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At least 757 records · Page 42Linked to original sources
PubMed2003

Quantitative gene expression analysis reveals transition of fetal liver progenitor cells to mature hepatocytes after transplantation in uPA/RAG-2 mice.

Therapies for liver diseases with stem and progenitor cells will require a detailed knowledge of the molecular mechanisms driving the in vivo differentiation process toward adult hepatic tissue. We applied quantitative gene expression methods to analyze the differentiation process of fetal liver progenitor cells after transplantation into an animal model of liver regeneration. Enhanced green fluorescent protein (EGFP)-transgenic liver progenitor cells were isolated from fetal mouse liver at stage embryonic day 13.5 and transplanted into uPA/RAG-2 mice. Two, 4, and 6 weeks after cell transplantation cryosections of liver tissue were analyzed for EGFP-positive regeneration nodules. RNA from laser-microdissected EGFP-positive tissue was isolated and used as template for quantitative real-time reverse transcriptase-polymerase chain reaction. Phenotypic differentiation was analyzed by staining of the canalicular marker enzyme dipeptidyl-peptidase IV. Proliferation in regenerative nodules and surrounding tissue was monitored with the BrdU incorporation assay. Alpha fetoprotein gene expression had already decreased 2 weeks after transplantation in EGFP-positive regeneration nodules compared to pretransplantation values and was not detectable after 4 and 6 weeks, whereas albumin slightly increased in transplanted cells indicating differentiation into a mature phenotype. The dipeptidyl-peptidase IV antigen was associated with some liver progenitor cells 2 weeks after transplantation and in virtually all cells after 4 and 6 weeks. Cell proliferation index in transplanted cells was maximally increased (4.8% BrdU-positive cells) after 2 weeks and decreased (0.4%) after 6 weeks to normal levels. Our results demonstrate that gene expression in liver progenitor cells changes from fetal to adult phenotype within 4 to 6 weeks after transplantation despite ongoing proliferation of the transplanted cells in a mouse model of liver regeneration. Quantitative gene expression profiles as shown here will have important implications in our understanding of the in vivo differentiation process of stem cells.

Animals↗
PubMed2001

Age and TP53 mutation frequency in childhood malignant gliomas: results in a multi-institutional cohort.

Malignant astrocytoma is one of the most deadly primary central nervous system tumors. Although significant progress has been made in understanding the molecular pathways that lead to the development of these tumors in adults, comparatively little analysis has been done in childhood astrocytomas, which are less common and have a more favorable prognosis. Our previous studies of an institutional cohort of children with malignant gliomas suggested the existence of distinct molecular pathways of tumorigenesis in younger versus older children, based on the finding of a high frequency of TP53 mutations in tumors from children >3 years of age at diagnosis, compared with those from younger children. In the current study, the association between TP53 mutations and age was examined in greater detail using the multi-institutional group of children enrolled in Children's Cancer Group Study 945, the largest cohort of childhood high-grade gliomas analyzed to date. Seventy-seven tumors with centrally reviewed diagnoses of anaplastic astrocytoma or glioblastoma multiforme had sufficient archival histopathological material for microdissection-based genotyping. Sections were examined histologically, and topographic targets that contained malignant tissue were isolated by microdissection and subjected to PCR-based amplification and sequencing of TP53 exons 5-8. Twenty-six tumors (33.8%) had mutations in those exons. Mutations were observed in 2 of 17 tumors (11.8%) from children <3 years of age at diagnosis versus 24 of 60 tumors (40%) from older children, a difference that was statistically significant (P = 0.04), in agreement with our previous results. Whereas malignant gliomas in older children have a frequency of mutations comparable to tumors that arise in young adults, those from children <3 years old do not. The association between age and frequency of TP53 mutations among pediatric malignant gliomas indicates the probable existence of two distinct pathways of molecular tumorigenesis in younger versus older children.

Adolescent↗
PubMed1999

Autoradiographic demonstration of quinuclidinyl benzilate binding sites in the vestibular organs of the gerbil.

Gerbil vestibular tissues were isolated by microdissection and incubated in vitro with 3H-quinuclidinyl benzilate (3H-QNB). Control tissues were incubated in medium containing unlabeled atropine to differentiate non-specific from specific binding. Autoradiographic grain densities were determined by morphometric techniques and evaluated by two-tailed t-test. The label densities of sensory epithelia from experimental preparations of ampulla, utricle and saccule were found to be significantly higher than those in the adjacent endolymphatic compartment and also higher than those of adjacent stromal tissue comprising connective tissue, nerve fibers and capillaries. In contrast, no tissue region in atropine controls showed label density significantly above that of the endolymphatic compartment. Label density of ampullar sensory epithelium incubated with 3H-QNB alone was significantly higher than that of sensory epithelium from utricle or saccule. Grain density was greater in the peripheral regions of the ampullar crista compared to the vertex. Appreciable label was also present in nerve bundles beneath the sensory epithelium of the ampulla. The current study demonstrates the existence of putative muscarinic neurotransmitter/neuromodulator receptor sites in mammalian vestibular sense organs at locations corresponding to efferent innervation, with particularly significant concentrations in the ampulla.

Animals↗
PubMed2004

Microarray analysis of fluoro-gold labeled rat dopamine neurons harvested by laser capture microdissection.

The cellular heterogeneity of brain tissue presents a challenge to gene expression profiling of specific neuronal cell types. The present study employed a fluorescent neural tracer to specifically label midbrain dopamine neurons and non-dopamine cortical neurons. The labeled cells were then used to visually guide harvesting of the cells by laser capture microdissection (LCM). RNA extracted from the two populations of harvested cells was then amplified, labeled and co-hybridized to high density cDNA microarrays for two-color differential expression profiling. Many of the genes most highly enriched in the dopamine neurons were found to be genes previously known to define the dopamine neuronal phenotype. However, results from the microarray were only partially validated by quantitative RT-PCR analysis. The results indicate that LCM harvesting of specific neuronal phenotypes can be effectively guided in a complex cellular environment by specific pre-labeling of the target cell populations and underlie the importance of independent validation of microarray results.

Animals↗
PubMed2003

Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications.

We previously reported that ethanol fixation and paraffin embedding of tissues produce excellent histomorphology and good preservation of macromolecules. Here, we present a detailed evaluation of ethanol-fixed tissues for proteomic initiatives. When proteins were extracted from ethanol-fixed, paraffin-embedded prostate tissue, resolved by two-dimensional gel electrophoresis (2-DE), and stained by standard methods, several hundred protein molecules could be detected and successfully analyzed by mass spectrometry. Protein profiles obtained from ethanol-fixed tissues were highly similar to those observed from frozen tissues, in contrast to the poor protein recovery from formalin-fixed material. The protein content of specific cells that were microdissected from ethanol-fixed tissue sections using laser capture microdissection could also be successfully analyzed by 2-DE. We observed that eosin staining of tissue sections had a detrimental effect on protein separation, whereas hematoxylin staining had minimal consequence. In order to illustrate the applicability of ethanol-fixed tissues for proteomic discovery studies, we compared the protein profiles of patient-matched, normal prostatic epithelial cells and invasive adenocarcinoma cells obtained from ethanol-fixed, paraffin-embedded tissues. A number of differentially expressed proteins was discovered and identified by mass spectrometry. Immunohistochemical analyses performed on ethanol-fixed tissue sections were in agreement with the proteomic discovery findings. In light of these results, we conclude that ethanol-fixed tissues can be successfully utilized for proteomic analyses.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.

Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-microm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.

Animals↗
PubMed2001

Diagnosis of aneuploidy in archival, paraffin-embedded pregnancy-loss tissues by comparative genomic hybridization.

OBJECTIVE: To evaluate the detection of aneuploidy in archival tissues from miscarriages by a method that uses microdissection and DNA extraction of villus cells from paraffin blocks, followed by universal DNA amplification and comparative genomic hybridization (CGH). DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Nine archival tissues from cases of spontaneous abortion with trisomy 16 (two cases), trisomy 21 (three cases), trisomy 22 (two cases), triploidy (one case), and monosomy X (one case). INTERVENTION(S): Villus DNA was extracted from microdissected, formalin-fixed, paraffin-embedded tissues. Aneuploidy was detected by CGH after universal amplification of the DNA with the use of degenerate oligonucleotide-primed polymerase chain reaction. MAIN OUTCOME MEASURE(S): Detection of aneuploidy in archival pregnancy-loss tissues using CGH. RESULT(S): In all nine cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH. In six of nine cases, the chromosomal abnormality detected by conventional cytogenetic analysis was identified by CGH: trisomy 16 (2/2), trisomy 21 (3/3), and trisomy 22 (1/2). One case of each of the following was not detectable: triploidy (1/1), monosomy X (1/1), and trisomy 22 (1/2). CONCLUSION(S): We propose CGH as a method for determination of aneuploidy in pregnancy-loss archival tissues when conventional cytogenetic analysis is unsuccessful or when it was not performed when fresh tissue was available.

Abortion, Spontaneous↗
PubMed2003

Gene expression profiles of microdissected pancreatic ductal adenocarcinoma.

In a search for new molecular markers of pancreatic ductal adenocarcinoma (PDAC), we compared the gene expression profiles of seven pancreatic carcinomas and one carcinoma of the papilla Vateri with those of duct cells from three non-neoplastic pancreatic tissues. In addition, the human pancreatic duct cell line and five PDAC cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF) were examined. RNA was extracted from microdissected tissue or cultured cell lines and analysed using a custom-made Affymetrix Chip containing 3023 genes, of which 1000 were known to be tumour associated. Hierarchical clustering revealed 81 differentially expressed genes. Of all the genes, 26 were downregulated in PDAC and 14 were upregulated in PDAC. In PDAC cell lines versus normal pancreatic duct cells, 21 genes were downregulated and 20 were upregulated. Of these 81 differentially expressed genes, 15 represented human genes previously implicated in the tumourigenesis of PDAC. From the genes that were so far not known to be associated with PDAC tumorigenesis, we selected ADAM9 for further validation because of its distinct overexpression in tumour tissue. Using immunohistochemistry, the over-expressed gene, ADAM9, was present in 70% of the PDACs analysed. In conclusion, using microarray technology we were able to identify a set of genes whose aberrant expression was associated with PDAC and may be used to target the disease.

Adenocarcinoma↗
PubMed2007

NanoLC-FT-ICR MS improves proteome coverage attainable for approximately 3000 laser-microdissected breast carcinoma cells.

Proteomics assays hold great promise for unraveling molecular events that underlie human diseases. Effective analysis of clinical samples is essential, but this task is considerably complicated by tissue heterogeneity. Laser capture microdissection (LCM) can be used to selectively isolate target cells from their native tissue environment. However, the small number of cells that is typically procured by LCM severely limits proteome coverage and biomarker discovery potential achievable by conventional proteomics platforms. Herein, we describe the use of nanoLC-FT-ICR MS for analyzing protein digests of 3000 LCM-derived tumor cells from breast carcinoma tissue, corresponding to 300 ng of total protein. A total of 2282 peptides were identified by matching LC-MS data to accurate mass and time (AMT) tag databases that were previously established for human breast (cancer) cell lines. One thousand and three unique proteins were confidently identified with two or more peptides. Based on gene ontology categorization, identified proteins appear to cover a wide variety of biological functions and cellular compartments. This work demonstrates that a substantial number of proteins can be detected and identified from limited number of cells using the AMT tag approach, and opens doors for high-throughput in-depth proteomics analysis of clinical samples.

Breast Neoplasms↗
PubMed2004

Tissue preparation for gene expression profiling of colorectal carcinoma: three alternatives to laser microdissection with preamplification.

Colorectal-carcinoma specimens are heterogeneous and include areas of nonmalignant mucosal and connective tissue. For those study designs in which laser microdissection and RNA preamplification are impracticable, the optimal yield of genuine cancer RNA is a key factor in gene-expression analysis. In this study we compared alternative methods of tissue purification. Three contiguous 0.5-cm(3) samples taken from an advanced primary adenocarcinoma of the sigmoid colon were processed immediately after surgery with the use of the following methods: (1) cryotomy after manual dissection (CMD), (2) microscopically assisted manual dissection (MAMD), and (3) tumor-cell isolation with the use of Ber-EP4 antibodies and Dynabeads (Dynal Biotech GmbH, Hamburg, Germany; technique abbreviated as DB). We generated gene-expression profiles with the use of GeneChip technology (Affymetrix, Santa Clara, Calif) and recorded preparation times, costs, and RNA quantity and quality. CMD took 60 minutes, MAMD 180 minutes, and DB 90 minutes to isolate 22, 8, and 23 microg of RNA, respectively. Expenses for materials amounted to 41, 23, and 91 US dollars for CMD, MAMD, and DB, respectively. The 3'/5' ratio, as determined with the GeneChips, for GAPDH/beta-actin was 1.01:1.03 for CMD, 1.13:1.28 for MAMD, 1.43:1.68 for DB, K-ras, APC, smad 2, transforming growth factor-beta, and p53 were marked as present in all cases, with the exception of APC, which was graded as marginal on DB. The correlation values of gene-expression profiles were 91% (CMD/DB), 93% (CMD/MAMD), and 97% (DB/MAMD). All 3 methods provided enough RNA, of sufficient quality, for gene-expression microarray analysis in colorectal carcinoma. Cross-methodologic analyses of array data should not be performed uncritically.

Adenocarcinoma↗
PubMed2003

Loss of heterozygosity analysis of mouse pulmonary adenomas induced by coal tar.

Manufactured gas plant (MGP) residues, commonly known as coal tars, were generated several decades ago as a byproduct of residential and industrial gas production from the distillation of coal. Previous short-term exposure studies have shown MGP residues to be tumorigenic in mouse liver and lung. In order to gain further insight into carcinogenesis by complex mixtures of environmental chemicals containing known carcinogenic polycyclic aromatic hydrocarbons, we investigated mouse pulmonary tumors for loss of heterozygosity (LOH) as a result of multiple exposure to MGP residues. Twenty mouse lung adenomas produced in (C57BL/6 x C3H)F1 hybrid mice and manually microdissected were selected to examine genome-wide allelic losses at 58 microsatellite loci. Regions of chromosomes 1, 4, 5, 8, and 11 were affected in 30-40% of tumors. The elevated rates of allelic imbalance in these chromosomes may indicate the location of unknown tumor suppressor genes significant to neoplastic transformation in mouse lung tissues. Laser capture microdissection-based LOH analysis of pulmonary adenomas showed that contamination of nonneoplastic tissues was not masking the allelic losses in the manually microdissected tumor analysis. The low frequency of chromosome instability in these tumors, measured by means of inter-simple sequence repeat PCR, suggests the presence of discrete regions of LOH instead of extensive structural rearrangements.

Adenoma↗
PubMed2005

Births using sperm retrieved via immediate microdissection of a solitary testis with cancer.

OBJECTIVE: To determine the feasibility of achieving births using sperm retrieved from a solitary testis with cancer. DESIGN: Prospective clinical study of azoospermic men with testis cancer in a solitary testis. SETTING: Infertility patients in an academic environment. PATIENT(S): Azoospermic men with previous history of orchiectomy and testis cancer in a remaining solitary testis. INTERVENTION(S): Viable sperm were retrieved by immediate microdissection of paratumor testicular tissue from orchiectomy specimen. MAIN OUTCOME MEASURE(S): Live births were achieved using sperm from immediate microdissection of orchiectomy specimen with testis cancer. CONCLUSION(S): Azoospermic men with cancer in a solitary testis have potential for fertility.

Adult↗
PubMed1999

A reliable PCR amplification method for microdissected tumor cells obtained from paraffin-embedded tissue.

We report a reliable method for PCR (polymerase chain reaction) amplification of genomic DNA from PET. This method uses DNA extraction with the QIAquick kit and amplification with AmpliTaq Gold. Amplification of up to 959 bp from PET was achieved with this combination which exceeds the current reported upper limit of 800 bp. In summary, the gradual activation of the AmpliTaq Gold during thermal cycling allows both for higher-fidelity and higher-throughput PCR amplification from PET. The use of the QIAquick kit for DNA purification of PET is sensitive, reproducible and suitable for management of a high number of samples.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1997

Microdissection and polymerase chain reaction amplification of genomic DNA from histological tissue sections.

Polymerase chain reaction (PCR)-based assays are being used increasingly to study the molecular genetic changes that occur in minute cellular lesions that are identified in histological sections. It is often desirable to microdissect the cells of interest in a lesion, isolating them from surrounding normal tissue to obtain the purest representation of genomic DNA possible. We present here an optimized microdissection and DNA extraction protocol that reliably produces PCR-amplifiable DNA from lesional tissue less than 0.1 mm in diameter. The utility of this technique is demonstrated by PCR amplification and sequencing of the K-ras gene and microsatellite PCR analysis of proliferative epithelial lesions in the small ducts of a human pancreas involved by cancer.

DNA↗
PubMed2005

Synaptic pathology, altered gene expression, and degeneration in photoreceptors impacted by drusen.

PURPOSE: Drusen are risk factors for age-related macular degeneration and have been shown to negatively impact cells of the RPE and retina. In this study, the effects of drusen on the synaptic machinery of retinal photoreceptors are investigated. METHODS: Human donor eye tissue containing retina, RPE, and choroid was processed for confocal immunofluorescence microscopy, laser capture microdissection, and light and electron microscopy. Tissue sections were immunostained with a panel of antibodies to synapse-associated proteins. Populations of photoreceptors over drusen and normal populations of photoreceptors were microdissected from fresh frozen tissue, RNA was purified, and quantitative PCR was performed to compare relative levels of gene expression. RESULTS: The number of photoreceptor synaptic terminals is reduced in regions of the outer plexiform layer over drusen, synaptic proteins are mislocalized in photoreceptor cells, and synaptic terminals are often observed within the outer nuclear layer. Photoreceptors over drusen also increase expression of the stress response proteins apolipoprotein E and alphaB-crystallin. Abnormal immunolabeling patterns are not restricted to photoreceptors directly over drusen but are also observed in cells flanking drusen. Gene expression analysis confirms reductions in the expression of genes coding for synapse-associated proteins and signal transduction proteins and increases in the expression of apolipoprotein E and alphaB-crystallingene transcripts. Ultrastructural analysis of photoreceptor synaptic terminals over drusen reveals significant abnormalities, and cell counts show a reduction in photoreceptor density directly over, and lateral to, drusen of all sizes. CONCLUSIONS: Photoreceptors overlying and flanking drusen exhibit morphologic and biochemical signs of degeneration. The expression of synapse-associated proteins decreases in photoreceptor synaptic terminals, whereas the expression of stress-response proteins increases. Reductions in photoreceptor cell densities over, and flanking, drusen suggest that these degenerative effects eventually result in the death of photoreceptors.

Aged↗
PubMed2004

Gene expression in TUNEL-positive neurons in human immunodeficiency virus-infected brain.

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) results in neuronal damage and apoptosis, and both in vitro models and pathological studies suggest that a variety of neurotoxins released by HIV-infected and -activated macrophages/microglia selectively damage susceptible subsets of neurons. Confirmation of in vitro findings of mechanisms of neurodegeneration and neuronal cell dysfunction in vivo has been approached through detailed pathological analysis of regional structural damage, immunohistochemical detection of selected antigens in damaged cells, and, more recently, analysis of gene expression in whole tissue blocks or pooled populations (hundreds/thousands) of microdissected cells. Recently developed techniques of gene expression analysis through antisense mRNA amplification (aRNA) at the single-cell level may offer the potential to study pathways of neuronal cell death and to determine patterns of coordinated gene expression that may more specifically identify susceptible neuronal subclasses in vivo. Utilizing this unique technique, the authors have demonstrated, for the first time, RNA amplification and gene expression profiling in individual deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-reactive neurons microdissected from fixed, archival human brain tissue. RNA amplification was successful in >80% of TUNEL-positive neurons, and quantitative aRNA/cDNA hybridization slot-blot analysis demonstrated similar levels of actin RNA but significant differences in caspase-2 RNA expression between TUNEL-reactive and -nonreactive neurons. Reliable quantitative comparisons were achieved with modest numbers of sampled neurons (approximately 10). These studies suggest that analysis of coordinated gene expression in individual damaged neurons in vivo can be reliably used to identify neuronal subclasses that express certain susceptibility- or survival-promoting genes that may be targeted for more specific neuroprotective strategies against HIV.

AIDS Dementia Complex↗