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1.
Cell Regen ; 13(1): 8, 2024 Apr 07.
Article in English | MEDLINE | ID: mdl-38583099

ABSTRACT

Continuous immunosuppression has been widely used in xenografts into non-human primate brains. However, how immune responses change after transplantation in host brains under continuous immunosuppressive administration and whether immunosuppression can be withdrawn to mitigate side effects remain unclear. Human induced neural stem/progenitor cells (iNPCs) have shown long-term survival and efficient neuronal differentiation in primate brains. Here, we evaluate the immune responses in primate brains triggered by human grafts. The results show that the immune responses, including the evident activation of microglia and the strong infiltration of lymphocytes (both T- and B-cells), are caused by xenografts at 4 months post transplantation (p.t.), but significantly reduced at 8 months p.t. under continuous administration of immunosuppressant Cyclosporin A. However, early immunosuppressant withdrawal at 5 months p.t. results in severe immune responses at 10 months p.t. These results suggest that continuous long-term immunosuppression is required for suppressing immune responses to xenografts in primate brains.

2.
Cell Regen ; 11(1): 28, 2022 Sep 02.
Article in English | MEDLINE | ID: mdl-36050613

ABSTRACT

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder associated with aging. Due to its insidious onset, protracted progression, and unclear pathogenesis, it is considered one of the most obscure and intractable brain disorders, and currently, there are no effective therapies for it. Convincing evidence indicates that the irreversible decline of cognitive abilities in patients coincides with the deterioration and degeneration of neurons and synapses in the AD brain. Human neural stem cells (NSCs) hold the potential to functionally replace lost neurons, reinforce impaired synaptic networks, and repair the damaged AD brain. They have therefore received extensive attention as a possible source of donor cells for cellular replacement therapies for AD. Here, we review the progress in NSC-based transplantation studies in animal models of AD and assess the therapeutic advantages and challenges of human NSCs as donor cells. We then formulate a promising transplantation approach for the treatment of human AD, which would help to explore the disease-modifying cellular therapeutic strategy for the treatment of human AD.

3.
iScience ; 24(10): 103207, 2021 Oct 22.
Article in English | MEDLINE | ID: mdl-34704001

ABSTRACT

As an insidious and slowly progressive neurodegenerative disorder, Alzheimer's disease (AD) uniquely develops in humans but fails in other species. Therefore, it has been challenged to rebuild human AD in animals, including in non-human primates. Here, we bilaterally delivered synthetic Aß oligomers (AßOs) into the cerebral parenchyma of cynomolgus monkeys, which rapidly drove the formation of massive Aß plaques and concomitant neurofibrillary tangles in the cynomolgus brain. The amyloid and tau pathology as well as their co-occurrence in AßO-monkeys were reminiscent of those in patients with AD. In addition, the activated astrocytes and microglia surrounding Aß plaques indicated the triggered neuroinflammation. The degenerative neurons and synapses around Aß plaques also emerged in cynomolgus brain. Together, soluble AßOs caused the cascade of pathologic events associated with AD in monkeys as occurred in patients at the early phase, which could facilitate the development of a promising animal model for human AD in non-human primates.

4.
STAR Protoc ; 2(1): 100346, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33665626

ABSTRACT

Generating induced neural stem/progenitor cells (iNPCs) from somatic cells for medical applications has remained challenging. Here, we describe a reliable protocol to make human iNPCs from a small volume of immobilized adult peripheral blood by direct reprogramming. We have verified that the integration-free human iNPCs can efficiently differentiate into mature neurons in mouse brain upon transplantation and display capacities to functionally replace the damaged neurons, suggesting their potential as donor cells in developing replacement medicine for neurodegenerative diseases. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2019).


Subject(s)
Adult Stem Cells/metabolism , Cells, Immobilized/metabolism , Induced Pluripotent Stem Cells/metabolism , Neural Stem Cells/metabolism , Stem Cell Transplantation , Animals , Heterografts , Humans , Mice
5.
J Mol Cell Biol ; 12(10): 807-816, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32492143

ABSTRACT

The induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to model and study Alzheimer's disease (AD) under patient-specific genetic background. The lower expression of transient receptor potential canonical 6 (TRPC6) was associated with AD patients, which might be involved in AD pathogenesis. However, the role of TRPC6 that played in AD process still needs more investigation in patient-relevant neurons. In this study, the iPSCs were generated from peripheral blood cells of sporadic AD patients and efficiently differentiated into mature cortical neurons. These sporadic AD-bearing neurons displayed higher levels of AD pathological markers Aß and phospho-tau, but lower levels of TRPC6, than those of control neurons. Treatment of AD neurons with TRPC6 protein fragment or agonist inhibited the elevation of Aß and phospho-tau. Our results in live AD neurons manifest that the compromised expression of TRPC6 substantially contributed to Aß pathology of sporadic AD, suggesting that targeting TRPC6 could help to develop novel therapeutic strategies for the treatments of AD.


Subject(s)
Alzheimer Disease/drug therapy , Induced Pluripotent Stem Cells/pathology , Neurons/pathology , TRPC6 Cation Channel/therapeutic use , Amyloid beta-Peptides/metabolism , Animals , Cell Differentiation , Humans , Mice , Neurons/metabolism , Phenotype , TRPC6 Cation Channel/agonists , tau Proteins/metabolism
6.
Stem Cell Reports ; 13(6): 1022-1037, 2019 12 10.
Article in English | MEDLINE | ID: mdl-31761676

ABSTRACT

Alzheimer's disease (AD) is characterized by memory impairments in its earliest clinical phase. The synaptic loss and dysfunction leading to failures of synaptic networks in AD brain directly cause cognitive deficits of patient. However, it remains unclear whether the synaptic networks in AD brain could be repaired. In this study, we generated functional human induced neural progenitor/stem cells (iNPCs) that had been transplanted into the hippocampus of immunodeficient wild-type and AD mice. The grafted human iNPCs efficiently differentiated into neurons that displayed long-term survival, progressively acquired mature membrane properties, formed graft-host synaptic connections with mouse neurons and functionally integrated into local synaptic circuits, which eventually reinforced and repaired the neural networks of host hippocampus. Consequently, AD mice with human iNPCs exhibited enhanced synaptic plasticity and improved cognitive abilities. Together, our results suggest that restoring synaptic failures by stem cells might provide new directions for the development of novel treatments for human AD.


Subject(s)
Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Cognitive Dysfunction , Hippocampus/metabolism , Neural Stem Cells/metabolism , Neuronal Plasticity , Synapses/metabolism , Alzheimer Disease/physiopathology , Animals , Biomarkers , Cell Survival , Disease Models, Animal , Fluorescent Antibody Technique , Hippocampus/physiopathology , Humans , Mice , Mice, Transgenic , Models, Biological , Neurons/metabolism
8.
Stem Cell Reports ; 5(5): 776-790, 2015 Nov 10.
Article in English | MEDLINE | ID: mdl-26489896

ABSTRACT

Degeneration of basal forebrain cholinergic neurons (BFCNs) is associated with cognitive impairments of Alzheimer's disease (AD), implying that BFCNs hold potentials in exploring stem cell-based replacement therapy for AD. However, studies on derivation of BFCNs from embryonic stem cells (ESCs) are limited, and the application of ESC-derived BFCNs remains to be determined. Here, we report on differentiation approaches for directing both mouse and human ESCs into mature BFCNs. These ESC-derived BFCNs exhibit features similar to those of their in vivo counterparts and acquire appropriate functional properties. After transplantation into the basal forebrain of AD model mice, ESC-derived BFCN progenitors predominantly differentiate into mature cholinergic neurons that functionally integrate into the endogenous basal forebrain cholinergic projection system. The AD mice grafted with mouse or human BFCNs exhibit improvements in learning and memory performances. Our findings suggest a promising perspective of ESC-derived BFCNs in the development of stem cell-based therapies for treatment of AD.


Subject(s)
Alzheimer Disease/therapy , Cholinergic Neurons/transplantation , Embryonic Stem Cells/cytology , Prosencephalon/cytology , Animals , Cell Line , Cells, Cultured , Cholinergic Neurons/cytology , Cognition , Humans , Memory , Mice , Neurogenesis
9.
Transl Neurodegener ; 4: 8, 2015.
Article in English | MEDLINE | ID: mdl-25954503

ABSTRACT

Alzheimer's disease (AD), a common neurodegenerative disorder associated with gradually to dramatic neuronal death, synaptic loss and dementia, is considered to be one of the most obscure and intractable brain disorders in medicine. Currently, there is no therapy clinically available to induce marked symptomatic relief in AD patients. In recent years, the proof-of-concept studies using stem cell-based approaches in transgenic AD animal models provide new hope to develop stem cell-based therapies for the effective treatment of AD. The degeneration of basal forebrain cholinergic neurons (BFCNs) and the resultant cholinergic abnormalities in the brain contribute substantially to the cognitive decline of AD patients. The approches using stem cell-derived BFCNs as donor cells need to be developed, and to provide proof of principle that this subtype-specific neurons can induce functional recovery of AD animal models. With the continuous scientific advances in both academic and industrial fields, the potentials of stem cells in cellular neuroprotection and cell replacement in vivo have been elucidated, and stem cell-based therapy for repairing degenerative brains of AD is promising.

10.
Nat Commun ; 6: 6830, 2015 Apr 23.
Article in English | MEDLINE | ID: mdl-25904100

ABSTRACT

Cell fate determination requires the cooperation between extrinsic signals and intrinsic molecules including transcription factors as well as epigenetic regulators. Nevertheless, how neural fate commitment is regulated by epigenetic modifications remains largely unclear. Here we show that transient histone deacetylation at epiblast stage promotes neural differentiation of mouse embryonic stem cells (mESCs). Histone deacetylase 1 (HDAC1) deficiency in mESCs partially phenocopies the inhibition of histone deacetylation in vitro, and displays reduced incorporation into neural tissues in chimeric mouse embryos in vivo. Mechanistic studies show that Nodal, which is repressed by histone deacetylation, is a direct target of HDAC1. Furthermore, the inhibition of histone deacetylation in the anterior explant of mouse embryos at E7.0 leads to Nodal activation and neural development repression. Thus, our study reveals an intrinsic mechanism that epigenetic histone deacetylation ensures neural fate commitment by restricting Nodal signalling in murine anterior epiblast ex vivo and mESC in vitro.


Subject(s)
Embryonic Stem Cells/metabolism , Endoderm/embryology , Gene Expression Regulation, Developmental , Histone Deacetylase 1/metabolism , Histones/metabolism , Mesoderm/embryology , Neurogenesis/genetics , Nodal Protein/genetics , RNA, Messenger/metabolism , Animals , Blotting, Western , CRISPR-Cas Systems , Chromatin Immunoprecipitation , Endoderm/metabolism , Epigenesis, Genetic , Germ Layers , In Vitro Techniques , Mesoderm/metabolism , Mice , Nodal Protein/metabolism , Oligonucleotide Array Sequence Analysis
11.
Methods Mol Biol ; 789: 89-110, 2011.
Article in English | MEDLINE | ID: mdl-21922402

ABSTRACT

Measurements of changes in pre-mRNA levels by intron-specific probes are generally accepted as more closely reflecting changes in gene transcription rates than are measurements of mRNA levels by exonic probes. This is, in part, because the pre-mRNAs, which include the primary transcript and various splicing intermediates located in the nucleus (also referred to as heteronuclear RNAs, or hnRNAs), are processed rapidly (with half-lives <60 min) as compared to neuropeptide mRNAs, which are then transferred to the cytoplasm and which have much longer half-lives (often over days). In this chapter, we describe the use of exon-and intron-specific probes to evaluate oxytocin (OT) and vasopressin (VP) neuropeptide gene expression by analyses of their mRNAs and hnRNAs by quantitative in situ hybridization (qISH) and also by using specific PCR primers in quantitative, real-time PCR (qPCR) procedures.


Subject(s)
Introns/genetics , Neuropeptides/genetics , Animals , Humans , In Situ Hybridization , Oxytocin/genetics , RNA Precursors/genetics , RNA, Heterogeneous Nuclear/genetics , Real-Time Polymerase Chain Reaction/methods , Vasopressins/genetics
12.
Exp Neurol ; 219(1): 212-22, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19463813

ABSTRACT

Acute increases in plasma osmotic pressure produced by intraperitoneal injection of hypertonic NaCl are sensed by osmoreceptors in the brain, which excite the magnocellular neurons (MCNs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in the hypothalamus inducing the secretion of vasopressin (VP) into the general circulation. Such systemic osmotic stimulation also causes rapid and transient increases in the gene expression of c-fos and VP in the MCNs. In this study we evaluated potential signals that might be responsible for initiating these gene expression changes during acute hyperosmotic stimulation. We use an in vivo paradigm in which we stereotaxically deliver putative agonists and antagonists over the SON unilaterally, and use the contralateral SON in the same rat, exposed only to vehicle solutions, as the control SON. Quantitative real time-PCR was used to compare the levels of c-fos mRNA, and VP mRNA and VP heteronuclear (hn)RNA in the SON. We found that the ionotropic glutamate agonists (NMDA plus AMPA) caused an approximately 6-fold increase of c-fos gene expression in the SON, and some, but not all, G-coupled protein receptor agonists (e.g., phenylephrine, senktide, a NK-3-receptor agonist, and alpha-MSH) increased the c-fos gene expression in the SON from between 1.5 to 2-fold of the control SONs. However, none of these agonists were effective in increasing VP hnRNA as is seen with acute salt-loading. This indicates that the stimulus-transcription coupling mechanisms that underlie the c-fos and VP transcription increases during acute osmotic stimulation differ significantly from one another.


Subject(s)
Gene Expression Regulation/genetics , Neurotransmitter Agents/metabolism , Proto-Oncogene Proteins c-fos/genetics , Supraoptic Nucleus/metabolism , Vasopressins/genetics , Water-Electrolyte Balance/genetics , Animals , Excitatory Amino Acid Agonists/pharmacology , Hypertonic Solutions/pharmacology , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/genetics
13.
J Neurosci Methods ; 178(1): 128-33, 2009 Mar 30.
Article in English | MEDLINE | ID: mdl-19118574

ABSTRACT

Organotypic cultures of mouse and rat magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) have served as important experimental models for the molecular and physiological study of this neuronal phenotype. However, it has been difficult to maintain significant numbers of the MCNs, particularly vasopressin MCNs, in these cultures for long periods. In this paper, we describe the use of the neurotrophic factors, leukemia inhibiting factor (LIF) and ciliary neurotrophic factor (CNTF) to rescue rat vasopressin (Avp)- and oxytocin (Oxt)-MCNs from axotomy-induced, programmed cell death in vitro. Quantitative data are presented for the efficacy of the LIF family of neurotrophic factors on the survival of MCNs in three nuclei, the paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei in the mouse and rat hypothalamus.


Subject(s)
Ciliary Neurotrophic Factor/pharmacology , Hypothalamus/cytology , Leukemia Inhibitory Factor/metabolism , Neurons/drug effects , Neurons/metabolism , Oxytocin/metabolism , Vasopressins/metabolism , Analysis of Variance , Animals , Animals, Newborn , Axotomy/methods , Cell Survival/drug effects , Mice , Neurophysins/metabolism , Organ Culture Techniques , Rats , Rats, Sprague-Dawley
14.
Physiol Genomics ; 35(3): 231-42, 2008 Nov 12.
Article in English | MEDLINE | ID: mdl-18765859

ABSTRACT

In this study, we test the hypothesis that there are differential splicing patterns between the expressed oxytocin (OT) and vasopressin (VP) genes in the rat supraoptic nucleus (SON). We quantify the low abundance, intron-containing heteronuclear RNAs (hnRNAs) and the higher abundance mRNAs in the SON using two-step, quantitative SYBR Green real-time reverse transcription (RT)-PCR and external standard curves constructed using synthetic 90 nt sense-strand oligonucleotides. The levels of OT and VP mRNA in the SON were found to be similar, approximately 10(8) copies/SON pair, whereas the copy numbers of VP hnRNAs containing intron 1 or 2 and the OT hnRNA containing intron 1 are much lower, i.e., approximately 10(2)-10(3) copies/rat SON pair. However, the estimated copy number of the intron 2-containing OT hnRNA is much larger, approximately 10(6) copies/SON pair. The relative distributions of all the OT and VP RNA species were invariant and independent of the physiological status of the rats (e.g., osmotically stimulated or lactating rats). Using intron-specific riboprobes against hnRNAs, we demonstrate by fluorescence in situ hybridization strong signals of OT hnRNA containing intron 2 predominantly in the cytoplasm, in contrast to the localization of the VP hnRNA found only in the nuclei. Taken together, these data support the view that the splicing patterns between OT and VP gene transcripts are different and show that there is a selective cytoplasmic retention of OT intron 2.


Subject(s)
Gene Expression Profiling , Oxytocin/genetics , RNA Splicing/genetics , Supraoptic Nucleus/metabolism , Vasopressins/genetics , Animals , Female , In Situ Hybridization , Male , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction
15.
Prog Brain Res ; 170: 43-51, 2008.
Article in English | MEDLINE | ID: mdl-18655870

ABSTRACT

Intron-specific probes measure heteronuclear RNA (hnRNA) levels and thus approximate the transcription rates of genes, in part because of the rapid turnover of this intermediate form of RNA in the cell nucleus. Previously, we used oxytocin (Oxt)- and vasopressin (Avp)- intron-specific riboprobes to measure changes in Oxt and Avp hnRNA levels in the supraoptic nucleus (SON) by quantitative in situ hybridization (ISH) after various classical physiological perturbations, including acute and chronic salt loading, and lactation. In the present experiments, we used a novel experimental model to study the neurotransmitter regulation of Oxt and Avp gene expression in the rat SON in vivo. Bilateral cannulae connected via tubing to Alzet osmotic mini-pumps were positioned over the SON. In every experiment, one SON was infused with PBS and served as the control SON in each animal, and the contralateral SON received infusions of various neurotransmitter agonists and antagonists. Using this approach, we found that Avp but not Oxt gene expression increased after acute (2-5h) combined excitatory amino acid agonist and GABA antagonist treatment, similar to what we found after an acute hyperosmotic stimulus. Since both OXT and AVP are known to be comparably and robustly secreted in response to acute osmotic stimuli in vivo and glutamate agonists in vitro, our results indicate a dissociation between OXT secretion and Oxt gene transcription in vivo.


Subject(s)
Central Nervous System/physiology , Oxytocin/genetics , Supraoptic Nucleus/physiology , Vasopressins/genetics , Animals , DNA Primers , In Situ Hybridization , Introns , Male , Oxytocin/metabolism , Polymerase Chain Reaction , RNA, Heterogeneous Nuclear/genetics , Rats , Rats, Sprague-Dawley , Transcription, Genetic , Vasopressins/metabolism
16.
J Neurosci Methods ; 164(1): 149-54, 2007 Aug 15.
Article in English | MEDLINE | ID: mdl-17540451

ABSTRACT

The hypothalamus contains distinct neuronal populations that express distinguishing neuropeptides. The supraoptic nucleus contains magnocellular neurons that predominantly express either vasopressin or oxytocin. Transcriptional activators of vasopressin and other neuropeptides have been the subject of much research. Here we present a method of measuring neuropeptide transcription by tailoring one-step quantitative real-time PCR (qRT-PCR) for the analysis of processed and pre-mRNA (heteronuclear RNA). Using moderate and strong hyperosmotic stimuli to induce transcription, we report an increase in vasopressin transcription (pre-mRNA) of 141% and 406% over control levels in response to a 2% injection of 900 mOsm saline or a 1% body weight i.p. injection of 2 M NaCl, respectively. These results agree with a host of studies employing the more labor-intensive method of in situ hybridization histochemistry by which investigators also measured intron-containing heteronuclear RNAs. Furthermore, these results confirm that qRT-PCR with intron-specific primers can be used to rapidly analyze transcription, and suggest an important further benefit of a real-time PCR analysis, such as the ability of measuring transcription of multiple neuropeptides along with other genes from a single sample.


Subject(s)
Introns/genetics , RNA Precursors/analysis , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Vasopressins/biosynthesis , Vasopressins/genetics , Animals , DNA Primers/genetics , Male , RNA Precursors/genetics , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Saline Solution, Hypertonic/pharmacology , Supraoptic Nucleus/drug effects , Supraoptic Nucleus/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/genetics , Water-Electrolyte Balance/drug effects , Water-Electrolyte Balance/genetics
17.
Cell Mol Neurobiol ; 26(4-6): 959-78, 2006.
Article in English | MEDLINE | ID: mdl-16699879

ABSTRACT

1. Hypoosmolality produces a dramatic inhibition of vasopressin (VP) and oxytocin (OT) gene expression in the supraoptic nucleus (SON). This study examines the effect of sustained hypoosmolality on global gene expression in the OT and VP magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS), in order to detect novel genes in this system that might be involved in osmoregulation in the MCNs. 2. For this purpose, we used Affymetrix oligonucleotide arrays to analyze the expression of specific genes in laser microdissected rat SONs, and their changes in expression during chronic hypoosmolality. We identified over 40 genes that had three-fold or more greater expression in the SON versus total hypothalamus, and that also changed more than two fold in expression as a result of the chronic hypoosmolar treatment. These genes contained both novel as well as genes previously known to be present in the SON. All of the raw data for the genes that are expressed in the SON and altered by hypoosmolality can be found on the following NINDS website URL address: http://data.ninds.nih.gov/Gainer/Publications.


Subject(s)
Oligonucleotide Array Sequence Analysis , Supraoptic Nucleus/metabolism , Water-Electrolyte Balance , Water-Electrolyte Imbalance , Animals , Male , Rats , Rats, Sprague-Dawley , Supraoptic Nucleus/drug effects , Transcription Factors/metabolism , Vasopressins/administration & dosage , Water-Electrolyte Balance/drug effects
18.
Am J Physiol Regul Integr Comp Physiol ; 290(5): R1233-41, 2006 May.
Article in English | MEDLINE | ID: mdl-16357095

ABSTRACT

To develop a comprehensive approach for the study of oxytocin (OT) and vasopressin (VP) gene expression in the rat hypothalamus, we first developed an intronic riboprobe to measure OT heteronuclear RNA (hnRNA) levels by in situ hybridization histochemistry (ISHH). Using this 84-bp riboprobe, directed against intron 2 of the OT gene, we demonstrate strong and specific signals in neurons confined to the supraoptic (SON) and paraventricular (PVN) nuclei of the rat hypothalamus. We used this new intronic OT probe, together with other well-established intronic and exonic OT and VP probes, to reevaluate OT and VP gene expression in the hypothalamus under two classical physiological conditions, acute osmotic stimulation, and lactation. We found that magnocellular neurons in 7- to 8-day lactating female rats exhibit increased OT but not VP hnRNA. Since VP mRNA is increased during lactation, this suggests that decreased VP mRNA degradation during lactation may be responsible for this change. In contrast, whereas there was the expected large increase in VP hnRNA after acute salt loading, there was no change in OT hnRNA, suggesting that acute hyperosmotic stimuli produce increased VP but not OT gene transcription. Hence, the use of both exon- and intron-specific probes, which distinguish the changes in hnRNA and mRNA levels, respectively, can provide insight into the relative roles of transcription and mRNA degradation processes in changes in gene expression evoked by physiological stimuli.


Subject(s)
Exons/genetics , Hypothalamus/metabolism , Introns/genetics , Oxytocin/biosynthesis , Oxytocin/genetics , Vasopressins/biosynthesis , Vasopressins/genetics , Animals , Base Sequence , Female , In Situ Hybridization , Lactation/physiology , Male , Molecular Sequence Data , Paraventricular Hypothalamic Nucleus/metabolism , RNA Probes/chemical synthesis , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Supraoptic Nucleus/metabolism
19.
Int J Cancer ; 108(2): 232-6, 2004 Jan 10.
Article in English | MEDLINE | ID: mdl-14639608

ABSTRACT

We have previously cloned and identified a novel esophageal cancer related gene 2 (ECRG2; GenBank Accession Number AF268198), which is down-regulated in esophageal squamous cell carcinoma (ESCC) and involved in the induction of the apoptosis in esophageal cancer cell lines. In the present study, we have found a short tandem repeat (STR) polymorphism in the noncoding region of the exon 4 of the ECRG2 gene by using PCR-denaturing high-performance liquid chromatography (DHPLC). Three STR genotypes, TCA3/TCA3, TCA3/TCA4 and TCA4/TCA4 were revealed and confirmed by DNA sequencing analysis. A total of 661 objects including 228 patients with ESCC and 373 normal controls were analyzed to investigate the impact of this ECRG2 STR polymorphism on risk of ESCC in case-control studies. Genotypes were determined in 231 controls and 162 cases from Beijing, which is a low risk area of ESCC, and in 142 controls and 126 cases from Linxian, a well-known high-risk area of ESCC. In both of the Beijing and Linxian population, subjects who carried the TCA3/TCA3 genotype were at an increased risk of ESCC compared to those carrying the TCA4/TCA4 genotype, with the adjusted odds ratios (ORs) being 2.05 [95% confidence interval (CI), 1.02-4.06] for the subjects from Beijing and 4.40 (95% CI, 1.93-10.01) for the subjects from Linxian. Furthermore, comparison of the genotype distributions among other cancer sites might suggest that risk of the ECRG2 STR polymorphism might be specific to the esophagus. These findings indicate for the first time that the ECRG2 STR is a genetic susceptibility factor for ESCC and the TCA3/TCA3 allele might play a role in the development of this cancer.


Subject(s)
Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Genetic Predisposition to Disease , Polymorphism, Genetic , Tandem Repeat Sequences/genetics , Tumor Suppressor Proteins/genetics , Aged , Alleles , Asian People , Carcinoma, Squamous Cell/ethnology , Case-Control Studies , China/epidemiology , Chromatography, High Pressure Liquid , Esophageal Neoplasms/ethnology , Esophagus/pathology , Exons/genetics , Female , Genotype , Humans , Male , Middle Aged , Proteinase Inhibitory Proteins, Secretory , Risk Factors , Serine Peptidase Inhibitors, Kazal Type
20.
World J Gastroenterol ; 9(6): 1174-8, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12800218

ABSTRACT

AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC). METHODS: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR. RESULTS: The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80 %) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing. CONCLUSION: These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.


Subject(s)
Carcinoma, Squamous Cell/genetics , CpG Islands/genetics , DNA Methylation , Down-Regulation , Esophageal Neoplasms/genetics , Gene Expression , Proteins/genetics , Humans , Neoplasm Proteins , Tumor Cells, Cultured , Tumor Suppressor Proteins
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