A large-scale screen reveals genes that mediate electrotaxis in Dictyostelium discoideum.

Directional cell migration in an electric field, a phenomenon called galvanotaxis or electrotaxis, occurs in many types of cells, and may play an important role in wound healing and development. Small extracellular electric fields can guide the migration of amoeboid cells, and we established a large-scale screening approach to search for mutants with electrotaxis phenotypes from a collection of 563 Dictyostelium discoideum strains with morphological defects. We identified 28 strains that were defective in electrotaxis and 10 strains with a slightly higher directional response. Using plasmid rescue followed by gene disruption, we identified some of the mutated genes, including some previously implicated in chemotaxis. Among these, we studied PiaA, which encodes a critical component of TORC2, a kinase protein complex that transduces changes in motility by activating the kinase PKB (also known as Akt). Furthermore, we found that electrotaxis was decreased in mutants lacking gefA, rasC, rip3, lst8, or pkbR1, genes that encode other components of the TORC2-PKB pathway. Thus, we have developed a high-throughput screening technique that will be a useful tool to elucidate the molecular mechanisms of electrotaxis.

Establishment and characterization of a highly tumorigenic African American prostate cancer cell line, E006AA-hT.

Genuine racial differences in prostate cancer (PCa) biology have been considered among the potential reasons to explain PCa disparities. There is no animal model to represent all aspects of human PCa and, more specifically, to be used for PCa disparity research. The lack of a spontaneously transformed in vitro cell-based model system has been a significant impediment to investigating and understanding potential molecular mechanisms, and the hormonal, genetic, and epigenetic factors underlying the biological and clinical aggressiveness of PCa in African American (AA) men. In this study, we established and characterized the E006AA-hT cell line as a highly tumorigenic subline of the previously characterized primary AA-PCa cell line, E006AA. Extensive characterization of the E006AA-hT cell line was accomplished using cytodifferentiation and prostate-specific markers, spectral karyotyping, cell line authentication assays, cell proliferation and migration assays, and in vitro tumorigenesis assays. Spectral karyotyping of E006AA-hT showed a hypertriploid chromosome complement and shared cytogenetic changes similar to its parental cells such as diploid X, absence of Y-chromosomes, numerical gains in chromosomes 5,6,8,10,17,20,21, and marker chromosomes of unknown origin. In addition, E006AA-hT also presented numerous clonal and structural aberrations such as insertion, deletion, duplication, and translocations in chromosomes 1-5, 8, 9, 11, 13, 14, 17, and 18. The E006AA-hT cell line was shown to be highly tumorigenic and produced tumors at an accelerated growth rate in both athymic nude and triple-deficient SCID mice. Silencing the mutated androgen receptor (AR-599 Ser>Gly) did not affect proliferation (loss-of-function), but decreased migration (gain-of-function) in E006AA-hT and its parental cell type. These data support that AR-point mutations may lead simultaneously to different "loss-of-function" and "gain-of-function" phenotypes in PCa cells. E006AA-Par and its subline as the only available spontaneously transformed low- and highly-tumorigenic primary AA-PCa cell lines could be used for basic and translational research aimed in supporting prostate cancer disparity research.

Glutamate, glutamate receptors, and downstream signaling pathways.

Glutamate is a nonessential amino acid, a major bioenergetic substrate for proliferating normal and neoplastic cells, and an excitatory neurotransmitter that is actively involved in biosynthetic, bioenergetic, metabolic, and oncogenic signaling pathways. Glutamate signaling activates a family of receptors consisting of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in chronic disabling brain disorders such as Schizophrenia and neurodegenerative diseases like Alzheimer's, Parkinson's, and multiple sclerosis. In this review, we discuss the structural and functional relationship of mGluRs and iGluRs and their downstream signaling pathways. The three groups of mGluRs, the associated second messenger systems, and subsequent activation of PI3K/Akt, MAPK, NFkB, PLC, and Ca/CaM signaling systems will be discussed in detail. The current state of human mGluR1a as one of the most important isoforms of Group I-mGluRs will be highlighted. The lack of studies on the human orthologues of mGluRs family will be outlined. We conclude that upon further study, human glutamate-initiated signaling pathways may provide novel therapeutic opportunities for a variety of non-malignant and malignant human diseases.

Glutamate signaling in benign and malignant disorders: current status, future perspectives, and therapeutic implications.

Glutamate, a nonessential amino acid, is the major excitatory neurotransmitter in the central nervous system. As such, glutamate has been shown to play a role in not only neural processes, such as learning and memory, but also in bioenergetics, biosynthetic and metabolic oncogenic pathways. Glutamate has been the target of intense investigation for its involvement not only in the pathogenesis of benign neurodegenerative diseases (NDDs) such as Parkinson's disease, Alzheimer's disease, schizophrenia, multiple sclerosis, and amyotropic lateral sclerosis (ALS), but also in carcinogenesis and progression of malignant diseases. In addition to its intracellular activities, glutamate in secreted form is a phylogenetically conserved cell signaling molecule. Glutamate binding activates multiple major receptor families including the metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in various signaling pathways in cancer. Inhibition of extracellular glutamate release or glutamate receptor activation via competitive or non-competitive antagonists decreases growth, migration and invasion and induces apoptosis in breast cancer, melanoma, glioma and prostate cancer cells. In this review, we discuss the current state of glutamate signaling research as it relates to benign and malignant diseases. In addition, we provide a synopsis of clinical trials using glutamate antagonists for the treatment of NDD and malignant diseases. We conclude that in addition to its potential role as a metabolic biomarker, glutamate receptors and glutamate-initiated signaling pathways may provide novel therapeutic opportunities for cancer.

Regulators of gene expression as biomarkers for prostate cancer.

Recent technological advancements in gene expression analysis have led to the discovery of a promising new group of prostate cancer (PCa) biomarkers that have the potential to influence diagnosis and the prediction of disease severity. The accumulation of deleterious changes in gene expression is a fundamental mechanism of prostate carcinogenesis. Aberrant gene expression can arise from changes in epigenetic regulation or mutation in the genome affecting either key regulatory elements or gene sequences themselves. At the epigenetic level, a myriad of abnormal histone modifications and changes in DNA methylation are found in PCa patients. In addition, many mutations in the genome have been associated with higher PCa risk. Finally, over- or underexpression of key genes involved in cell cycle regulation, apoptosis, cell adhesion and regulation of transcription has been observed. An interesting group of biomarkers are emerging from these studies which may prove more predictive than the standard prostate specific antigen (PSA) serum test. In this review, we discuss recent results in the field of gene expression analysis in PCa including the most promising biomarkers in the areas of epigenetics, genomics and the transcriptome, some of which are currently under investigation as clinical tests for early detection and better prognostic prediction of PCa.

Serum glutamate levels correlate with Gleason score and glutamate blockade decreases proliferation, migration, and invasion and induces apoptosis in prostate cancer cells.

PURPOSE: During glutaminolysis, glutamine is catabolized to glutamate and incorporated into citric acid cycle and lipogenesis. Serum glutamate levels were measured in patients with primary prostate cancer or metastatic castrate-resistant prostate cancer (mCRPCa) to establish clinical relevance. The effect of glutamate deprivation or blockade by metabotropic glutamate receptor 1 (GRM1) antagonists was investigated on prostate cancer cells' growth, migration, and invasion to establish biologic relevance. EXPERIMENTAL DESIGN: Serum glutamate levels were measured in normal men (n = 60) and patients with primary prostate cancer (n = 197) or mCRPCa (n = 109). GRM1 expression in prostatic tissues was examined using immunohistochemistry (IHC). Cell growth, migration, and invasion were determined using cell cytotoxicity and modified Boyden chamber assays, respectively. Apoptosis was detected using immunoblotting against cleaved caspases, PARP, and γ-H2AX. RESULTS: Univariate and multivariate analyses showed significantly higher serum glutamate levels in Gleason score ≥ 8 than in the Gleason score ≤ 7 and in African Americans than in the Caucasian Americans. African Americans with mCRPCa had significantly higher serum glutamate levels than those with primary prostate cancer or benign prostate. However, in Caucasian Americans, serum glutamate levels were similar in normal research subjects and patients with mCRPC. IHC showed weak or no expression of GRM1 in luminal acinar epithelial cells of normal or hyperplastic glands but high expression in primary or metastatic prostate cancer tissues. Glutamate deprivation or blockade decreased prostate cancer cells' proliferation, migration, and invasion and led to apoptotic cell death. CONCLUSIONS: Glutamate expression is mechanistically associated with and may provide a biomarker of prostate cancer aggressiveness.

Tumor suppressor Hippo/MST1 kinase mediates chemotaxis by regulating spreading and adhesion.

Chemotaxis depends on a network of parallel pathways that coordinate cytoskeletal events to bias cell movement along a chemoattractant gradient. Using a forward genetic screen in Dictyostelium discoideum, we identified the Ste20 kinase KrsB, a homolog of tumor suppressors Hippo and MST1/2, as a negative regulator of cell spreading and substrate attachment. The excessive adhesion of krsB(-) cells reduced directional movement and prolonged the streaming phase of multicellular aggregation. These phenotypes depended on an intact kinase domain and phosphorylation of a conserved threonine (T176) within the activation loop. Chemoattractants triggered a rapid, transient autophosphorylation of T176 in a heterotrimeric G protein-dependent and PI3K- and TorC2-independent manner. The active phosphorylated form of KrsB acts to decrease adhesion to the substrate. Taken together these studies suggest that cycling between active and inactive forms of KrsB may provide the dynamic regulation of cell adhesion needed for proper cell migration and chemotaxis. KrsB interacts genetically with another D. discoideum Hippo/MST homolog, KrsA, but the two genes are not functionally redundant. These studies show that Hippo/MST proteins, like the tumor suppressor PTEN and oncogenes Ras and PI3K, play a key role in cell morphological events in addition to their role in regulating cell growth.

Signaling pathways mediating chemotaxis in the social amoeba, Dictyostelium discoideum.

Chemotaxis, or cell migration guided by chemical cues, is critical for a multitude of biological processes in a diverse array of organisms. Dictyostelium discoideum amoebae rely on chemotaxis to find food and to survive starvation conditions, and we have taken advantage of this system to study the molecular regulation of this vital cell behavior. Previous work has identified phosphoinositide signaling as one mechanism which may contribute to directional sensing and actin polymerization during chemotaxis; a mechanism which is conserved in mammalian neutrophils. In this review, we will discuss recent data on genes and pathways governing directional sensing and actin polymerization, with a particular emphasis on contributions from our laboratory.

Chronic cocaine exposure in Drosophila: life, cell death and oogenesis.

Developmental signaling cascades that can be perturbed by cocaine and other drugs of abuse have been difficult to study in humans and vertebrate models. Although numerous direct neural targets of cocaine have been elucidated at the molecular level, little is known about the specific cellular events that are impacted indirectly as a result of the drug's perturbation of neural circuits. We have developed oogenesis in Drosophila melanogaster as a model in which to identify downstream biochemical and/or cellular processes that are disrupted by chronic cocaine exposure. In this model, cocaine feeding resulted not only in expected reductions in viability, but also in unanticipated developmental defects during oogenesis, including aberrant follicle morphogenesis and vitellogenic follicle degeneration. To identify mechanisms through which cocaine exerted its deleterious effects on oogenesis, we examined candidate components of neural and hormonal signaling pathways. Cocaine-induced disruptions in follicle formation were enhanced by juvenile hormone exposure and phenocopied by serotonin feeding, while cocaine-activated follicle apoptosis was enhanced by concomitant dopamine feeding. HPLC analysis of dopamine and serotonin in the ovary suggests that these neurotransmitters could variably mediate cocaine's effects on oogenesis indirectly in the brain and/or directly in the ovary itself. We confirmed the involvement of hormone signaling by measuring ecdysteroids, which increase following cocaine exposure, and by demonstrating suppression of cocaine-induced follicle loss by hormone receptor mutants. Cocaine-induced ovarian follicle apoptosis and adult lethality appear to be caused by modulation of dopamine levels, while morphological defects during follicle formation likely result from perturbing serotonin signaling during cocaine exposure. Our work suggests not only a new role for juvenile hormone and/or serotonin in Drosophila ovarian follicle formation, but also a cocaine-sensitive role for dopamine in modulating hormone levels in the female fly.

Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility.

The trace biogenic amine tyramine is present in the nervous systems of animals ranging in complexity from nematodes to mammals. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC), a member of the aromatic amino acid family, but this enzyme has not been identified in Drosophila or in higher animals. To further clarify the roles of tyramine and its metabolite octopamine, we have cloned two TDC genes from Drosophila melanogaster, dTdc1 and dTdc2. Although both gene products have TDC activity in vivo, dTdc1 is expressed nonneurally, whereas dTdc2 is expressed neurally. Flies with a mutation in dTdc2 lack neural tyramine and octopamine and are female sterile due to egg retention. Although other Drosophila mutants that lack octopamine retain eggs completely within the ovaries, dTdc2 mutants release eggs into the oviducts but are unable to deposit them. This specific sterility phenotype can be partially rescued by driving the expression of dTdc2 in a dTdc2-specific pattern, whereas driving the expression of dTdc1 in the same pattern results in a complete rescue. The disparity in rescue efficiencies between the ectopically expressed Tdc genes may reflect the differential activities of these gene products. The egg retention phenotype of the dTdc2 mutant and the phenotypes associated with ectopic dTdc expression contribute to a model in which octopamine and tyramine have distinct and separable neural activities.

stall-mediated extrinsic control of ovarian follicle formation in Drosophila.

Complex patterns of morphogenesis require intricate coordination of multiple, regulatory processes that control cellular identities, shapes, and behaviors, both locally and over vast distances in the developing organism or tissue. Studying Drosophila oogenesis as a model for tissue morphogenesis, we have discovered extraovarian regulation of follicle formation. Clonal analysis and ovary transplantation have demonstrated that long-range control of follicle individualization requires stall gene function in cells outside of the ovary. Although tissue nonautonomous regulation has been shown to govern follicle maturation and survival, this is the first report of an extraovarian pathway involved in normal follicle formation.