Re-expression of miR-200s in claudin-low mammary tumor cells alters cell shape and reduces proliferation and invasion potentially through modulating other miRNAs and SUZ12 regulated genes.

BACKGROUND: MicroRNAs are a class of non-coding RNAs that regulate gene expression through binding to mRNAs and preventing their translation. One family of microRNAs known as the miR-200 family is an important regulator of epithelial identity. The miR-200 family consists of five members expressed in two distinct clusters; the miR-200c/141 cluster and the miR-200b/200a/429 cluster. We have found that murine and human mammary tumor cells with claudin-low characteristics are associated with very low levels of all five miR-200s. METHODS: To determine the impact of miR-200s on claudin-low mammary tumor cells, the miR-200c/141 cluster and the miR-200b/200a/429 cluster were stably re-expressed in murine (RJ423) and human (MDA-MB-231) claudin-low mammary tumor cells. Cell proliferation and migration were assessed using BrdU incorporation and transwell migration across Matrigel coated inserts, respectively. miRNA sequencing and RNA sequencing were performed to explore miRNAs and mRNAs regulated by miR-200 re-expression while Enrichr-based pathway analysis was utilized to identify cellular functions modified by miR-200s. RESULTS: Re-expression of the miR-200s in murine and human claudin-low mammary tumor cells partially restored an epithelial cell morphology and significantly inhibited proliferation and cell invasion in vitro. miRNA sequencing and mRNA sequencing revealed that re-expression of miR-200s altered the expression of other microRNAs and genes regulated by SUZ12 providing insight into the complexity of miR-200 function. SUZ12 is a member of the polycomb repressor complex 2 that suppresses gene expression through methylating histone H3 at lysine 27. Flow cytometry confirmed that re-expression of miR-200s increased histone H3 methylation at lysine 27. CONCLUSIONS: Re-expression of miR-200s in claudin-low mammary tumor cells alters cell morphology and reduces proliferation and invasion, an effect potentially mediated by SUZ12-regulated genes and other microRNAs.

MMP inhibitors augment fibroblast adhesion through stabilization of focal adhesion contacts and up-regulation of cadherin function.

Increased pericellular proteolysis due to an imbalance between MMPs (matrix metalloproteinases) and TIMPs (tissue inhibitors of metalloproteinases) promotes early stages of tumorigenesis. We have reported that TIMP-1 down-regulation confers tumorigenicity on immortal Swiss 3T3 fibroblasts. In pursuit of the mechanism involved in this transformation, we asked whether MMP inhibitors modulate contact inhibition and cell adhesion, because the dysregulation of these events is essential for cellular transformation. Using both genetic and biochemical means, we demonstrate that MMP inhibitors regulate fibroblast cell adhesion. TIMP-1 down-regulated cells formed dense, multilayered colonies, suggesting a loss of contact inhibition. Recombinant TIMP-1 and synthetic MMP inhibitors (MMPi) restored normal cell contact and density of these cells in a dose-dependent manner. Consequently, the effect of MMPi on both cell-extracellular matrix (ECM) and cell-cell adhesion were investigated. Upon MMPi treatment, p125(FAK) was redistributed, together with vinculin, to points of cell-ECM contact. Furthermore, phosphorylation of p125(FAK) was restored to levels similar to that of wild type. In parallel, MMPi treatment increased cadherin levels and stabilized cadherin-mediated cell-cell contacts. Moreover, enhanced cadherin function was evident as increased calcium-dependent cell-cell aggregation and co-localization of cadherin and beta-catenin at the cell membrane. We also obtained independent evidence of altered cadherin function using timp-1(-/-) mouse embryonic fibroblasts. Our data provide provocative evidence that increased pericellular proteolysis impacts cell adhesion systems to offset normal contact inhibition, with subsequent effects on cell transformation and tumorigenesis.

Detecting bouts of physical activity in a field setting.

The purposes of this study were to assess the TRITRAC and CSA for: (a) interaccelerometer agreement; (b) agreement in detecting patterns of moderate-intensity physical activity; and (c) agreement in detecting walking patterns recorded in a diary. Thirty-one women wore both the TRITRAC and CSA accelerometers for three consecutive days. Interaccelerometer agreement (measured with generalizability coefficients) ranged from .88 to .99. In total, 71.3% of the accelerometers' patterns agreed in length, with CSA patterns being on average significantly longer. Interaccelerometer agreement in detecting patterns of brisk walking, as recorded in a diary, was comparable (69.4%). Interaccelerometer discrepancies may be related in part to the threshold employed by each instrument for classifying moderate intensity patterns.

The trithorax group gene moira encodes a brahma-associated putative chromatin-remodeling factor in Drosophila melanogaster.

The genes of the trithorax group (trxG) in Drosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes. The precise role of each of the diverse trxG members and the functional relationships among them are not well understood. Here, we report on the isolation of the trxG gene moira (mor) and its molecular characterization. mor encodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3. mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM. MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes. These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.

A cytoplasmic protein, bystin, interacts with trophinin, tastin, and cytokeratin and may be involved in trophinin-mediated cell adhesion between trophoblast and endometrial epithelial cells.

Trophinin and tastin form a cell adhesion molecule complex that potentially mediates an initial attachment of the blastocyst to uterine epithelial cells at the time of implantation. Trophinin and tastin, however, do not directly bind to each other, suggesting the presence of an intermediary protein. The present study identifies a cytoplasmic protein, named bystin, that directly binds trophinin and tastin. Bystin consists of 306 amino acid residues and is predicted to contain tyrosine, serine, and threonine residues in contexts conforming to motifs for phosphorylation by protein kinases. Database searches revealed a 53% identity of the predicted peptide sequence with the Drosophila bys (mrr) gene. Direct protein-protein interactions of trophinin, tastin, and bystin analyzed by yeast two-hybrid assays and by in vitro protein binding assays indicated that binding between bystin and trophinin and between bystin and tastin is enhanced when cytokeratin 8 and 18 are present as the third molecule. Immunocytochemistry of bystin showed that bystin colocalizes with trophinin, tastin, and cytokeratins in a human trophoblastic teratocarcinoma cell, HT-H. It is therefore possible that these molecules form a complex and thus are involved in the process of embryo implantation.

Opioid modulation of attention-related responses: peripheral-to-central progression and development of mu influence as learning occurs.

Endogenous opioids modulate attention-related bradycardiac responses evoked by novel stimuli and Pavlovian conditioned signals, and these effects are distinct from those of endogenous opioids on memory. We investigated the role of peripheral opioid receptors in modulating attention and Pavlovian learning, in rabbits tested for bradycardiac orienting responses to novel tones, and for Pavlovian conditioning and extinction of cardiac discrimination. Pretraining, IV treatment with the opiate antagonist naloxone-HCl (0.1-0.5 mg/kg) facilitated initial development of Pavlovian conditioned discrimination and delayed its later extinction, compared to saline vehicle, as previously observed. Pretraining treatment with its peripherally acting analog, quaternary naloxone-methiodide (1.29-6.47 mg/kg), also promoted initial development, but not extinction, of discrimination, and it reduced the magnitude of bradycardiac orienting responses and of tachycardiac unconditioned responses. Treatment with the selective mu-antagonist peptide CTOP (10-30 microg/kg) facilitated conditioned responses and reduced unconditioned responses, somewhat later during training, but it did not reliably affect extinction or orienting responses. These results confirm an important role of peripheral opioids in regulating attentional and associative functions involved in orienting and the earliest stage of Pavlovian learning, prior to development of central opioid regulation of later associative, hedonic and mnemonic functions. These findings also suggest that cardiovascular opioid receptors might mediate peripheral opioid influences on attention and early association formation, via modulation of cardiac responses to stimuli and autonomic sensory feedback to the brain.

Opioid modulation of attention-related responses: delta-receptors modulate habituation and conditioned bradycardia.

Endogenous opioids modulate attention-related heart rate responses evoked by novel stimuli and conditioned signals in ways that differ from their better-known effects on motivation and memory functions. We investigated the role of delta-opioids in modulating bradycardiac orienting and Pavlovian conditioned responses in rabbits, following i.v. treatment with the highly selective delta-receptor antagonist naltrindole (NTI; 0.037-0.370 mg/kg). When administered immediately before testing, NTI induced modest but detectable effects: the lowest dose increased cardiac discrimination near the end of the first training session, whereas the higher doses of NTI impaired discrimination, compared to saline-treated controls. NTI treatment immediately before testing also appeared to promote habituation of bradycardiac orienting responses elicited by novel tones, but NTI did not alter unconditioned heart rate responses following tone-shock pairs or extinction of conditioned responses. In contrast, the low dose of NTI administered 20 min, rather than immediately before testing, facilitated conditioned bradycardia during extinction, as well as during training. These results provide evidence that endogenous delta-opioid modulators normally delay the disappearance of bradycardiac orienting responses during habituation, inhibit or promote the development of bradycardiac conditioned responses during Pavlovian training depending on dose, and promote the disappearance of conditioned responses during extinction. These findings suggest that endogenous delta-opioid activity, probably involving both peripheral and central systems, coincides with, and may reflect, uncertainty about stimulus significance.

A Drosophila gene structurally and functionally homologous to the mammalian 70-kDa s6 kinase gene.

cDNAs encoding the Drosophila 70-kDa S6 kinase (S6K) were isolated by low-stringency hybridization with mammalian p70S6k probes. Conceptual translation of S6k cDNA sequences yields a product containing all of the canonical features typical of serine/threonine kinases and has 78% amino acid identity in the catalytic domain with the human p70S6k homologue. The S6k gene, located at polytene chromosome site 65D, gives rise to two predominant transcripts of 3.0 and 5.0 kb and at least two smaller transcripts (< 3.0 kb) that are found in whole-animal RNAs at all stages of development. Blood cells derived from the hematopoietic organs of ribosomal protein S6 (RpS6air8) mutant animals express higher levels of the smaller S6k transcripts, suggesting tissue- or genotype-specific differences in the regulation of the S6k gene. Drosophila S6K expressed in COS or NIH 3T3 cells phosphorylates mammalian RPS6 in a mitogen-dependent wortmannin- and rapamycin-sensitive manner, suggesting that its regulation is similiar to mammalian p70S6k.

Drosophila warts--tumor suppressor and member of the myotonic dystrophy protein kinase family.

Tumor suppressor genes represent a broad class of genes that normally function in the negative regulation of cell proliferation. Loss-of-function mutations in these genes lead to unrestrained cell proliferation and tumor formation. A fundamental understanding of how tumor suppressor genes regulate cell proliferation and differentiation should reveal important aspects of signalling pathways and cell cycle control. A recent report describing the Drosophila tumor suppressor gene warts has implications in the study of the human myotonic dystrophy gene. These genes encode members of a cyclic AMP-dependent protein kinase subfamily that includes other plant and animal orthologues.

Drosophila in cancer research: the first fifty tumor suppressor genes.

In Drosophila, over 50 genes have been identified in which loss-of-function mutations lead to excess cell proliferation in the embryo, in the central nervous system, imaginal discs or hematopoietic organs of the larva, or in the adult gonads. Twenty-two of these genes have been cloned and characterized at the molecular level, and nine of them show clear homology to mammalian genes. Most of these mammalian genes had not been previously implicated in cell proliferation control. Overgrowth in some of the mutants involves conversion to a cell type that, in normal development, shows more cell proliferation than the original cell type. Thus the neurogenic mutants, including Notch, show conversion of epidermal cells to neuroblasts, leading to the 'neurogenic' phenotype of excess nervous tissue. The ovarian tumor mutants show conversion of the female germ line to a cell type resembling the male germ line, which undergoes more proliferation than the female germ line. Mutations of the fat locus cause hyperplastic overgrowth of imaginal discs, in which the epithelial structure is largely intact. The predicted fat protein product is a giant relative of cadherins, supporting indications from human cancer that cadherins play an important role in tumor suppression. Mutations in the lethal(2)giant larvae and lethal(1)discs large genes cause neoplastic overgrowth of imaginal discs as well as the larval brain. The dlg gene encodes a membrane-associated guanylate kinase homolog that is localized at septate junctions between epithelial cells. This protein is a member of a family of homologs that also includes two proteins found at mammalian tight junctions (ZO-1 and ZO-2) and a protein found at mammalian synaptic junctions (PSD-95/SAP90). Genes in which mutations cause blood cell overproduction include aberrant immune response-8, which encodes the RpS6 ribosomal protein and hopscotch, which encodes a putative non-receptor protein tyrosine kinase. The gene products identified by ovarian tumor mutants do not show clear amino acid sequence homology to known proteins. Drosophila provides an opportunity to rapidly identify and characterize tumor suppressor genes, many of which have mammalian homologs that might also be involved in cell proliferation control and tumor suppression.

Tumor suppressor genes encoding proteins required for cell interactions and signal transduction in Drosophila.

Tumor suppressor genes, whose products are required for the control of cell proliferation, have been identified by their mutant phenotype of tissue overgrowth. Here we describe recent work on the molecular identification of tumor suppressor genes that function in two different cell types of the Drosophila larva: the blood cells, and the undifferentiated epithelial cells of developing imaginal discs. Mutations in the aberrant immune response8 (air8) gene lead to overproduction and precocious differentiation of blood cells. This gene encodes the Drosophila homolog of human ribosomal protein S6. The mutant phenotype is consistent with a role for S6 in the control of cell proliferation, and is compatible with findings from mammalian cells where alterations in S6 expression and phosphorylation are associated with changes in cell proliferation. Mutations in the discs large (dlg) gene cause neoplastic overgrowth of imaginal discs in the larva. The mutant discs show loss of septate junctions and of apical-basal cell polarity, and they also lose the ability to differentiate cuticular structures. The dlg protein product (DlgA) is localized at septate junctions between epithelial cells, and cDNA sequencing indicates that the gene product includes a domain with homology to guanylate kinase (GUK). Two mammalian homologs of this gene have been identified, and one of them (PSD-95/SAP90) encodes a component of synaptic densities in the brain; this protein therefore resembles the DlgA protein in being located in a specialized cell junction that functions in information transfer between cells. Mutations in the fat gene cause hyperplastic imaginal disc overgrowth, in which the overgrowing disc tissue retains its epithelial structure and its ability to differentiate. Some of the excess disc tissue is shed as vesicles suggesting a loss of cell adhesion. In support of this hypothesis, the predicted gene product shows homology to cadherins in its extracellular domain. However, the fat protein is much larger than known cadherins. As in human cancer, somatic loss of the normal alleles of tumor suppressor genes can lead to tumor formation in Drosophila; an example of this is provided by the warts (wts) locus. The wts gene was identified by the dramatic overgrowth of mitotic recombination clones that are homozygous for a wts deletion. In these clones the cuticle intrudes between epithelial cells, suggesting an alteration in cell adhesion. The study of these and other tumor suppressor genes in Drosophila is providing new evidence supporting the critical role of cell interactions and specialized apical junctions in controlling epithelial cell proliferation.

Drosophila homolog of the human S6 ribosomal protein is required for tumor suppression in the hematopoietic system.

The tumor suppressor gene lethal(1)aberrant immune response 8 (air8) of Drosophila melanogaster encodes a homolog of the human S6 ribosomal protein. P element insertions that prevent expression of this gene cause overgrowth of the lymph glands (the hematopoietic organs), abnormal blood cell differentiation, and melanotic tumor formation. They also cause delayed development, inhibit growth of most of the larval organs, and lead to larval lethality. Mitotic recombination experiments indicate that the normal S6 gene is required for clone survival in the germ line and imaginal discs. The S6 gene produces a 1.1-kilobase transcript that is abundant throughout development in wild-type animals and in revertants derived from the insertional mutants but is barely detectable in the mutant larvae. cDNAs corresponding to this transcript show a 248-amino acid open reading frame with 75.4% identity and 94.8% similarity to both human and rat S6 ribosomal protein sequences. The results reveal a regulatory function of this ribosomal protein in the hematopoietic system of Drosophila that may be related to its developmentally regulated phosphorylation.

Lethal(1) aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster.

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tuml and tu(1)Szts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from "autoimmune responses" or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.

Neuronal activity in the mediodorsal and intralaminar nuclei of the dorsal thalamus during classical heart rate conditioning.

Multiple unit activity (MUA) was recorded from chronically implanted electrodes in either the mediodorsal (MD) or the intralaminar (IL) nuclei of the dorsal thalamus in separate groups of rabbits during (a) habituation of the cardiac orienting reflex, (b) Pavlovian heart rate (HR) conditioning, and (c) extinction of the HR conditioned response (CR). Other animals with similar recording electrodes received explicitly unpaired presentations of the conditioned stimulus (CS) and unconditioned stimulus (US). The cardiac orienting reflex and the HR CR consisted of bradycardia. However, tone-evoked tachycardia was obtained in animals that received CS/US unpaired presentations. MUA evoked by the CS consisted of a short latency (20-40 ms) increase under all conditions, which reached its maximum 200-300 ms after CS onset. This response habituated greatly during tone-alone pretraining, but was considerably greater in the paired than unpaired group during the later trials of conditioning in animals with MD, but not IL, placements. Instead, a longer latency increase (greater than 500 ms) in MUA occurred in the paired but not in the unpaired animals in the IL group. The MUA increases in both instances, including the early, short latency increase in the MD group, and the longer latency increase in the IL group, were trial-related, and declined to pretraining levels during extinction, indicating that these neuronal changes had an associative basis. These findings suggest that neuronal activity in both MD and IL is related to the early events involved in Pavlovian conditioning, but that the relative roles of these two closely related thalamic nuclei in associative learning must be somewhat different.

Cardiac rehabilitation: evaluation and intervention less than 6 weeks after myocardial infarction.

Twenty-nine patients having myocardial infarction (MI) of recent origin (6 weeks or less) were evaluated and treated using a program in which assessment and progression were based not on generalized data but on the individual responses of each patient. Fourteen patients had complications including left ventricular impairment, continuing ischemia or rhythm disturbances, and 15 did not. Cardiac tolerance for the common self-care activities, walking and light exercise, was objectively defined by 3 levels of functional monitoring. The physician, occupational therapist and physical therapist evaluated the patient's responses to mild exertion, utilizing a 12-lead electrocardiogram (ecg), physical examination, 24-hour monitoring with a portable ecg, self-care evaluation and a modified treadmill screening test. Patient performance was assessed at each stage of testing and individualized activity levels were determined in accordance with cardiac responses. Thus patients progressed at their optimum rate with safety and without loss of time, ie, artificially induced invalidism. Following the 1st self-care evaluation, 3 patients were ordered to bedrest for further medical treatment while 26 were cleared for ward activity. When their conditions improved the 3 patients were reevaluated and began the program. The 15 patients without complications had appropriate responses to activity and proceeded to a mild exercise program with minimal observation. Of the 14 with complications, 5 experienced a temporary program interruption and 2 were dropped from the program secondary to severity of complications. The remaining 7 were progressed in the same manner as those without complication.