Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine.
Identifying mechanisms to enhance neuroprotection holds tremendous promise in developing new treatments for neuropsychiatric and neurodegenerative diseases. We sought to determine the potential role for microRNAs (miRNAs) in neuroprotection following neuronal death. A neuronal culture system of rat cerebellar granule cells was used to examine miRNA expression changes following glutamate-induced excitotoxicity and neuroprotective treatments. Combination treatment with the mood stabilizers lithium and valproic acid provided near-complete protection from glutamate excitotoxicity. Numerous miRNAs were detected by microarrays to be regulated by the combined lithium and valproic acid treatment, and the following candidates were confirmed using real-time PCR: miR-34a, miR-147b, miR-182, miR-222, miR-495, and miR-690. We then verified the apoptotic actions of miR-34a mimic in a human neuroblastoma cell line (SH-SY5Y) under basal conditions and following endoplasmic reticulum stress. To gain insight into the function of these mood stabilizer-regulated miRNAs, we performed two separate analyses: a candidate approach using Ingenuity Pathway Analysis that was restricted to only our PCR-verified miRNAs, and a global approach using DIANA-mirPath that included all significantly regulated miRNAs. It was observed that the pathways associated with mood stabilizer-regulated miRNAs in our study (global approach) are strongly associated with pathways implicated in neuropsychiatric diseases such as schizophrenia. We also observed an overlap in the mood stabilizer-regulated miRNAs identified from our study along with dysregulated miRNAs in both neuropsychiatric and neurodegenerative disorders. We anticipate that these associations and overlaps implicate critical pathways and miRNAs in disease mechanisms for novel therapeutic treatments that may hold potential for many neurological diseases.
microRNA; neuroprotection; glutamate excitotoxicity; lithium; valproic acid; mood stabilizers
Maternal immune activation (MIA) is a risk factor for the development of schizophrenia and autism. Infections during pregnancy activate the mother’s immune system and alter the fetal environment, with consequential effects on CNS function and behavior in the offspring, but the cellular and molecular links between infection-induced altered fetal development and risk for neuropsychiatric disorders are unknown. We investigated the immunological, molecular, and behavioral effects of MIA in the offspring of pregnant Sprague-Dawley rats given an intraperitoneal (0.25 mg/kg) injection of lipopolysaccharide (LPS) on gestational day 15. LPS significantly elevated pro-inflammatory cytokine levels in maternal serum, amniotic fluid, and fetal brain at 4 h, and levels decreased but remained elevated at 24 h. Offspring born to LPS-treated dams exhibited reduced social preference and exploration behaviors as juveniles and young adults. Whole genome microarray analysis of the fetal brain at 4 h post maternal LPS was performed to elucidate the possible molecular mechanisms by which MIA affects the fetal brain. We observed dysregulation of 3,285 genes in restricted functional categories, with increased mRNA expression of cellular stress and cell death genes and reduced expression of developmentally-regulated and brain-specific genes, specifically those that regulate neuronal migration of GABAergic interneurons, including the Distal-less (Dlx) family of transcription factors required for tangential migration from progenitor pools within the ganglionic eminences into the cerebral cortex. Our results provide a novel mechanism by which MIA induces the widespread down-regulation of critical neurodevelopmental genes, including those previously associated with autism.
Maternal immune activation; Inflammation; Cytokine; Lipopolysaccharide; Schizophrenia; Autism; Microarray; Bioinformatics; Cortical interneurons; GABA
Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. c-Myc, Klf4, Oct4, and Sox2 expression is elevated in CD47-null endothelial cells, in several tissues of CD47- and thrombospondin-1-null mice, and in a human T cell line lacking CD47. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells and in vivo, whereas CD47 ligation by thrombospondin-1 suppresses c-Myc expression. The inhibitory effects of increasing CD47 levels can be overcome by maintaining c-Myc expression and are absent in cells with dysregulated c-Myc. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.
Pheochromocytomas (PHEOs) and paragangliomas (PGLs) related to mutations in the mitochondrial succinate dehydrogenase (SDH) subunits A, B, C, and D, SDH complex assembly factor 2, and the von Hippel-Lindau (VHL) genes share a pseudohypoxic expression profile. However, genotype-specific differences in expression have been emerging. Development of effective new therapies for distinctive manifestations, e.g., a high rate of malignancy in SDHB- or predisposition to multifocal PGLs in SDHD patients, mandates improved stratification. To identify mutation/location-related characteristics among pseudohypoxic PHEOs/PGLs, we used comprehensive microarray profiling (SDHB: n = 18, SDHD-abdominal/thoracic (AT): n = 6, SDHD-head/neck (HN): n = 8, VHL: n = 13). To avoid location-specific bias, typical adrenal medulla genes were derived from matched normal medullas and cortices (n = 8) for data normalization. Unsupervised analysis identified two dominant clusters, separating SDHB and SDHD-AT PHEOs/PGLs (cluster A) from VHL PHEOs and SDHD-HN PGLs (cluster B). Supervised analysis yielded 6937 highly predictive genes (misclassification error rate of 0.175). Enrichment analysis revealed that energy metabolism and inflammation/fibrosis-related genes were most pronouncedly changed in clusters A and B, respectively. A minimum subset of 40 classifiers was validated by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction vs. microarray: r = 0.87). Expression of several individual classifiers was identified as characteristic for VHL and SDHD-HN PHEOs and PGLs. In the present study, we show for the first time that SDHD-HN PGLs share more features with VHL PHEOs than with SDHD-AT PGLs. The presented data suggest novel subclassification of pseudohypoxic PHEOs/PGLs and implies cluster-specific pathogenic mechanisms and treatment strategies.
The human genome contains approximately 50 copies of the replication-defective human endogenous retrovirus 9 (ERV-9) and thousands of copies of its solitary long term repeat (sLTR) element. While some sLTRs are located upstream of critical genes and have enhancer activity, other sLTRs are located within introns and may be transcribed as RNAs. We found that intronic RNAs arising from U3 sLTRs of ERV-9 were expressed as both sense (S) and antisense (AS) transcripts in all human cells tested but that expression levels differed in malignant versus nonmalignant cells. In nonmalignant cells, AS was expressed at higher levels than S and at higher levels than in malignant cells; in malignant cells, AS was expressed at amounts equivalent to those of S RNA. Critically, U3 AS RNA was found to physically bind to key transcription factors for cellular proliferation, including NF-Y, p53, and sp1, indicating that such RNA transcripts may function as decoy targets or traps for NF-Y and thus inhibit the growth of human cancer cells. Indeed, short U3 oligodeoxynucleotides (ODNs) based on these RNA sequences ably inhibited proliferation of cancer cell lines driven by cyclins B1/B2, the gene targets of NF-Y.
Previous investigations have combined transcriptional and genetic analyses in human cell lines1-3, but few have applied these techniques to human neural tissue4-8. To gain a global molecular perspective on the role of the human genome in cortical development, function and ageing, we explore the temporal dynamics and genetic control of transcription in human prefrontal cortex in an extensive series of post-mortem brains from fetal development through ageing. We discover a wave of gene expression changes occurring during fetal development which are reversed in early postnatal life. One half-century later in life, this pattern of reversals is mirrored in ageing and in neurodegeneration. Although we identify thousands of robust associations of individual genetic polymorphisms with gene expression, we also demonstrate that there is no association between the total extent of genetic differences between subjects and the global similarity of their transcriptional profiles. Hence, the human genome produces a consistent molecular architecture in the prefrontal cortex, despite millions of genetic differences across individuals and races. To enable further discovery, this entire data set is freely available (from Gene Expression Omnibus: accession GSE30272; and dbGaP: accession phs000417.v1.p1) and can also be interrogated via a biologist-friendly stand-alone application (http://www.libd.org/braincloud).
Follicular T helper cells (Tfh) provide critical help to B cells for germinal center (GC) formation. Mutations affecting SLAM-associated Protein (SAP) prevent GC formation due to defective T-B cell interactions, yet effects on Tfh cell differentiation remain unclear. We describe the in vitro differentiation of functionally-competent “Tfh-like” cells that expressed Interleukin-21, Tfh markers, and Bcl6, and rescued GC formation in SAP-deficient hosts better than other T helper (Th) cells. SAP-deficient Tfh-like cells appeared virtually indistinguishable from wildtype, yet failed to support GCs in vivo. Interestingly, both Tfh-like and in vivo-derived Tfh cells could produce effector cytokines in response to polarizing conditions. Moreover, Th1, Th2 and Th17 cells could be reprogrammed to obtain Tfh characteristics. ChIP-Seq analyses revealed positive epigenetic markings on Tbx21, Gata3 and Rorc in Tfh-like and ex vivo Tfh cells, and Bcl6 in non-Tfh cells, supporting the concept of plasticity between Tfh and other Th cell populations.
Entamoeba histolytica, a protozoan parasite, is an important cause of diarrhea and colitis in the developing world. Amebic colitis is characterized by ulceration of the intestinal mucosa. We performed microarray analysis of intestinal biopsies during acute and convalescent amebiasis in order to identify genes potentially involved in tissue injury or repair. Colonic biopsy samples were obtained from 8 patients during acute E. histolytica colitis and again 60 days after recovery. Gene expression in the biopsies was evaluated using microarray, and confirmed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). REG 1A and REG 1B were the most up-regulated of all genes in the human intestine in acute versus convalescent E. histolytica disease: as determined by microarray, the levels of induction were 7.4-fold and 10.7 fold for REG1A and B; p=0.003 and p=0.006 respectively. Increased expression of REG 1A and REG 1B protein in the colonic crypt epithelial cells during acute amebiasis was similarly observed by immunohistochemistry. Because REG 1 protein is anti-apoptotic and pro-proliferative, and since E. histolytica induces apoptosis of the intestinal epithelium as part of its disease process, we next tested if REG 1 might be protective during amebiasis by preventing parasite-induced apoptosis. Intestinal epithelial cells from REG 1 -/- mice were found to be more susceptible to spontaneous, and parasite-induced, apoptosis in vitro (p=0.03). We concluded that REG 1A and REG 1B were upregulated during amebiasis and may function to protect the intestinal epithelium from parasite-induced apoptosis.
Entamoeba histolytica; Regenerating gene (REG) 1; apoptosis
Stroke is a devastating brain injury that is a leading cause of adult disability with limited treatment options. Using a rat model of middle cerebral artery occlusion (MCAO) to induce cerebral ischemia, we profiled microRNAs (miRNAs), small non-protein coding RNAs, in the ischemic cortex. Many miRNAs were confirmed by qPCR to be robustly upregulated 24 hours following MCAO surgery including miR-155, miR-297a, miR-466f, miR-466h, and miR-1224. In addition, we treated MCAO rats with valproic acid (VPA), a mood stabilizer and histone deacetylase inhibitor. This post-insult treatment was shown to improve neurological deficits and motor performance following MCAO. To provide mechanistic insight into the potential targets and pathways that may underlie these benefits, we profiled miRNAs regulated following this VPA treatment. Two promising post-insult VPA-regulated candidates were miR-331 and miR-885-3p. miR-331 was also regulated by VPA pre-treatment in rat cortical neuronal cultures subjected to oxygen-glucose deprivation, an in vitro ischemic model. The predicted targets of these miRNAs analyzed by Ingenuity Pathway Analysis (IPA) identified networks involved in hematological system development, cell death, and nervous system development. These predicted networks were further filtered using IPA and showed significant associations with neurological diseases including movement disorders, neurodegenerative disorders, damage to cerebral cortex, and seizure disorders among others. Collectively, these data support common disease mechanisms that may be under miRNA control and provide exciting directions for further investigations aimed at elucidating the miRNA mechanisms and targets that may yield new therapies for neurological disorders.
Cerebral ischemia; valproic acid; microRNA; oxygen-glucose deprivation; neuroprotection
Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIAs) are analogues of the products of PI3K that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of 5 active PIAs and the PI3K inhibitor LY294002 (LY) was performed in NSCLC cells using high-density oligonucleotide arrays. Gene ontology analysis revealed genes involved in apoptosis, wounding response, and angiogenesis were upregulated by PIAs, while genes involved in DNA replication, repair and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into 3 groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3 and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB) and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity.
Akt; microarray; PIA; RhoB; NSCLC
A series of isogenic, basal-like human mammary epithelial cells (HMECs) with altered TGF-β sensitivity and different malignancy is used to elucidate molecular mechanisms that evade oncogenic Ras-induced growth arrest and promote transformation. Attenuation of TGF-β signaling is found to cause metastatic progression of Ras-transformed HMECs.
The molecular mechanisms that drive triple-negative, basal-like breast cancer progression are elusive. Few molecular targets have been identified for the prevention or treatment of this disease. Here we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered transforming growth factor-β (TGF-β) sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative type II receptor (DNRII) of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-Ras-V12–induced senescence-like growth arrest (SLGA). Furthermore, coexpression of DNRII and H-Ras-V12 rendered HMECs highly tumorigenic and metastatic in vivo in comparison with H-Ras-V12–transformed HMECs that spontaneously escaped H-Ras-V12–induced SLGA. Microarray analysis revealed that p21 was the major player mediating Ras-induced SLGA, and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling was blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-Ras-V12–induced SLGA. Our results identify that autocrine TGF-β signaling is an integral part of the cellular anti-transformation network by suppressing the expression of a host of genes, including p21-regulated genes, that mediate oncogene-induced transformation in basal-like breast cancer.
Most cancer cells are characterized by aneuploidy, an abnormal number of chromosomes. We have identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors. A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer.
Mucosal tolerance to E-selectin prevents stroke and protects against ischemic brain damage in experimental models of stroke studying healthy animals or spontaneously hypertensive stroke-prone rats. A reduction in inflammation and neural damage was associated with immunomodulatory or “tolerogenic” responses to E-selectin. The purpose of the current study on ApoE deficient mice is to assess the capacity of this stroke prevention innovation to influence atherosclerosis, a major underlying cause for ischemic strokes; human E-selectin is being translated as a potential clinical prevention strategy for secondary stroke. Female ApoE−/− mice received intranasal delivery of E-selectin prior to (pre-tolerization) or simultaneously with initiation of a high-fat diet. After 7 weeks on the high-fat diet, lipid lesions in the aorta, serum triglycerides, and total cholesterol were assessed as markers of atherosclerosis development. We also assessed E-selectin-specific antibodies and cytokine responses, in addition to inflammatory responses that included macrophage infiltration of the aorta and altered gene expression profiles of aortic mRNA. Intranasal delivery of E-selectin prior to initiation of high-fat chow decreased atherosclerosis, serum total cholesterol, and expression of the leucocyte chemoattractant CCL21 that is typically upregulated in atherosclerotic lesions of ApoE−/− mice. This response was associated with the induction of E-selectin specific cells producing the immunomodulatory cytokine IL-10 and immunosuppressive antibody isotypes. Intranasal administration of E-selectin generates E-selectin specific immune responses that are immunosuppressive in nature and can ameliorate atherosclerosis, a major risk factor for ischemic stroke. These results provide additional preclinical support for the potential of induction of mucosal tolerance to E-selectin to prevent stroke.
The origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other's metastatic behavior.
ER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC.
The presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines.
Spontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity.
Coat color and type are essential characteristics of domestic dog breeds. Although the genetic basis of coat color has been well characterized, relatively little is known about the genes influencing coat growth pattern, length, and curl. We performed genome-wide association studies of more than 1000 dogs from 80 domestic breeds to identify genes associated with canine fur phenotypes. Taking advantage of both inter- and intrabreed variability, we identified distinct mutations in three genes, RSPO2, FGF5, and KRT71 (encoding R-spondin–2, fibroblast growth factor–5, and keratin-71, respectively), that together account for most coat phenotypes in purebred dogs in the United States. Thus, an array of varied and seemingly complex phenotypes can be reduced to the combinatorial effects of only a few genes.
Initial PACAP-regulated transcriptomes of PACAP-treated cultured chromaffin cells, and the adrenal gland of wild-type versus PACAP-deficient mice, have been assembled using microarray analysis. These were compared to previously acquired PACAP-regulated transcriptome sets from PC12 cells and mouse central nervous system, using the same microarray platform. The Ingenuity Pathways Knowledge Base was then employed to group regulated transcripts into common first and second messenger regulatory clusters. The purpose of our meta-analysis was to identify sets of genes regulated distinctly or in common by the neurotransmitter/neurotrophin PACAP in specific physiological contexts. Results suggest that PACAP participates in both the basal differentiated expression, and the induction upon physiological stimulation, of distinct sets of transcripts in neuronal and endocrine cells. PACAP in both developmental and acute regulatory paradigms acts on target genes regulated by either TNFα or TGFβ, two first messengers acting on transcription mainly through NFκB and Smads, respectively.
adrenal gland; bovine chromaffin cells; Btg2; C/EBP; Ier3; meta-analysis; microarray; middle cerebral artery occlusion; PACAP; PC12; Scmh1; secretogranin; TGFβ; TNFα; YWAHZ
The Forkhead Box H1 (FoxH1) protein is a co-transcription factor recruited by phosphorylated Smad2 downstream of several TGFβs, including Nodal-related proteins. We have reassessed the function of zebrafish FoxH1 using antisense morpholino oligonucleotides (MOs). MOs targeting translation of foxH1 disrupt embryonic epiboly movements during gastrulation and cause death on the first day of development. The FoxH1 morphant phenotype is much more severe than that of zebrafish carrying foxh1/schmalspur (sur) DNA-binding domain mutations, FoxH1 splice-blocking morphants or other Nodal pathway mutants, and it cannot be altered by concomitant perturbations in Nodal signaling. Apart from disrupting epiboly, FoxH1 MO treatment disrupts convergence and internalization movements. Late gastrula-stage FoxH1 morphants exhibit delayed mesoderm and endoderm marker gene expression and failed patterning of the central nervous system. Probing FoxH1 morphant RNA by microarray, we identified a cohort of five keratin genes – cyt1, cyt2, krt4, krt8 and krt18 - that are normally transcribed in the embryo’s enveloping layer (EVL) and which have significantly reduced expression in FoxH1-depleted embryos. Simultaneously disrupting these keratins with a mixture of MOs reproduces the FoxH1 morphant phenotype. Our studies thus point to an essential role for maternal FoxH1 and downstream keratins during gastrulation that is epistatic to Nodal signaling.
FoxH1; Gastrulation; Enveloping Layer; Keratin
Phaeochromocytomas in patients with multiple endocrine neoplasia type 2 (MEN 2) produce adrenaline, whereas those with von Hippel-Lindau (VHL) syndrome do not. This study assessed whether these distinctions relate to differences in expression of the transporters responsible for uptake and storage of catecholamines - the noradrenaline transporter and the vesicular monoamine transporters (VMAT 1 and VMAT 2).
Tumour tissue and plasma samples were obtained from 31 patients with hereditary phaeochromocytoma -18 with VHL syndrome and 13 with MEN 2. We used quantitative PCR, Western blotting, electron microscopy, immunohistochemistry and measurements of plasma and tumour catecholamines to assess differences in expression of the transporters in noradrenaline-producing vs adrenaline-producing hereditary tumours. These differences were compared with those in a further group of 26 patients with non-syndromic phaeochromocytoma.
Adrenaline-producing phaeochromocytomas in MEN 2 patients expressed more noradrenaline transporter mRNA and protein than noradrenaline-producing tumours in VHL patients. In contrast, there was greater expression of VMAT 1 in VHL than MEN 2 tumours, while expression of VMAT 2 did not differ significantly. These differences were associated with larger numbers of storage vesicles and higher tissue contents of catecholamines in MEN 2 than in VHL tumours. Differences in expression of the noradrenaline transporter were weaker, and those of VMAT 1 and VMAT 2 stronger, in noradrenaline and adrenaline-producing non-syndromic than in hereditary tumours.
The findings show that, in addition to differences in catecholamine biosynthesis, phaeochromocytomas in MEN 2 and VHL syndrome also differ in expression of the transporters responsible for uptake and vesicular storage of catecholamines.
Selective COX-2 inhibitors reduce adenoma formation and cancer progression in rodent models of colorectal cancer. To assess the therapeutic activity of selective COX-2 inhibitors, we tested the effect of SC-58125 treatment on the growth of human colon carcinoma cells in nude mice. Delaying treatment by 2, 4, or 7 weeks following implantation of the carcinoma cells resulted in a significant inhibition of tumor growth. Furthermore, short-term (48 hours) treatment with SC-58125 was sufficient to attenuate tumor growth for up to 15 days. SC-58125 treatment did not alter the rate at which cells underwent apoptosis, but did result in a delayed progression through the cell cycle at the G2/M transition. Accordingly, p34cdc2 protein levels and activity were decreased following SC-58125 treatment. We conclude that SC-58125 primarily exerts a cytostatic effect in vivo, which is likely to be mediated through inhibition of progression through the G2/M phase of the cell cycle.
colorectal cancer; COX-2; cancer prevention; prostaglandins; cell cycle arrest
Pheochromocytomas are chromaffin cell-derived neuroendocrine tumors. There is presently no cure for metastatic pheochromocytoma and no reliable way to distinguish malignant from benign tumors before the development of metastases. In order to successfully manage pheochromocytoma, it is necessary to better understand the biological determinants of tumor behavior. For this purpose, we have recently established a mouse model of metastatic pheochromocytoma using tail vein injection of mouse pheochromocytoma (MPC) cells. We optimized this model modifying the number of cells injected, length of trypsin pre-treatment, and incubation temperature and duration for the MPC cells before injection, and by serial passage and re-selection of tumors exhibiting the metastatic phenotype. We evaluated the effect of these modifications on tumor growth using serial in vivo Magnetic Resonance Imaging studies. These results show that number of cells injected, the pre-injection incubation temperature, and duration of trypsin treatment are important factors to produce faster growing, more aggressive tumors that yielded secondary metastatic lesions. Serial harvest, culture and re-selection of metastatic liver lesions produced even more aggressive pheochromocytoma cells that retained their biochemical phenotype. Microarray gene expression comparison and quantitative real-time PCR of these more aggressive cells to the MPC-parental cell line identified genes that may be important for the metastatic process.
Animal model; Cell line; Pheochromocytoma; Magnetic resonance imaging; Microarray; Quantitative real-time PCR
The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery.
The largest genetic study to date of morphology in domestic dogs identifies genes
controlling nearly 100 morphological traits and identifies important trends in
phenotypic variation within this species.
Domestic dogs exhibit tremendous phenotypic diversity, including a greater
variation in body size than any other terrestrial mammal. Here, we generate a
high density map of canine genetic variation by genotyping 915 dogs from 80
domestic dog breeds, 83 wild canids, and 10 outbred African shelter dogs across
60,968 single-nucleotide polymorphisms (SNPs). Coupling this genomic resource
with external measurements from breed standards and individuals as well as
skeletal measurements from museum specimens, we identify 51 regions of the dog
genome associated with phenotypic variation among breeds in 57 traits. The
complex traits include average breed body size and external body dimensions and
cranial, dental, and long bone shape and size with and without allometric
scaling. In contrast to the results from association mapping of quantitative
traits in humans and domesticated plants, we find that across dog breeds, a
small number of quantitative trait loci (≤3) explain the majority of
phenotypic variation for most of the traits we studied. In addition, many
genomic regions show signatures of recent selection, with most of the highly
differentiated regions being associated with breed-defining traits such as body
size, coat characteristics, and ear floppiness. Our results demonstrate the
efficacy of mapping multiple traits in the domestic dog using a database of
genotyped individuals and highlight the important role human-directed selection
has played in altering the genetic architecture of key traits in this important
Dogs offer a unique system for the study of genes controlling morphology. DNA
from 915 dogs from 80 domestic breeds, as well as a set of feral dogs, was
tested at over 60,000 points of variation and the dataset analyzed using novel
methods to find loci regulating body size, head shape, leg length, ear position,
and a host of other traits. Because each dog breed has undergone strong
selection by breeders to have a particular appearance, there is a strong
footprint of selection in regions of the genome that are important for
controlling traits that define each breed. These analyses identified new regions
of the genome, or loci, that are important in controlling body size and shape.
Our results, which feature the largest number of domestic dogs studied at such a
high level of genetic detail, demonstrate the power of the dog as a model for
finding genes that control the body plan of mammals. Further, we show that the
remarkable diversity of form in the dog, in contrast to some other species
studied to date, appears to have a simple genetic basis dominated by genes of