Profound Cell Death Research Articles

MTOR Kinase Inhibition Causes Feedback-Dependent Biphasic Regulation of AKT Signaling

mTOR kinase inhibitors block mTORC1 and mTORC2 and thus do not cause the mTORC2 activation of AKT observed with rapamycin. We now show, however, that these drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT serine 473 (S473) dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. However, inhibition of mTOR kinase also relieves feedback inhibition of receptor tyrosine kinases (RTK), leading to subsequent phosphoinositide 3-kinase activation and rephosphorylation of AKT T308 sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound suppression of mTORC1 and accumulation of activated AKT phosphorylated on T308, but not S473. Combined inhibition of mTOR kinase and the induced RTKs fully abolishes AKT signaling and results in substantial cell death and tumor regression in vivo. These findings reveal the adaptive capabilities of oncogenic signaling networks and the limitations of monotherapy for inhibiting feedback-regulated pathways. The results of this study show the adaptive capabilities of oncogenic signaling networks, as AKT signaling becomes reactivated through a feedback-induced AKT species phosphorylated on T308 but lacking S473. The addition of RTK inhibitors can prevent this reactivation of AKT signaling and cause profound cell death and tumor regression in vivo, highlighting the possible need for combinatorial approaches to block feedback-regulated pathways.

Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.

Abstract LB-222: Identifying cFLIP as a marker and also a potentially “druggable” target of SAHA+TRAIL (TNF-Related Apoptosis Inducing Ligand), cytotoxicity in malignant pleural mesothelioma (MPM)

Abstract Background: Despite expressing adequate levels of receptors for TRAIL significant percentages of cancer cells are resistant to TRAIL-induced apoptosis. We have previously reported that histone deacetylase inhibitor SAHA (vorinostat) + TRAIL combination induces profound supra-additive cytotoxicity in MPM in vitro. We observe that only MPM cells with some TRAIL sensitivity are very susceptible to SAHA+TRAIL cytotoxicity. We hypothesize that TRAIL-initiated death signal at the membrane level determine cellular susceptibility to SAHA+TRAIL. The objective of this study is to identify the molecular marker that predicts TRAIL and thus TRAIL+SAHA sensitivity in MPM cells. Materials and methods: Intrinsic sensitivity of 8 MPM and primary normal endothelial cells to TRAIL and SAHA+TRAIL is determined by cell viability assay; basal expression of cFLIP, caspase 8, DR4/DR5, FADD, RIP, TRADD in these cells are evaluated by western blots. Selective knockdown of FLIP is achieved by siRNA. FLIP mRNA levels were determined by quantitative RT-PCR. Results: 5/8 MPM cells are very suceptible to SAHA+TRAIL cytotoxicity (combination-sensitive cells) while 3 others are classified as combination-resistant. Western blot analysis identifies an inverse relationship between cFLIP as well as procaspase 8 expression and sensitivity to SAHA+TRAIL with only the difference in cFLIP levels as quantified by densitometric analysis being distinctive between two groups: 0.87±0.02 for 5 combination-sensitive MPM cells versus 0.15±0.05 for 3 combination-resistant MPM cells. siRNA-mediated partial cFLIP knockdown restores DISC activity in high cFLIP expressing cells as evidenced by caspase 8 and 3 catalytic processing with stronger caspase activation being noted in combination-treated cells. Quantitative RT-PCR demonstrates high level of cFLIP messenger RNA in high cFLIP expressors indicating that cFLIP is transcriptionally upregulated in these cells. Selective cFLIP downregulation in high cFLIP expressing cells restores susceptibility to TRAIL and strongly sensitizes them to SAHA+TRAIL cytotoxicity. Additionally, selective complete cFLIP knockdown abrogates the need for SAHA to achieve profound TRAIL-mediated cell death in MPM cells regardless of their intrinsic cFLIP epxression. Conclusdion: Our study identifies cFLIP expression as a marker of cellular sensitivity to SAHA+TRAIL in MPM in vitro. More importantly, gene knockdown experiments provide the proof of concept that cFLIP is a pontential “druggable” target and downregulation of which sensitizes resistant cancer cells to TRAIL and SAHA+TRAIL. Ongoing works aim to validate this observation in a larger panel of novel MPM cells and to define treatments strategies to downregulate cFLIP in tumor cells expressing high levels of this antiapoptotic protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-222. doi:10.1158/1538-7445.AM2011-LB-222

Abstract 1126: Gene expression profiling reveals that WT1 and WTX control cell growth through similar gene networks but different specific genes

Abstract The WTX Wilms tumor suppressor is present on the X chromosome, and hence a single WTX suppression may be sufficient to promote carcinogenesis in a subset of Wilms tumors. Thought WTX mutations were only occasionally found in other tumors, the study of this protein may give new insights into growth suppressive pathways. Unlike the WT1 tumor suppressor gene, WTX lacks known functional protein domains. Previous biochemical studies suggested that WTX inhibits WNT signaling by degrading beta-catenin and similarly, WT1 inhibits WNT signaling. The study of WTX function was hampered by the fact that transient expression of WTX but not WT1 caused profound cell death. Hence we constructed inducible cell lines expressing WTX and tumor associated WTX mutants in HEK293 cells. Similar to our results with WT1, induction of WTX resulted in a 40% increase in G1/G0 cells and a concomitant decrease of cells in S phase. A short, tumor-associated truncation mutant of WTX (Arg358ter) led to a more modest 15-20% increase in G1 while a longer truncation mutant (Leu561ter) led to cell cycle arrest to a similar extent as wild type WTX. Wild-type WTX decreased cell growth by nearly 50% while Arg358ter led to only a 7% decrease in growth and Leu561 led to a 22% decrease. WT1 and WTX both slow growth and cause cell cycle arrest, but gene expression profiling showed that only a few specific target genes were regulated in common by the proteins. Nevertheless, the two tumor suppressors converged on similar pathways as Ingenuity Pathway Analysis showed that gene networks implicated in the cell cycle, cell death, cancer, cellular growth, and cardiovascular system development were both affected by both WT1 and WTX. Gene networks involved in gastrointestinal disease, nucleic acid metabolism, and reproductive system disease were uniquely enriched in WTX targets, while lipid metabolism, molecular transport, endocrine system disorders, cellular assembly and organization, and gene expression functions were enriched among WT1 target genes. When gene expression changes mediated by wild-type WTX and the Leu561Ter were compared, there was a 50% overlap of WTX up-regulated genes and 30% overlap in down-regulated genes. By contrast, the Arg358Ter regulated few genes in common with wild-type WTX, implying that a domain containing amino acid residues from 358 and 561 may be critical for tumor suppressor function of WTX. Collectively these data indicate that although both associated with tumor development, Arg 358 is a near complete loss of function mutant while Arg 561 is a hypomorphic form of WTX. WTX regulates similar sets of genes as WT1 to suppress cell growth through cell cycle arrest and induction of apoptosis. Whether patients harboring the shorter truncation mutations of WTX have a distinct phenotype remains to be determined. Further study will be necessary to determine how these genes are directly or indirectly regulated by WTX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1126.

Abstract 284: FGFR2 is required for growth and survival in a rare subset of FGFR2 amplified colorectal cancer

Abstract FGFR2 amplification has been reported in subsets of gastric cancer and breast cancer. We describe FGFR2 amplification in a cell line derived from a colorectal carcinoma arising in a young patient with ulcerative colitis. The cell line has features of endocrine differentiation, representing a rare (< 1%) subset of colorectal cancer. FGFR2 amplification in this cell line results in an overexpressed and highly activated FGFR2 kinase. The FGFR selective inhibitor PD173074 blocked FGFR2 phosphorylation and inhibited Erk, Akt, and S6 ribosomal protein phosphorylation. FGFR2 inhibition resulted in profound cell death as measured by robust accumulation of cells in subG1 by flow cytometry and PARP cleavage by western analysis. A structurally unrelated FGFR2 inhibitor, MK2461, also caused cell death and inhibited downstream signaling pathways. RTK arrays revealed coactivation of EGFR family kinases, and a functional role for these kinases is being investigated. Interestingly, an FGFR2 isoform in this cell line migrated at 160kD, in contrast to the 140kD isoform observed in SNU16 and other cell lines. However, C-terminal FGFR2 antibodies did not react with the 160kD FGFR2 isoform, suggesting that it has a C-terminal deletion similar to the FGFR2 isoform in OCUM2M and KatoIII cell lines. We are currently defining the frequency of FGFR2 overexpression and activation in colon cancer tissue microarrays. The strong growth inhibition and induction of cell death observed in vitro suggests that colorectal cancer tumor cells harboring FGFR2 amplification / activation may show similar response to FGFR inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 284.

GSK-3β promotes cell survival by modulating Bif-1-dependent autophagy and cell death

Glycogen synthase kinase 3 beta (GSK-3beta) is constantly active in cells and its activity increases after serum deprivation, indicating that GSK-3beta might play a major role in cell survival under serum starvation. In this study, we attempted to determine how GSK-3beta promotes cell survival after serum depletion. Under full culture conditions (10% FBS), GSK-3beta inhibition with chemical inhibitors or siRNAs failed to induce cell death in human prostate cancer cells. By contrast, under conditions of serum starvation, a profound necrotic cell death was observed as evidenced by cellular morphologic features and biochemical markers. Further analysis revealed that GSK-3beta-inhibition-induced cell death was in parallel with an extensive autophagic response. Interestingly, blocking the autophagic response switched GSK-3beta-inhibition-induced necrosis to apoptotic cell death. Finally, GSK-3beta inhibition resulted in a remarkable elevation of Bif-1 protein levels, and silencing Bif-1 expression abrogated GSK-3beta-inhibition-induced autophagic response and cell death. Taken together, our study suggests that GSK-3beta promotes cell survival by modulating Bif-1-dependent autophagic response and cell death.

Mesoporosity and Functional Group Dependent Endocytosis and Cytotoxicity of Silica Nanomaterials

We report different mesoporosity-dependent and functional group-dependent cytotoxicity and endocytosis of various silica nanomaterials on suspended and adherent cells. This dependency further varied with incubation time and particle dosage, and appeared to be associated with the particles' endocytotic efficiency and their chemical and physical properties. We studied two common mesoporous nanomaterials (MSNs), MCM-41 and SBA-15, and one type of solid-cored silica microsphere, paralleled by their quaternary amine functionalized counterparts. Compared to SBA-15, MCM-41 has a larger surface area but smaller pore size, whereas SMS exhibits low surface area and poor porosity. In Jurkat cells, SBA-15 and MCM-41 exhibited different cytotoxicity profiles. However, no significant cell death was detected when treated with the aminated MSNs, indicating that the positively charged quaternary amines prevented cellular injury from mesoporous nanoparticles. Furthermore, the effective internalization of MSN but not aminated-MSNs was clearly observed, in line with their consequent cytotoxicity. SK-N-SH (human neuroblastoma) cells were found to be more resistant to the treatment of MSN, whether aminated or not. Incubation with either SBA-15 or MCM-41 over time showed a recovery in cell viability, while exposure to MSN-N particles did not induce a noticeable cell death until longer incubation with high dosage of 200 microg/mL was applied. Both aminated and nonaminated silica spheres exhibited instant and constant toxicity on Jurkat (human T-cell lymphoma) cells. TEM images revealed successful endocytosis of SMS and SMS-N, although SMS-N appeared to accumulate more in the nucleus. For SK-N-SH cells, low dosage of SMS was found to be less toxic, whereas high dosage produced profound cell death.

Induction of apoptosis by the retinoid inducible growth regulator RIG1 depends on the NC motif in HtTA cervical cancer cells

BackgroundRetinoid-inducible gene 1 (RIG1), also known as tazarotene-induced gene 3 or retinoic-acid receptor responder 3, is a growth regulator, which induces apoptosis and differentiation. RIG1 is classified into the NC protein family. This study investigated functional domains and critical amino acids associated with RIG1-mediated cell death and apoptosis.ResultsUsing enhanced green fluorescence protein (EGFP)-tagged RIG1 variants, RIG1 proteins with deletion at the NC domain significantly decreased cell death induced by RIG1, and fusion variants containing only the NC domain significantly induced apoptosis of HtTA cervical cancer cells. The EGFP-RIG1-induced apoptosis was significantly decreased in cells expressing N112C113 motif double- (NC→FG) or triple- (NCR→FGE) mutated RIG1 variants. Using dodecapeptides, nuclear localization and profound cell death was observed in HtTA cells expressing wild type RIG1111–123 or Leu121-mutated RIG1111–123:L→ C peptide, but peptides double- or triple-mutated at the NC motif alone, RIG1111–123:NC→FG or RIG1111–123:NCR→FGE, were cytoplasmically localized and did not induce apoptosis. The RIG1111–123 also induced apoptosis of A2058 melanoma cells but not normal human fibroblasts.ConclusionThe NC domain, especially the NC motif, plays the major role in RIG1-mediated pro-apoptotic activity. The RIG1111–123 dodecapeptide exhibited strong pro-apoptotic activity and has potential as an anticancer drug.

Targeting HMG-CoA Reductase in Acute Lymphoblastic Leukemia (ALL): Statins Inhibit Proliferation and Induce Apoptosis in ALL Cells

HMG-CoA reductase inhibitors (statins) are commonly used, FDA-approved agents for the treatment of hypercholesterolemia. In addition to reducing serum cholesterol, statins have been shown to have anti-inflammatory properties and anti-proliferative effects on a number of cell types, including lymphocytes. Many of these effects occur because inhibition of HMG-CoA reductase results in depletion of a number of important cellular intermediates required for signal transduction through the AKT, Ras, and Erk pathways. Statins have been demonstrated to have anti-proliferative effects in a number of malignancies, including acute myeloid leukemia using preclinical models; however, there are no published reports evaluating the efficacy of these agents against acute lymphoblastic leukemia. Large population studies have also suggested that statins may reduce the incidence of a number of cancers, furthering interest in the use of these agents in malignancies, especially since the dose-limiting toxicities (hepatitis and rhabdomyolysis) do not overlap with the majority of commonly used cytotoxic agents. We hypothesized that ALL cells are dependent on the function of HMG-CoA reductase for survival. We tested this hypothesis by studying the efficacy of statins against ALL cells. We studied the effects of 5 different compounds (mevastatin, pravastatin, fluvastatin, lovastatin, and simvastatin) against 7 ALL cell lines (4 human and 3 murine). We found that the more potent 2nd generation statins (fluvastatin, lovastatin, and simvastatin) had marked effects on ALL cells, whereas the less potent first generation statins (mevastatin and pravastatin) had less significant effects. We found that fluvastatin, lovastatin, and simvastatin inhibited proliferation of all 7 ALL cell lines using MTT assay (p <0.05). We also found these three statins induced apoptosis, leading to profound cell death in all 7 cell lines (p<0.05) as assessed by flow cytometry for Annenix-V staining and 7-AAD. IC50 dosing for the three agents ranged between 500nM and 5uM depending on the cell line, levels easily obtainable in humans. The most likely explanation for this remarkable, single-agent effect on ALL is that ALL cells are indeed dependent on cholesterol biosynthesis. Other potential explanations include the possibility that ALL cells are dependant on one of the signal transduction pathways affected by targeting HMG-CoA reductase, or the formal possibility of an off-target effect of statins separate from the inhibition of cholesterol biosynthesis. To determine if the effects of the statins were due to a direct effect on HMG-CoA reductase, we treated cells with melavonolactone, the product of conversion of HMG-CoA by HMGCoA reductase. We found that the addition of melavonolactone completely reversed the effects of all statins even at high doses (>10uM) in all cell lines, making the explanation of an off-target, non-HMG-CoA reductase-based mechanism unlikely. Ongoing work includes the testing of statins in NOD/SCID xenograft models of primary human ALL models, assessing the effects of statins on AKT, Ras, and Erk in ALL cells, and assessing the combination of statins with cytotoxic agents in ALL. In conclusion, we found that ALL cells are dependent of HMG-CoA reductase for survival. Since statins are safe and well-tolerated, this class of agents should be further explored in patients with ALL.

Dicer loss in striatal neurons produces behavioral and neuroanatomical phenotypes in the absence of neurodegeneration

MicroRNAs (miRNAs) are small noncoding RNAs that can act to repress target mRNAs by suppressing translation and/or reducing mRNA stability. Although it is clear that miRNAs and Dicer, an RNase III enzyme that is central to the production of mature miRNAs, have a role in the early development of neurons, their roles in the postmitotic neuron in vivo are largely unknown. To determine the roles of Dicer in neurons, we ablated Dicer in dopaminoceptive neurons. Mice that have lost Dicer in these cells display a range of phenotypes including ataxia, front and hind limb clasping, reduced brain size, and smaller neurons. Surprisingly, dopaminoceptive neurons without Dicer survive over the life of the animal. The lack of profound cell death contrasts with other mouse models in which Dicer has been ablated. These studies highlight the complicated nature of Dicer ablation in the brain and provide a useful mouse model for studying dopaminoceptive neuron function.

Mild traumatic brain injury to the infant mouse causes robust white matter axonal degeneration which precedes apoptotic death of cortical and thalamic neurons

The immature brain in the first several years of childhood is very vulnerable to trauma. Traumatic brain injury (TBI) during this critical period often leads to neuropathological and cognitive impairment. Previous experimental studies in rodent models of infant TBI were mostly concentrated on neuronal degeneration, while axonal injury and its relationship to cell death have attracted much less attention. To address this, we developed a closed controlled head injury model in infant (P7) mice and characterized the temporospatial pattern of axonal degeneration and neuronal cell death in the brain following mild injury. Using amyloid precursor protein (APP) as marker of axonal injury we found that mild head trauma causes robust axonal degeneration in the cingulum/external capsule as early as 30 min post-impact. These levels of axonal injury persisted throughout a 24 h period, but significantly declined by 48 h. During the first 24 h injured axons underwent significant and rapid pathomorphological changes. Initial small axonal swellings evolved into larger spheroids and club-like swellings indicating the early disconnection of axons. Ultrastructural analysis revealed compaction of organelles, axolemmal and cytoskeletal defects. Axonal degeneration was followed by profound apoptotic cell death in the posterior cingulate and retrosplenial cortex and anterior thalamus which peaked between 16 and 24 h post-injury. At early stages post-injury no evidence of excitotoxic neuronal death at the impact site was found. At 48 h apoptotic cell death was reduced and paralleled with the reduction in the number of APP-labeled axonal profiles. Our data suggest that early degenerative response to injury in axons of the cingulum and external capsule may cause disconnection between cortical and thalamic neurons, and lead to their delayed apoptotic death.

Androgen receptor‐dependent regulation of Bcl‐xL expression: Implication in prostate cancer progression

Recently we reported that silencing the androgen receptor (AR) gene reduced Bcl-xL expression that was associated with a profound apoptotic cell death in prostate cancer cells. In this study we further investigated AR-regulated Bcl-xL expression. Prostate cancer cell line LNCaP and its sublines, LNCaP/PURO and LNCaP/Bclxl, were used for cell proliferation assay and xenograft experiments in nude mice. Luciferase gene reporters driven by mouse or human bcl-x gene promoter were used to determine androgen regulation of Bcl-xL expression. RT-PCR and Western blot assays were conducted to assess Bcl-xL gene expression. Chromatin immunoprecipitation assay was performed to determine AR interaction with Bcl-xL promoter. Bcl-xL-induced alteration of gene expression was examined using cDNA microarray assay. In cultured prostate cancer LNCaP cells, androgen treatment significantly increased Bcl-xL expression at mRNA and protein levels via an AR-dependent mechanism. Promoter analyses demonstrated that the AR mediated androgen-stimulated bcl-x promoter activation and that the AR interacted with bcl-x promoter. Enforced expression of Bcl-xL gene dramatically increased cell proliferation in vitro and promoted xenograft tumor growth in vivo. Genome-wide gene profiling analysis revealed that Bcl-xL expression was significantly higher in metastatic and castration-resistant diseases compared to normal prostate tissues or primary cancers. Bcl-xL overexpression significantly increased the expression of cyclin D2, which might be responsible for Bcl-xL-induced cell proliferation and tumor growth. Taken together, our data strongly suggest that androgen stimulates Bcl-xL expression via the AR and that increased Bcl-xL expression plays a versatile role in castration-resistant progression of prostate cancer.

Murine Wee1 Plays a Critical Role in Cell Cycle Regulation and Pre-Implantation Stages of Embryonic Development

Wee1 kinase regulates the G2/M cell cycle checkpoint by phosphorylating and inactivating the mitotic cyclin-dependent kinase 1 (Cdk1). Loss of Wee1 in many systems, including yeast and drosophila, leads to premature mitotic entry. However, the developmental role of Wee1 in mammals remains unclear. In this study, we established Wee1 knockout mice by gene targeting. We found that Wee-/- embryos were defective in the G2/M cell cycle checkpoint induced by gamma-irradiation and died of apoptosis before embryonic (E) day 3.5. To study the function of Wee1 further, we have developed MEF cells in which Wee1 is disrupted by a tamoxifen inducible Cre-LoxP approach. We found that acute deletion of Wee1 resulted in profound growth defects and cell death. Wee1 deficient cells displayed chromosome aneuploidy and DNA damage as revealed by gamma-H2AX foci formation and Chk2 activation. Further studies revealed a conserved mechanism of Wee1 in regulating mitotic entry and the G2/M checkpoint compared with other lower organisms. These data provide in vivo evidence that mammalian Wee1 plays a critical role in maintaining genome integrity and is essential for embryonic survival at the pre-implantation stage of mouse development.

Dissecting independent channel and scaffolding roles of the Drosophila transient receptor potential channel

Drosophila transient receptor potential (TRP) serves dual roles as a cation channel and as a molecular anchor for the PDZ protein, INAD (inactivation no afterpotential D). Null mutations in trp cause impairment of visual transduction, mislocalization of INAD, and retinal degeneration. However, the impact of specifically altering TRP channel function is not known because existing loss-of-function alleles greatly reduce protein expression. In the current study we describe the isolation of a set of new trp alleles, including trp 14 with an amino acid substitution juxtaposed to the TRP domain. The trp 14 flies stably express TRP and display normal molecular anchoring, but defective channel function. Elimination of the anchoring function alone in trp Δ 1272, had minor effects on retinal morphology whereas disruption of channel function caused profound light-induced cell death. This retinal degeneration was greatly suppressed by elimination of the Na+/Ca2+ exchanger, CalX, indicating that the cell death was due primarily to deficient Ca2+ entry rather than disruption of the TRP-anchoring function.

Transient exposure of mammary tumors to PD184352 and UCN-01 causes tumor cell death in vivo and prolonged suppression of tumor re-growth

Previous studies from our group have demonstrated in vitro that UCN-01 (7-hydroxystaurosporine) and inhibitors of MEK1/2 interact to cause tumor cell death in a wide variety of malignant, but not in non-transformed, cell types. The present studies determined whether UCN-01 and MEK1/2 inhibitors interacted to cause tumor cell death in vivo. In vitro colony formation studies demonstrated that UCN-01 and the MEK1/2 inhibitor PD184352 interacted to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) with similar combination index values. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of ~100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), UCN-01 (0.1-0.2 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following ~30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to either PD184352 or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo ~15-30 days after drug administration. In contrast, combined treatment with PD184352 and UCN-01 significantly reduced MDA-MB-231, and largely abolished MCF7 tumor growth. Tumor control values for both cell lines were 0.36. Tumor cells isolated ~30 days after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Reduced tumor growth correlated with profound tumor cell death within 5 days of combined drug exposure, which was also evident ~30 days after exposure. In addition, tumor cell death correlated with a reduction in the phosphorylation of ERK1/2 and the immuno-reactivity of Ki67 and of CD31. Collectively, these findings argue that UCN-01 and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.

Astrocyte–neurone communication following oxygen–glucose deprivation

We looked at the possible interactions between astrocytes and neurones during reperfusion using an in vitro model of ischaemia-reperfusion injury, as a controlled environment that lends itself easily to manipulation of the numerous variables involved in such an insult. We constructed a chamber in which O2 can be lowered to a concentration of 1 microm and developed a primary cortical neuronal culture that is 99% pure and can survive to at least 10 days in vitro. We also established a novel system for the co-culture of astrocytes and neurones in order to study the communication between these cells in a manner that allows the complete separation of one cell type from another. Neurone cultures showed profound cell death following an ischaemic period of only 15 min. We co-cultured neurones that had been subjected to a 15-min ischaemic insult with either non-insulted astrocytes or astrocyte-conditioned medium during the reperfusion stage. Both astrocytes and astrocyte-conditioned medium enhanced neuronal survival. Our data also suggest that astrocyte-sourced neuronal glutathione synthesis may play a role in preventing neuronal death.

Differential Effects of Gqα, G14α, and G15α on Vascular Smooth Muscle Cell Survival and Gene Expression Profiles

Gqalpha family members (Gqalpha, G11alpha, G14alpha, and G15/16alpha) stimulate phospholipase Cbeta (PLCbeta) and inositol lipid signaling but differ markedly in amino acid sequence and tissue distribution predicting unappreciated functional diversity. To examine functional differences, we compared the signaling properties of Gqalpha, G14alpha, and G15alpha and their cellular responses in vascular smooth muscle cells (VSMC). Constitutively active forms of Gqalpha, G14alpha, or G15alpha elicit markedly different responses when introduced to VSMC. Whereas each Galpha stimulated PLCbeta to similar extents when expressed at equal protein levels, Gqalpha and G14alpha but not G15alpha initiated profound cell death within 48 h. This response was the result of activation of apoptotic pathways, because Gqalpha and G14alpha, but not G15alpha, stimulated caspase-3 activation and did not alter phospho-Akt, a regulator of cell survival pathways. Gqalpha and G14alpha stimulate nuclear factor of activated T cell (NFAT) activation in VSMC, but Galpha-induced cell death seems independent of PKC, InsP(3)/Ca(2+), and NFAT, in that pharmacological inhibitors of these pathways did not block cell death. Gene expression analysis indicates that Gqalpha, G14alpha, and G15alpha each elicit markedly different profiles of altered gene sets in VSMC after 24 h. Whereas all three Galpha stimulated changes (> or =2-fold) in 50 shared mRNA, Gqalpha and G14alpha (but not G15alpha) stimulated changes in 221 shared mRNA, many of which are reported to be pro-apoptotic and/or involved with TNF-alpha signaling. We were surprised to find that each Galpha also stimulated changes in nonoverlapping Galpha-specific gene sets. These findings demonstrate that Gqalpha family members activate both overlapping and distinct signaling pathways and are more functionally diverse than previously thought.

Chemosensitization of carmustine with maitake beta-glucan on androgen-independent prostatic cancer cells: involvement of glyoxalase I.

To improve the poor efficacy (< 10%) of chemotherapy for patients with hormone-refractory prostate cancer, we investigated a possible cytotoxic effect of carmustine/beta-glucan combination on prostatic cancer PC-3 cells, focusing on a glutathione-dependent detoxifying enzyme, glyoxalase I (Gly-I). Carmustine (BCNU) is an anticancer agent and a putative inhibitor of Gly-I, while beta-glucan is a unique, nontoxic polysaccharide extracted from maitake mushrooms. The cytotoxic effects of BCNU or other anticancer agents with beta-glucan on PC-3 cells were assessed by cell-viability testing and Gly-I activity was measured using the spectrophotometric method. BCNU, 5-fluorouracil (5-FU), and methotrexate (MTX) were capable of inducing approximately a 50% reduction in cell viability at 72 hours, while etoposide, cisplatin, and mitomycin C were all ineffective. Only the combination of BCNU (50 micro ;mol) and beta-glucan (60 micro g/mL) exhibited an enhanced cytotoxicity with an approximate 90% cell viability reduction, but little improvement was seen with any combinations of 5-FU, MTX, or beta-glucon. Gly-I assays revealed that such a profound (approximately 90%) cell death was accompanied by an approximate 80% reduction in Gly-I activity by 6 hours. This study demonstrates a sensitized cytotoxic effect of BCNU with beta-glucan in PC-3 cells, which was associated with a drastic (approximately 80%) inactivation of Gly-I. Therefore, the BCNU/beta-glucan combination may help to improve current treatment efficacy by targeting Gly-I, which appears to be critically involved in prostate cancer viability.

A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis.

Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.

2-5A Antisense Directed against Telomerase RNA Produces Apoptosis in Ovarian Cancer Cells

Objective. RNase L is converted to an active form upon binding short 2′,5′-oligoadenylates (2-5A). To direct RNase L to an RNA target, 2-5A is attached to an antisense oligonucleotide (2-5A antisense). This chimera can be directed against telomerase—an RNA–protein complex that elongates telomeric DNA and is involved in cellular immortalization. Our objective is to investigate the effect of 2-5A antisense by targeting telomerase RNA (hTR) in the ovarian cancer cell line, HEY-1B.Methods. Baseline RNase L levels and telomerase activities were measured in both HEY-1B and normal ovarian epithelial cells (NOE). Cells were treated daily with chimeric oligonuclotides (ODN) directed against four different hTR sites, or control ODNs including nonchimeric antisense, 2-5A fused to a mismatched sequence, or inactive 2-5A fused to antisense. At 48 h, apoptosis was evaluated using the TUNEL assay. After six daily ODN administrations, telomerase activity was redetermined, and at 7 days viability counts were obtained.Results. Both cell lines expressed similar levels of RNase L. Hey-1B displayed telomerase activity while NOE did not. After 7 days of transfection, 2-5A antisense ODNs caused profound cell death in the HEY-1B cells, but not in the NOE cells. This effect was seen regardless of hTR target site, and ODN controls showed no significant decrease in cell viability in either cell line. HEY1B cells treated with 2-5A antisense against hTR showed a decrease in telomerase activity and a profound induction of programmed cell death.Conclusions. The results suggest that 2-5A antisense directed against telomerase RNA results in apoptotic cell death in ovarian cancer cells, but not normal ovarian epithelial cells. The 2-5A antisense strategy may hold a considerable advantage over the conventional antisense approach in targeting cancer-causing genes.