Contractile function of rat myocardium is less susceptible to hypoxia/reoxygenation after acute infarction

The FHIT (fragile histidine triad) gene located at chromosome 3p14.2 has been proposed as a candidate tumor suppressor gene in human cancers. Fhit protein with the diadenosine 5',5'-P¹,P³-triphosphate (Ap₃A) hydrolase activity is the protein product of FHIT gene. The way in which Fhit exerts its tumor suppressor activity and the relationship of the Ap₃A hydrolase activity to tumor suppression are not known. As a step toward understanding of the Fhit function in the cell we have explored its intracellular localization and distribution in the rat tissues. Data obtained from immunoblot analysis showed that Fhit protein was most abundant in spleen and brain. Moderate amount of Fhit was detected in kidney and liver, whereas the level of Fhit protein in heart, skeletal muscle and kidney glomeruli was undetectable. RT-PCR performed on RNA isolated from these tissues showed no product, whereas the level of Fhit mRNA in spleen, brain, kidney, liver and lung correlated with the Fhit protein level. The immunoblot analysis performed on subcellular fractions of various rat tissues obtained by differential and density-gradient centrifugation showed that Fhit protein was localized exclusively in nucleus and at the plasma membrane. Presented data showing nuclear and plasma membrane localization of Fhit may support the hypothesis concerning Fhit as a signaling molecule.     

Activation of Gαq subunits up-regulates the expression of the tumor suppressor Fhit

The tumor suppressor Fhit protein is defective or absent in many tumor cells due to methylation, mutation or deletion of the FHIT gene. Despite numerous attempts to unravel the functions of Fhit, the mechanisms by which the function and expression of Fhit are regulated remain poorly understood. We have recently shown that activated Gα_q subunits interact directly with Fhit and enhance its inhibitory effect on cell growth. Here we investigated the regulation of Fhit expression by G_q. Our results showed that Fhit was up-regulated specifically by activating Gα subunits of the G_q subfamily but not by those of the other G protein subfamilies. This up-regulation effect was mediated by a PKC/MEK pathway independent of Src-mediated Fhit Tyr¹¹⁴ phosphorylation. We further demonstrated that elevated Fhit expression was due to the specific regulation of Fhit protein synthesis in the ribosome by activated Gα_q, where the regulations of cap-dependent protein synthesis were apparently not required. Moreover, we showed that activated Gα_q could increase cell–cell adhesion through Fhit. These findings provide a possible handle to modulate the level of the Fhit tumor suppressor by manipulating the activity of G_q-coupled receptors.     

Wogonin Induced Calreticulin/Annexin A1 Exposure Dictates the Immunogenicity of Cancer Cells in a PERK/AKT Dependent Manner

In response to ionizing irradiation and certain chemotherapeutic agents, dying tumor cells elicit a potent anticancer immune response. However, the potential effect of wogonin (5,7-dihydroxy-8-methoxyflavone) on cancer immunogenicity has not been studied. Here we demonstrated for the first time that wogonin elicits a potent antitumor immunity effect by inducing the translocation of calreticulin (CRT) and Annexin A1 to cell plasma membrane as well as the release of high-mobility group protein 1 (HMGB1) and ATP. Signal pathways involved in this process were studied. We found that wogonin-induced reactive oxygen species (ROS) production causes an endoplasmic reticulum (ER) stress response, including the phosphorylation of PERK (PKR-like endoplasmic reticulum kinase)/PKR (protein kinase R) and eIF2α (eukaryotic initiation factor 2α), which served as upstream signal for the activation of phosphoinositide 3-kinase (PI3K)/AKT, inducing calreticulin (CRT)/Annexin A1 cell membrane translocation. P22/CHP, a Ca²⁺-binding protein, was associated with CRT and was required for CRT translocation to cell membrane. The releases of HMGB1 and ATP from wogonin treated MFC cells, alone or together with other possible factors, activated dendritic cells and induced cytokine releases. In vivo study confirmed that immunization with wogonin-pretreated tumor cells vaccination significantly inhibited homoplastic grafted gastric tumor growth in mice and a possible inflammatory response was involved. In conclusion, the activation of PI3K pathway elicited by ER stress induced CRT/Annexin A1 translocation (“eat me” signal) and HMGB1 release, mediating wogonin-induced immunity of tumor cell vaccine. This indicated that wogonin is a novel effective candidate of immunotherapy against gastric tumor.     

Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.     

S100 and annexin proteins identify cell membrane damage as the Achilles heel of metastatic cancer cells

Mechanical activity of cells and the stress imposed on them by extracellular environment is a constant source of injury to the plasma membrane (PM). In invasive tumor cells, increased motility together with the harsh environment of the tumor stroma further increases the risk of PM injury. The impact of these stresses on tumor cell plasma membrane and mechanism by which tumor cells repair the PM damage are poorly understood. Ca²⁺ entry through the injured PM initiates repair of the PM. Depending on the cell type, different organelles and proteins respond to this Ca²⁺ entry and facilitate repair of the damaged plasma membrane. We recently identified that proteins expressed in various metastatic cancers including Ca²⁺-binding EF hand protein S100A11 and its binding partner annexin A2 are used by tumor cells for plasma membrane repair (PMR). Here we will discuss the involvement of S100, annexin proteins and their regulation of actin cytoskeleton, leading to PMR. Additionally, we will show that another S100 member – S100A4 accumulates at the injured PM. These findings reveal a new role for the S100 and annexin protein up regulation in metastatic cancers and identify these proteins and PMR as targets for treating metastatic cancers.     

Increased Spinal Cord Na+-K+-2Cl? Cotransporter-1 (NKCC1) Activity Contributes to Impairment of Synaptic Inhibition in Paclitaxel-induced Neuropathic Pain

Microtubule-stabilizing agents, such as paclitaxel (Taxol), are effective chemotherapy drugs for treating many cancers, and painful neuropathy is a major dose-limiting adverse effect. Cation-chloride cotransporters, such as Na⁺-K⁺-2Cl⁻ cotransporter-1 (NKCC1) and K⁺-Cl⁻ cotransporter-2 (KCC2), critically influence spinal synaptic inhibition by regulating intracellular chloride concentrations. Here we show that paclitaxel treatment in rats significantly reduced GABA-induced membrane hyperpolarization and caused a depolarizing shift in GABA reversal potential of dorsal horn neurons. However, paclitaxel had no significant effect on AMPA or NMDA receptor-mediated glutamatergic input from primary afferents to dorsal horn neurons. Paclitaxel treatment significantly increased protein levels, but not mRNA levels, of NKCC1 in spinal cords. Inhibition of NKCC1 with bumetanide reversed the paclitaxel effect on GABA-mediated hyperpolarization and GABA reversal potentials. Also, intrathecal bumetanide significantly attenuated hyperalgesia and allodynia induced by paclitaxel. Co-immunoprecipitation revealed that NKCC1 interacted with β-tubulin and β-actin in spinal cords. Remarkably, paclitaxel increased NKCC1 protein levels at the plasma membrane and reduced NKCC1 levels in the cytosol of spinal cords. In contrast, treatment with an actin-stabilizing agent had no significant effect on NKCC1 protein levels in the plasma membrane or cytosolic fractions of spinal cords. In addition, inhibition of the motor protein dynein blocked paclitaxel-induced subcellular redistribution of NKCC1, whereas inhibition of kinesin-5 mimicked the paclitaxel effect. Our findings suggest that increased NKCC1 activity contributes to diminished spinal synaptic inhibition and neuropathic pain caused by paclitaxel. Paclitaxel disrupts intracellular NKCC1 trafficking by interfering with microtubule dynamics and associated motor proteins.     

Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors

Background

Cell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged.

Methodology/Principal Findings

Cell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ET_BR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence.

Conclusions/Significance

Although aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application.     

Identification of an AHNAK binding motif specific for the Annexin2/S100A10 tetramer

The Annexin2 tetramer (A2t), which consists of two Annexin2 molecules bound to a S100A10 dimer, is implicated in membrane-trafficking events. Here, we showed using a yeast triple-hybrid experiment and in vitro binding assay that Annexin2 is required for strong binding of S100A10 to the C-terminal domain of the protein Ahnak. We also revealed that this effect involves only the Annexin2 N-terminal tail, which is implicated in S100A10/Annexin2 tetramerization. The minimal A2t binding motif (A2tBP1) in Ahnak was mapped to a 20-amino acid peptide, and this peptide is highly specific for A2t. We also identified a second A2t binding motif (A2tBP2) present in the N-terminal domain of Ahnak, which binds to A2t, albeit with less affinity. When overexpressed as an EGFP fusion protein in MDCK cells, A2tBPs cofractionate in a calcium-dependent manner and co-immunoprecipitate with S100A10 and Annexin2. In living cells, A2tBPs target EGFP to the cytoplasm as does Annexin2. In response to oxidative and mechanical stress, EGFP-A2tBPs relocalize within minutes to the plasma membrane; a behavior shared with Annexin2-GFP. These results suggest that the A2t complex exists within the cytoplasm of resting living cells and that its localization at the plasma membrane relies on cellular signaling. Together, our data demonstrate that A2tBP1 is a specific A2t complex binding domain and may be a powerful tool to help elucidate A2t structure and cellular functions.     

Annexin VI-mediated Loss of Spectrin during Coated Pit Budding Is Coupled to Delivery of LDL to Lysosomes

Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH₂-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI–dependent budding is accompanied by the loss of ∼50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI–dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.     

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability^1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells³. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells ^4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane ^1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases^7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence ^1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment¹⁰. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence¹⁰. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).     

Decrease of Let-7f in Low-Dose Metronomic Paclitaxel Chemotherapy Contributed to Upregulation of Thrombospondin-1 in Breast Cancer

Low-dose metronomic (LDM) paclitaxel therapy displayed a stronger anti-angiogenic activity on breast tumors with fewer side effects. Upregulation of anti-angiogenic factor Thrombospondin-1 (TSP-1) accords for therapeutic potency of LDM paclitaxel, but its molecular mechanism has not been elucidated yet. microRNAs (miRNAs) have emerged as new important regulators of tumor growth and metastasis. Here, we hypothesize that miRNAs are involved in TSP-1 overexpression in paclitaxel LDM therapy of breast tumors. The miRNA profile of tumor tissues from control, LDM and MTD groups in 4T1 mouse breast cancer model was detected by microarray, and then verified by quantitative real-time PCR (qRT-PCR). Luciferase assay and western blot were employed to explore the mechanisms of miRNAs involved in this process. We found that let-7f, let-7a, miR-19b and miR-340-5p were reduced by >2 fold, and miR-543* and miR-684 were upregulated by at least 50% in paclitaxel LDM therapy. qRT-PCR verification revealed that let-7f level was reduced most significantly in LDM therapy. Computational prediction using TargetScan and miRanda suggested THBS1 which encodes TSP-1 as a potential target for let-7f. Luciferase activity assay further confirmed that let-7f may bind to 3''UTR of THBS1 gene and inhibit its activity. Moreover, forced expression of let-7f led to a decrease of TSP-1 at both mRNA and protein levels in MCF-7 cells. Contrastly, let-7f inhibition induced an increased expression of THBS1 mRNA and TSP-1 protein, but did not affect the proliferation and apoptosis of MCF-7 cells. Paclitaxel LDM therapy led to a decrease of let-7f and the elevation of TSP-1 protein expression in MCF-7 cells, while overexpression of let-7f may abolish LDM-induced the upregulation of TSP-1 in MCF-7 cells. In summary, let-7f inhibition contributed to the upregulation of TSP-1 in paclitaxel LDM therapy, independently of proliferation, cell cycle arrest and apoptosis of breast cancer. This study indicates let-7f as a potential therapeutic target for breast tumor.     

Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy

KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors.     

Phosphatidylserine Asymmetry Promotes the Membrane Insertion of a Transmembrane Helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. Here, we studied the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.     

Protein expression profiling identifies cyclophilin A as a molecular target in Fhit-mediated tumor suppression

Loss of fragile histidine triad (Fhit) expression is often associated with human malignancies, and Fhit functions as a tumor suppressor in controlling cell growth and apoptosis, although specific signal pathways are still undefined. We have used a proteomic approach to define proteins in the Fhit-mediated tumor suppression pathway. Because substitution of Tyr(114) (Y114) with phenylalanine (Y114F) diminishes Fhit functions, we did protein expression profiling to identify proteins differentially expressed in Fhit-negative H1299 lung cancer cells infected with wild-type (Ad-FHIT-wt) and Y114 mutant FHIT-expressing (Ad-FHIT-Y114F) adenoviruses. Among 12 distinct proteins that exhibited 4-fold differences in expression on comparison of the two infected cell lysates, cyclophilin A, the intracellular reporter of the immunosuppressive drug cyclosporine A, showed a remarkably decreased protein level in cells infected with Ad-FHIT-wt versus Ad-FHIT-Y114F. Conversely, loss of Fhit expression resulted in increased cyclophilin A expression in mouse tissues and cell lines. Restoration of Fhit expression led to down-regulated cyclophilin A protein expression and subsequently prevented cyclophilin A-induced up-regulation of cyclin D1, Cdk4, and resultant cell cycle progression (G(1)-S transition), which was independent of Ca(2+)/calmodulin-dependent kinase inhibitor, KN-93. Interestingly, Fhit down-modulation of phosphatase activity of calcineurin, which controls cyclin D1/Cdk4 activation, was reversed by cyclophilin A treatment in a concentration-dependent manner, a reversal that was inhibited by additional cyclosporine A treatment. Thus, cyclophilin A is a downstream target in Fhit-mediated cessation of cell cycle progression at late G(1) phase. Elucidation of the protein effectors of Fhit signaling may lead to identification of targets for lung cancer therapy.     

The Roles of Phosphorylation and SHAGGY-Like Protein Kinases in Geminivirus C4 Protein Induced Hyperplasia

Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.     

Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca²⁺-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca²⁺-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.     

Suppression of histone deacetylase 3 (HDAC3) enhances apoptosis induced by paclitaxel in human maxillary cancer cells in vitro and in vivo

Inclusion of chemotherapeutic drugs in treatment of patients with newly diagnosed head and neck cancer has improved response rates and prolonged median survival. Nevertheless, most patients with advanced head and neck cancer are destined to relapse and to develop resistance to initially used drugs such as paclitaxel. Consequently, it has been more important in cancer therapy to determine the molecular mechanisms that are related to cell-killing effects of anti-cancer agents or cancer resistance against them. Consequently, we examined whether abrogation of histone deacetylase 3 (HDAC3) expression by anti-sense oligonucleotides (ASOs) potentiates the efficacy of paclitaxel in human maxillary cancer IMC-3 cells. Here, we showed that paclitaxel-induced apoptosis was enhanced significantly by addition of ASOs for HDAC3 in cultured cells. Furthermore, paclitaxel-induced apoptosis in IMC-3 tumors transplanted in nude mice was enhanced significantly by administration of ASOs for HDAC3, thereby suppressing tumor growth. We provide new evidence that HDAC3 is a novel molecular target whose inactivation can potentiate the efficacy of anti-cancer drugs disrupting microtubules such as paclitaxel.