl-2/Bcl-x(L) antagonism is sufficient to induce neuronal apoptosis, Bim likely promotes neuronal death by interacting with additional proteins besides Bcl-2/Bcl-x(L).

Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (alpha9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.

Glycogen synthase kinase-3beta (GSK-3beta) is a critical activator of neuronal apoptosis induced by a diverse array of neurotoxic insults. However, the downstream substrates of GSK-3beta that ultimately induce neuronal death are unknown. Here, we show that GSK-3beta phosphorylates and regulates the activity of Bax, a pro-apoptotic Bcl-2 family member that stimulates the intrinsic (mitochondrial) death pathway by eliciting cytochrome c release from mitochondria. In cerebellar granule neurons undergoing apoptosis, inhibition of GSK-3beta suppressed both the mitochondrial translocation of an expressed green fluorescent protein (GFP)-Bax(alpha) fusion protein and the conformational activation of endogenous Bax. GSK-3beta directly phosphorylated Bax(alpha) on Ser163, a residue found within a species-conserved, putative GSK-3beta phosphorylation motif. Coexpression of GFP-Bax(alpha) with a constitutively active mutant of GSK-3beta, GSK-3beta(Ser9Ala), enhanced the in vivo phosphorylation of wild-type Bax(alpha), but not a Ser163Ala mutant of Bax(alpha), in transfected human embryonic kidney 293 (HEK293) cells. Moreover, cotransfection with constitutively active GSK-3beta promoted the localization of Bax(alpha) to mitochondria and induced apoptosis in both transfected HEK293 cells and cerebellar granule neurons. In contrast, neither a Ser163Ala point mutant of Bax(alpha) nor a naturally occurring splice variant that lacks 13 amino acids encompassing Ser163 (Bax(sigma)) were driven to mitochondria in HEK293 cells coexpressing constitutively active GSK-3beta. In a similar manner, either mutation or deletion of the identified GSK-3beta phosphorylation motif prevented the localization of Bax to mitochondria in cerebellar granule neurons undergoing apoptosis. Our results indicate that GSK-3beta exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.

Peroxisome proliferator activated receptors are nuclear hormone receptors that regulate the expression of genes containing a peroxisome proliferator activated receptor response element. We report here that the human bcl-2 gene contains a functional peroxisome proliferator activated receptor response element in the 3' untranslated region. Peroxisome proliferator activated receptor gamma bound the human bcl-2 peroxisome proliferator activated receptor response element in gel shift assays and co-transfection of this receptor led to increased luciferase activity from a reporter plasmid containing the human bcl-2 peroxisome proliferator activated receptor response element. Examination of peroxisome proliferator activated receptor gamma-transfected cells demonstrated an increased amount of bcl-2 message compared to empty vector-transfected cells. Confocal analyses confirmed that more Bcl-2 protein was present in peroxisome proliferator activated receptor gamma-transfected cells compared to control-transfected cells. The functionality of the increased Bcl-2 protein was examined using resistance to bile salt-induced apoptosis as the endpoint. Peroxisome proliferator activated receptor gamma-transfected cells were almost twice as resistant as control-transfected cells. These data show that PPARgamma can mediate transcription of bcl-2, resulting in an increase in Bcl-2 protein and protection from apoptosis. We discuss these findings with regards to their potential implications for colon carcinogenesis.

Cultured rat cerebellar granule neurons (CGNs) require depolarization-mediated calcium influx for survival. Calcium regulates the activity of the pro-survival transcription factor, myocyte enhancer factor 2D (MEF2D). MEF2D is hyperphosphorylated and degraded in CGNs undergoing apoptosis induced by lowering the extracellular potassium concentration from 25 mM to 5 mM. Since insulin-like growth factor-I (IGF-I) is known to protect CGNs from apoptotic cell death, we investigated the effects of IGF-I on MEF2D processing during apoptosis. IGF-I administered during the apoptotic insult did not prevent the hyperphosphorylation of MEF2D and consequential loss of DNA binding. However, IGF-I significantly blocked the degradation of MEF2D. Furthermore, IGF-I had no effect on the initial loss of MEF2 transcriptional activity following hyperphosphorylation, but the recovery of MEF2 activity following restoration of intracellular calcium was significantly increased by IGF-I. We conclude that IGF-I blocks the degradation of MEF2D and enhances recovery of MEF2 activity by protecting MEF2D from caspase-dependent cleavage during apoptosis. These results suggest that IGF-I can prolong the time of commitment to irreversible cell death and enhance the recovery of neurons subjected to an acute apoptotic stimulus by preserving the activity of the pro-survival factor MEF2D.

Resistance to apoptosis may be a critical phenotype that cells must acquire during skin carcinogenesis. The Akt kinase is a known upstream regulator of apoptosis in many cell types and has been shown to be activated by increased reactive oxygen species (ROS). We have previously demonstrated that two malignant variants (6M90 and 6R90) of the mouse keratinocyte 308 cell line have elevated ROS because of loss of catalase activity, and that this elevated ROS confers a growth advantage. We report here that in addition to a growth advantage, chronically increased ROS in the variants resulted in an increase in resistance to ultraviolet (UV) B-induced apoptosis. This resistance was due to basal increases of Akt phosphorylation in the malignant variants compared to the 308 cells. Modulation of ROS in 6M90 and 6R90 cells by catalase overexpression or antioxidant treatment resulted in decreased levels of Akt phosphorylation and subsequent loss of resistance to UVB-induced apoptosis. Conversely, treatment of 308 cells with hydrogen peroxide caused increases in Akt phosphorylation and increased apoptosis resistance. These results indicate that the chronically elevated ROS often observed in tumors may contribute to a malignant phenotype by keeping Akt in a phosphorylated state, resulting in increased apoptosis resistance., (Copyright 2003 Wiley-Liss, Inc.)

Evidence from a number of studies suggests that the mechanism by which tumor necrosis factor (TNF) kills transformed cells involves oxidative stress. NAD(P)H:(quinone acceptor) oxidoreductase (NQO1) is an antioxidant enzyme with particular relevance to cancer. The MCF-7 breast cancer cell line was stably transfected with rat NQO1 cDNA to determine whether increased NQO1 activity alters sensitivity to TNF-induced apoptosis. Five clones, with a range of NQO1 enzyme activities from 5- to 50-fold greater than the MCF-7 line, and two control transfectants were examined. Northern blot hybridization analyses and reverse transcription-PCR demonstrated that the increase in NQO1 activity in the transfectants was attributable to expression from the transfected rat sequence. Based on sulforhodamine B assays for the number of viable cells, the NQO1 clones showed increased sensitivity to EO9, an indoloquinone that undergoes bioactive reduction by NQO1. Viability studies also demonstrated that the NQO1 transfectants were significantly more sensitive to TNF than the control transfectants or MCF-7 parent. This increased sensitivity could not be explained by changes in superoxide dismutase or catalase activity or to increased sensitivity to oxidative stress in general, as assessed by response to hydrogen peroxide and paraquat treatment. Using dichlorodihydrofluorescein diacetate as a probe, we found that the NQO1 transfectants had no difference in baseline level of oxidative stress compared to the control cells but did exhibit greater intracellular oxidative stress after TNF treatment. We conclude that NQO1 can affect the TNF-mediated pathway to apoptosis.