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1.
Learn Mem ; 31(5)2024 May.
Article in English | MEDLINE | ID: mdl-38862165

ABSTRACT

In this review, we aggregated the different types of learning and memory paradigms developed in adult Drosophila and attempted to assess the similarities and differences in the neural mechanisms supporting diverse types of memory. The simplest association memory assays are conditioning paradigms (olfactory, visual, and gustatory). A great deal of work has been done on these memories, revealing hundreds of genes and neural circuits supporting this memory. Variations of conditioning assays (reversal learning, trace conditioning, latent inhibition, and extinction) also reveal interesting memory mechanisms, whereas mechanisms supporting spatial memory (thermal maze, orientation memory, and heat box) and the conditioned suppression of innate behaviors (phototaxis, negative geotaxis, anemotaxis, and locomotion) remain largely unexplored. In recent years, there has been an increased interest in multisensory and multicomponent memories (context-dependent and cross-modal memory) and higher-order memory (sensory preconditioning and second-order conditioning). Some of this work has revealed how the intricate mushroom body (MB) neural circuitry can support more complex memories. Finally, the most complex memories are arguably those involving social memory: courtship conditioning and social learning (mate-copying and egg-laying behaviors). Currently, very little is known about the mechanisms supporting social memories. Overall, the MBs are important for association memories of multiple sensory modalities and multisensory integration, whereas the central complex is important for place, orientation, and navigation memories. Interestingly, several different types of memory appear to use similar or variants of the olfactory conditioning neural circuitry, which are repurposed in different ways.


Subject(s)
Memory , Animals , Memory/physiology , Drosophila/physiology , Mushroom Bodies/physiology , Behavior, Animal/physiology
2.
Mol Psychiatry ; 2024 Mar 26.
Article in English | MEDLINE | ID: mdl-38532011

ABSTRACT

Recent and pioneering animal research has revealed the brain utilizes a variety of molecular, cellular, and network-level mechanisms used to forget memories in a process referred to as "active forgetting". Active forgetting increases behavioral flexibility and removes irrelevant information. Individuals with impaired active forgetting mechanisms can experience intrusive memories, distressing thoughts, and unwanted impulses that occur in neuropsychiatric diseases. The current evidence indicates that active forgetting mechanisms degrade, or mask, molecular and cellular memory traces created in synaptic connections of "engram cells" that are specific for a given memory. Combined molecular genetic/behavioral studies using Drosophila have uncovered a complex system of cellular active-forgetting pathways within engram cells that is regulated by dopamine neurons and involves dopamine-nitric oxide co-transmission and reception, endoplasmic reticulum Ca2+ signaling, and cytoskeletal remodeling machinery regulated by small GTPases. Some of these molecular cellular mechanisms have already been found to be conserved in mammals. Interestingly, some pathways independently regulate forgetting of distinct memory types and temporal phases, suggesting a multi-layering organization of forgetting systems. In mammals, active forgetting also involves modulation of memory trace synaptic strength by altering AMPA receptor trafficking. Furthermore, active-forgetting employs network level mechanisms wherein non-engram neurons, newly born-engram neurons, and glial cells regulate engram synapses in a state and experience dependent manner. Remarkably, there is evidence for potential coordination between the network and cellular level forgetting mechanisms. Finally, subjects with several neuropsychiatric diseases have been tested and shown to be impaired in active forgetting. Insights obtained from research on active forgetting in animal models will continue to enrich our understanding of the brain dysfunctions that occur in neuropsychiatric diseases.

3.
Nature ; 591(7850): 426-430, 2021 03.
Article in English | MEDLINE | ID: mdl-33473212

ABSTRACT

Active forgetting is an essential component of the memory management system of the brain1. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. Here we identify a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval.


Subject(s)
Dopamine/metabolism , Dopaminergic Neurons/physiology , Drosophila melanogaster/physiology , Mental Recall/physiology , Animals , Central Nervous System/cytology , Central Nervous System/physiology , Conditioning, Psychological , Dendrites/physiology , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Electric Stimulation , Female , Memory, Long-Term/physiology , Mushroom Bodies/cytology , Mushroom Bodies/physiology , Odorants , Receptors, Dopamine D1/metabolism , Time Factors
4.
Front Cell Neurosci ; 14: 258, 2020.
Article in English | MEDLINE | ID: mdl-33061890

ABSTRACT

Active memory forgetting is a well-regulated biological process thought to be adaptive and to allow proper cognitive functions. Recent efforts have elucidated several molecular players involved in the regulation of olfactory forgetting in Drosophila, including the small G protein Rac1, the dopamine receptor DAMB, and the scaffold protein Scribble. Similarly, we recently reported that dopaminergic neurons mediate both learning- and forgetting-induced plasticity in the mushroom body output neuron MBON-γ2α'1. Two open questions remain: how does forgetting affect plasticity in other, highly plastic, mushroom body compartments and how do genes that regulate forgetting affect this cellular plasticity? Here, we show that forgetting reverses short-term synaptic depression induced by aversive conditioning in the highly plastic mushroom body output neuron MBON-γ1pedc>α/ß. In addition, our results indicate that genetic tampering with normal forgetting by inhibition of small G protein Rac1 impairs restoration of depressed odor responses to learned odor by intrinsic forgetting through time passing and forgetting induced acutely by shock stimulation after conditioning or reversal learning. These data further indicate that some forms of forgetting truly erase physiological changes generated by memory encoding.

5.
Neuron ; 101(1): 103-118.e5, 2019 01 02.
Article in English | MEDLINE | ID: mdl-30503644

ABSTRACT

Stromalin, a cohesin complex protein, was recently identified as a novel memory suppressor gene, but its mechanism remained unknown. Here, we show that Stromalin functions as a negative regulator of synaptic vesicle (SV) pool size in Drosophila neurons. Stromalin knockdown in dopamine neurons during a critical developmental period enhances learning and increases SV pool size without altering the number of dopamine neurons, their axons, or synapses. The developmental effect of Stromalin knockdown persists into adulthood, leading to strengthened synaptic connections and enhanced olfactory memory acquisition in adult flies. Correcting the SV content in dopamine neuron axon terminals by impairing anterograde SV trafficking motor protein Unc104/KIF1A rescues the enhanced-learning phenotype in Stromalin knockdown flies. Our results identify a new mechanism for memory suppression and reveal that the size of the SV pool is controlled genetically and independent from other aspects of neuron structure and function through Stromalin.


Subject(s)
Drosophila Proteins/deficiency , Memory/physiology , Nuclear Proteins/deficiency , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructure , Amino Acid Sequence , Animals , Animals, Genetically Modified , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/ultrastructure , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Nuclear Proteins/genetics , Synapses/genetics , Synapses/metabolism , Synapses/ultrastructure , Synaptic Vesicles/genetics
6.
Cell Rep ; 25(3): 651-662.e5, 2018 10 16.
Article in English | MEDLINE | ID: mdl-30332645

ABSTRACT

It remains unclear how memory engrams are altered by experience, such as new learning, to cause forgetting. Here, we report that short-term aversive memory in Drosophila is encoded by and retrieved from the mushroom body output neuron MBOn-γ2α'1. Pairing an odor with aversive electric shock creates a robust depression in the calcium response of MBOn-γ2α'1 and increases avoidance to the paired odor. Electric shock after learning, which activates the cognate dopamine neuron DAn-γ2α'1, restores the response properties of MBOn-γ2α'1 and causes behavioral forgetting. Conditioning with a second odor restores the responses of MBOn-γ2α'1 to a previously learned odor while depressing responses to the newly learned odor, showing that learning and forgetting can occur simultaneously. Moreover, optogenetic activation of DAn-γ2α'1 is sufficient for the bidirectional modulation of MBOn-γ2α'1 response properties. Thus, a single DAn can drive both learning and forgetting by bidirectionally modulating a cellular memory trace.


Subject(s)
Avoidance Learning/physiology , Dopaminergic Neurons/physiology , Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Memory, Short-Term/physiology , Mushroom Bodies/physiology , Smell/physiology , Animals , Behavior, Animal , Drosophila Proteins/genetics , Female
7.
Cell Rep ; 21(8): 2074-2081, 2017 Nov 21.
Article in English | MEDLINE | ID: mdl-29166600

ABSTRACT

Prior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cyclic AMP (cAMP) accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. Here, we surveyed the spectrum of G protein coupling of dDA1 and Damb, and we confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca2+ signaling with greater efficiency and dopamine sensitivity. The knockdown of Gαq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. Our findings identify a Damb/Gq-signaling pathway that stimulates forgetting and resolves the opposing effects of dopamine on acquisition and forgetting.


Subject(s)
Drosophila Proteins/metabolism , Memory/physiology , Receptors, Dopamine D1/metabolism , Receptors, Dopamine/metabolism , Animals , Behavior, Animal/physiology , Conditioning, Classical/physiology , Cyclic AMP/metabolism , Dopamine/metabolism , Dopaminergic Neurons/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Mushroom Bodies/metabolism , Receptors, Dopamine/genetics , Receptors, Dopamine D1/genetics , Smell/physiology
8.
Cell ; 161(7): 1656-67, 2015 Jun 18.
Article in English | MEDLINE | ID: mdl-26073942

ABSTRACT

Early studies from psychology suggest that sleep facilitates memory retention by stopping ongoing retroactive interference caused by mental activity or external sensory stimuli. Neuroscience research with animal models, on the other hand, suggests that sleep facilitates retention by enhancing memory consolidation. Recently, in Drosophila, the ongoing activity of specific dopamine neurons was shown to regulate the forgetting of olfactory memories. Here, we show this ongoing dopaminergic activity is modulated with behavioral state, increasing robustly with locomotor activity and decreasing with rest. Increasing sleep-drive, with either the sleep-promoting agent Gaboxadol or by genetic stimulation of the neural circuit for sleep, decreases ongoing dopaminergic activity, while enhancing memory retention. Conversely, increasing arousal stimulates ongoing dopaminergic activity and accelerates dopaminergic-based forgetting. Therefore, forgetting is regulated by the behavioral state modulation of dopaminergic-based plasticity. Our findings integrate psychological and neuroscience research on sleep and forgetting.


Subject(s)
Drosophila melanogaster/physiology , Learning , Memory , Models, Animal , Sleep , Animals , Arousal , Behavior, Animal , Dopamine/metabolism , Drosophila melanogaster/cytology , GABA Agonists/pharmacology , Isoxazoles/pharmacology , Neuronal Plasticity , Neurons/metabolism , Sleep/drug effects
9.
Prog Brain Res ; 208: 39-62, 2014.
Article in English | MEDLINE | ID: mdl-24767478

ABSTRACT

Failure to remember, or forgetting, is a phenomenon familiar to everyone and despite more than a century of scientific inquiry, why we forget what we once knew remains unclear. If the brain marshals significant resources to form and store memories, why is it that these memories become lost? In the last century, psychological studies have divided forgetting into decay theory, in which memory simply dissipates with time, and interference theory, in which additional learning or mental activity hinders memory by reducing its stability or retrieval (for review, Dewar et al., 2007; Wixted, 2004). Importantly, these psychological models of forgetting posit that forgetting is a passive property of the brain and thus a failure of the brain to retain memories. However, recent neuroscience research on olfactory memory in Drosophila has offered evidence for an alternative conclusion that forgetting is an "active" process, with specific, biologically regulated mechanisms that remove existing memories (Berry et al., 2012; Shuai et al., 2010). Similar to the bidirectional regulation of cell number by mitosis and apoptosis, protein concentration by translation and lysosomal or proteomal degradation, and protein phosphate modification by kinases and phosphatases, biologically regulated memory formation and removal would be yet another example in biological systems where distinct and separate pathways regulate the creation and destruction of biological substrates.


Subject(s)
Memory Disorders/physiopathology , Memory/physiology , Olfactory Pathways/physiology , Smell/physiology , Animals , Drosophila
10.
J Neurosci ; 33(23): 9846-54, 2013 Jun 05.
Article in English | MEDLINE | ID: mdl-23739981

ABSTRACT

System consolidation, as opposed to cellular consolidation, is defined as the relatively slow process of reorganizing the brain circuits that maintain long-term memory. This concept is founded in part on observations made in mammals that recently formed memories become progressively independent of brain regions initially involved in their acquisition and retrieval and dependent on other brain regions for their long-term storage. Here we present evidence that olfactory appetitive and aversive memories in Drosophila evolve using a system-like consolidation process. We show that all three classes of mushroom body neurons (MBNs) are involved in the retrieval of short- and intermediate-term memory. With the passage of time, memory retrieval becomes independent of α'/ß' and γ MBNs, and long-term memory becomes completely dependent on α/ß MBNs. This shift in neuronal dependency for behavioral performance is paralleled by shifts in the activity of the relevant neurons during the retrieval of short-term versus long-term memories. Moreover, transient neuron inactivation experiments using flies trained to have both early and remote memories showed that the α'/ß' MBNs have a time-limited role in memory processing. These results argue that system consolidation is not a unique feature of the mammalian brain and memory systems, but rather a general and conserved feature of how different temporal memories are encoded from relatively simple to complex brains.


Subject(s)
Avoidance Learning/physiology , Memory/physiology , Mushroom Bodies/physiology , Odorants , Smell/physiology , Animals , Animals, Genetically Modified , Drosophila melanogaster
11.
Neuron ; 74(3): 530-42, 2012 May 10.
Article in English | MEDLINE | ID: mdl-22578504

ABSTRACT

Psychological studies in humans and behavioral studies of model organisms suggest that forgetting is a common and biologically regulated process, but the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the bidirectional modulation of a small subset of dopamine neurons (DANs) after olfactory learning regulates the rate of forgetting of both punishing (aversive) and rewarding (appetitive) memories. Two of these DANs, MP1 and MV1, exhibit synchronized ongoing activity in the mushroom body neuropil in alive and awake flies before and after learning, as revealed by functional cellular imaging. Furthermore, while the mushroom-body-expressed dDA1 dopamine receptor is essential for the acquisition of memory, we show that the dopamine receptor DAMB, also highly expressed in mushroom body neurons, is required for forgetting. We propose a dual role for dopamine: memory acquisition through dDA1 signaling and forgetting through DAMB signaling in the mushroom body neurons.


Subject(s)
Avoidance Learning/physiology , Dopamine/metabolism , Dopaminergic Neurons/physiology , Memory Disorders/physiopathology , Mushroom Bodies/cytology , Analysis of Variance , Animals , Animals, Genetically Modified , Appetite/genetics , Behavior, Animal , Biophysics , Conditioning, Classical/physiology , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Dynamins/genetics , Electroshock/adverse effects , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Ion Channels , Male , Memory Disorders/genetics , Models, Biological , Mushroom Bodies/physiology , Odorants , Receptors, Dopamine D1/metabolism , Repressor Proteins/genetics , Retention, Psychology/physiology , TRPA1 Cation Channel , TRPC Cation Channels/genetics , Temperature , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Tyrosine 3-Monooxygenase/genetics
12.
J Surg Res ; 147(2): 240-6, 2008 Jun 15.
Article in English | MEDLINE | ID: mdl-18498876

ABSTRACT

INTRODUCTION: Cyclooxygenase (COX2) expression in primary breast cancer correlates with a worse prognosis. We reported previously that COX2 expression in MCF10A breast epithelial cells of basal subtype induces genomic instability. To understand the role of COX2 in estrogen receptor-positive (non-basal) breast cancer, we transfected the MCF7 cell line with COX2 and analyzed its chromosomal profile, BCL2 protein expression, and resistance to doxorubicin. We also analyzed cell cultures grown as mammospheres to determine whether COX2 expression affects the cancer-initiating ("stem") cell phenotype in MCF7 cells. METHODS: MCF7 Tet On cells (obtained from Clontech) were stably transfected with pTRE2pur-COX2 or pTRE2pur-COX2-GFP to produce COX2 or COX2-GFP protein, respectively. BCL2 protein was detected by Western blotting. Sensitivity of cells to drug treatment was analyzed by MTT assay. Groups were compared using Chi-Square test. We analyzed the genomic instability phenotype by chromosome analysis of control and COX2 transfected metaphase-arrested MCF7 cells after Giemsa staining. We assessed the tumorigenic potential of cells grown as mammospheres with a clonogenic assay. RESULTS: Cytogenetic analysis of early passage COX2 transfected MCF7 cells demonstrated significant genomic instability as compared to parental MCF7 cells. COX2 overexpression was associated with a significant increase in chromosomal aberrations (chromatid breaks, chromosome fusions, C anaphase). COX2 transfected MCF7 cells produced a significantly higher level of the anti-apoptotic protein BCL2 than the parental cells. In a functional assay, we found that COX2 expression correlated with increased resistance to doxorubicin. In a complimentary approach to determine tumorigenic potential of cells, we found that COX2 increased the ability of MCF7 cells to grow as mammospheres in culture, which correlated with an increase in clonogenic efficiency. CONCLUSIONS: We found that COX2 expression in MCF7 breast cancer cells induced genomic instability, BCL2 expression, and doxorubicin resistance, thus making them significantly more tumorigenic. This data suggests that COX-2 may be an important target for breast cancer treatment.


Subject(s)
Adenocarcinoma/enzymology , Breast Neoplasms/enzymology , Cell Transformation, Neoplastic , Cyclooxygenase 2/metabolism , Genomic Instability/physiology , Antibiotics, Antineoplastic/administration & dosage , Cell Culture Techniques , Cell Line, Tumor , Cell Transformation, Neoplastic/drug effects , Colony-Forming Units Assay , Cyclooxygenase 2/genetics , Cytogenetic Analysis , DNA, Complementary , Doxorubicin/administration & dosage , Drug Resistance, Neoplasm/physiology , Female , Humans , Phenotype , Proto-Oncogene Proteins c-bcl-2/metabolism , Transfection
13.
J Surg Res ; 140(2): 220-6, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17418864

ABSTRACT

BACKGROUND: Cyclooxygenase-2 (COX-2) is induced in many breast cancers and COX-2 expression correlates with a worse outcome in the clinic. We hypothesized that the induction of genomic instability is a major mechanism through which COX-2 contributes to breast cancer progression. METHODS: We transfected a normal immortalized breast epithelial cell line of Basal B subtype, MCF10A, with the pSG5-COX-2 vector and established the stably transfected cell line MCF10A/COX-2. We analyzed the genomic instability phenotype by chromosomal analysis of metaphase-arrested MCF10A and MCF10A/COX-2 cells after Giemsa staining. Groups were compared using chi(2) tests. To investigate the DNA damage checkpoint signaling, we analyzed the phosphorylation status of CHK1 protein with a phospho-specific antibody. RESULTS: Cytogenetic analysis of early passage transfected cells showed that COX-2 expression increased genomic instability compared with the MCF10A cells transfected with a luciferase vector alone. COX-2 overexpression was associated with a significant increase in chromosomal aberrations (fusions, breaks, and tetraploidy). There was a statistically significant increase in the number of polyploid cells in the COX-2 transfected cells versus the control (P=0.004). We also found that an inhibitory CHK1 phosphorylation at Ser-280 was dramatically increased upon COX-2 overexpression in MCF10A cells, thus explaining the mechanism of inactivation of an important cell cycle checkpoint. Further analysis of the MCF10A/COX-2 cells showed that these cells have acquired a premalignant phenotype characterized by a morphological transformation, a resistance to anoikis, a reduced requirement of epidermal growth factor for growth in culture, but their inability to establish tumors in a nude mouse model of malignancy. CONCLUSION: We found that COX-2 expression in MCF10A breast epithelial cells confers a premalignant phenotype that includes enhanced genomic instability and altered cell-cycle regulation.


Subject(s)
Breast/enzymology , Cyclooxygenase 2/metabolism , Epithelial Cells/enzymology , Genomic Instability/genetics , Animals , Anoikis/genetics , Breast/cytology , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Cycle , Cell Line , Checkpoint Kinase 1 , Cyclooxygenase 2/genetics , DNA Damage , Disease Progression , Epidermal Growth Factor/physiology , Epithelial Cells/cytology , Female , Gene Expression Regulation, Enzymologic , Humans , Mice , Mice, Nude , Phenotype , Precancerous Conditions/enzymology , Precancerous Conditions/genetics , Precancerous Conditions/pathology , Protein Kinases/genetics , Protein Kinases/metabolism , Transfection , Transplantation, Heterologous
14.
J Surg Res ; 134(1): 44-51, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16678856

ABSTRACT

BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression by a primary tumor correlates with poor prognosis in breast cancer, including early spread to bone. Interleukin-8 (IL-8) stimulates osteoclastogenesis and resorption of bone, and elevated IL-8 levels predict early metastatic spread of breast cancer. The purpose of this study was to test our hypothesis that tumors that overexpress COX-2 induce IL-8 production. MATERIALS AND METHODS: We cotransfected MCF-10A (nonmalignant breast epithelial) cells, as well as MDA-231 (highly metastatic human breast cancer) cell lines with a pSG5-COX-2 vector and pEF1a-Luc-IRES-Neo vector (luciferase reporter). COX-2 overexpression was confirmed by Western blot and PGE2 (a product of the COX-2 pathway) immunoassay. IL-8 production was measured by immunoassay. In vivo testing used a nude mouse model to measure COX-2 and IL-8 production from breast cancer cells that had metastasized to bone (bone-seeking clones (BSCs)). Long bone metastases were localized and quantified by luciferase imaging (Xenogen IVIS system) and X-ray. BSCs were isolated and cultured and then tested for the production of PGE2 and IL-8. RESULTS: COX-2 overexpression caused a 4- to 5-fold increase in IL-8 production in both MCF-10A and MDA-231 cells in vitro. In vivo, we observed that the MDA-231-BSC (metastatic cells isolated from bone metastases) produced significantly greater levels of both PGE2 and IL-8 compared to the parental MDA-231 cells (P < 0.01). In contrast to the results obtained with these estrogen receptor-negative cell lines, COX-2 expression failed to induce IL-8 in the MCF-7 estrogen receptor-positive breast cancer cell line. Treatment with the COX-2 inhibitor NS-398 at a low 1-mu[scap]M dose reduced the production of IL-8 in COX-2-transfected MDA-231 cells by 30%, thus confirming the involvement of COX-2 in IL-8 induction. CONCLUSION: COX-2 expression induced formation of PGE2 and IL-8 in breast cancer cells. Since PGE2 and IL-8 stimulate osteoclasts to resorb bone, COX-2 inhibition is a potential target for treatment to prevent bone metastases.


Subject(s)
Bone Neoplasms/secondary , Breast Neoplasms/pathology , Cyclooxygenase 2/physiology , Interleukin-8/physiology , Animals , Cell Line, Tumor , Cyclooxygenase 2 Inhibitors/pharmacology , Female , Humans , Interleukin-8/biosynthesis , Mice , Mice, Nude , Protein Processing, Post-Translational
15.
J Surg Res ; 131(2): 267-75, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16457848

ABSTRACT

BACKGROUND: Cyclooxygenase-2 (COX-2) is overexpressed in 40% of human invasive breast cancers. Interleukin-11 (IL-11), a potent mediator of osteoclastogenesis, is involved in breast cancer metastasis to bone. Since breast cancers that overexpress COX-2 are associated with a higher rate of metastasis to bone, we hypothesized that COX-2 expression in tumor cells would induce IL-11. MATERIALS AND METHODS: We transfected MCF-7 (poorly metastatic) and MDA-231 (highly metastatic) human breast cancer cell lines with COX-2 expression vectors. COX-2 overexpression was confirmed by Western blot and PGE(2) immunoassay, and IL-11 production was measured by immunoassay. We also used a nude mouse model to study COX-2 and IL-11 production from breast cancer cells that metastasized to bone. The bone-seeking clones (BSC) were isolated and cultured from the long bone metastases. RESULTS: COX-2 transfection caused an approximately 5- to 6-fold increase in IL-11 production in both MCF-7 and MDA-231 cells. MDA-435S-COX2-BSC (cells isolated from bone metastasis) produced elevated levels of IL-11 and PGE2 (an important mediator of COX-2) as compared to the parental MDA-435S-COX2 cells. Furthermore, a treatment with low 1- to 2-microm concentration NS-398 or Celecoxib significantly reduced the production of IL-11 in COX-2-transfected MDA-231 cells, thus confirming the involvement of COX-2 in IL-11 induction. CONCLUSION: COX-2-mediated production of IL-11 in breast cancer cells may be vital to the development of osteolytic bone metastases in patients with breast cancer, and a COX-2 inhibitor may be useful in inhibiting this process.


Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2/physiology , Interleukin-11/biosynthesis , Neoplasm Metastasis/physiopathology , Animals , Blotting, Western , Bone Neoplasms/secondary , Female , Humans , Immunoassay , Mice , Mice, Nude , Transfection , Tumor Cells, Cultured , Up-Regulation
16.
Int J Oncol ; 26(5): 1393-9, 2005 May.
Article in English | MEDLINE | ID: mdl-15809733

ABSTRACT

Cyclooxygenase-2 (COX-2), an inducible enzyme involved in prostaglandin (including PGE(2)) biosynthesis, is overexpressed in several epithelial malignancies including breast cancer. We tested the hypothesis that COX-2 overexpression in breast cancer cells results in increased cell motility and invasion. COX-2 overproducing cells were generated by stable transfection of several human breast cancer cells with pSG5-COX2 vector. We confirmed the overexpression of COX-2 protein by western blotting, and by measuring PGE(2) in the medium with an immunoassay. We measured cell motility by counting the number of cells crossing an 8-micron pore size PET membrane, and cell invasion by counting the number of cells invading through a Matrigel-coated membrane that simulates basement membrane. COX-2 transfected MDA-231 cells produced 30-43-fold more PGE2 as compared to parental cells. COX-2 overexpression increased cell migration approximately 2.2-fold and cell invasion through Matrigel approximately 5.1-fold. Addition of 50 microM NS-398, a COX-2 inhibitor, inhibited Matrigel invasion of MDA-231 cells by 54% as compared to solvent confirming the role of COX-2 in cell invasion. It is known that an increase in cell migration and invasion can be brought about by cytoskeletal alterations and basement membrane degradation due to increased expression of pro-urokinase plasminogen activator (pro-uPA). To investigate the mechanism of our observed increase in cell invasion by COX-2, we found by western blotting that the level of pro-uPA was significantly higher (approximately 5-fold) in COX-2 transfected MDA-231 cells than untransfected MDA-231 cells. Similar to our observations in cell culture, we found evidence that increased COX-2 activity correlates with uPA in a mouse model of breast cancer metastasis to bone. In this study, we conclude that COX-2 overexpression in human breast cancer cells enhances cell motility and invasiveness thus suggesting a mechanism of COX-2 mediated metastasis.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement , Neoplasm Invasiveness , Prostaglandin-Endoperoxide Synthases/biosynthesis , Prostaglandin-Endoperoxide Synthases/metabolism , Animals , Blotting, Western , Cyclooxygenase 2 , Dinoprostone/biosynthesis , Disease Models, Animal , Female , Humans , Immunoassay , Membrane Proteins , Mice , Transfection , Tumor Cells, Cultured
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