RESUMO
BACKGROUND: Identification and characterization of the prostate stem cell is important for understanding normal prostate development and carcinogenesis. The flow cytometry-based side population (SP) technique has been developed to isolate putative adult stem cells in several human tissue types including the prostate. This phenotype is mainly mediated by the ATP-binding cassette membrane transporter ABCG2. METHODS: Immunolocalization of ABCG2 was performed on normal prostate tissue obtained from radical prostatectomies. Normal human prostate SP cells and ABCG2+ cells were isolated and gene expression was determined with DNA array analysis and RT-PCR. Endothelial cells were removed by pre-sorting with CD31. RESULTS: ABCG2 positive cells were localized to the prostate basal epithelium and endothelium. ABCG2+ cells in the basal epithelium constituted less than 1% of the total basal cell population. SP cells constituted 0.5-3% of the total epithelial fraction. The SP transcriptome was essentially the same as ABCG2+ and both populations expressed genes indicative of a stem cell phenotype, however, the cells also expressed many genes in common with endothelial cells. CONCLUSION: These results provide gene expression profiles for the prostate SP and ABCG2+ cells that will be critical for studying normal development and carcinogenesis, in particular as related to the cancer stem cell concept.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Perfilação da Expressão Gênica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Próstata/química , Próstata/citologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Linhagem Celular , Células Epiteliais , Humanos , Masculino , Células-TroncoRESUMO
BACKGROUND: The gene expression profiles of most human tissues have been studied by determining the transcriptome of whole tissue homogenates. Due to the solid composition of tissues it is difficult to study the transcriptomes of individual cell types that compose a tissue. To overcome the problem of heterogeneity we have developed a method to isolate individual cell types from whole tissue that are a source of RNA suitable for transcriptome profiling. RESULTS: Using monoclonal antibodies specific for basal (integrin beta4), luminal secretory (dipeptidyl peptidase IV), stromal fibromuscular (integrin alpha 1), and endothelial (PECAM-1) cells, respectively, we separated the cell types of the prostate with magnetic cell sorting (MACS). Gene expression of MACS-sorted cell populations was assessed with Affymetrix GeneChips. Analysis of the data provided insight into gene expression patterns at the level of individual cell populations in the prostate. CONCLUSION: In this study, we have determined the transcriptome profile of a solid tissue at the level of individual cell types. Our data will be useful for studying prostate development and cancer progression in the context of single cell populations within the organ.
Assuntos
Perfilação da Expressão Gênica , Próstata/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Humanos , Separação Imunomagnética , Masculino , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Fenótipo , Neoplasias da Próstata/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
We describe the creation process of the Minimum Information Specification for In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE). Modeled after the existing minimum information specification for microarray data, we created a new specification for gene expression localization experiments, initially to facilitate data sharing within a consortium. After successful use within the consortium, the specification was circulated to members of the wider biomedical research community for comment and refinement. After a period of acquiring many new suggested requirements, it was necessary to enter a final phase of excluding those requirements that were deemed inappropriate as a minimum requirement for all experiments. The full specification will soon be published as a version 1.0 proposal to the community, upon which a more full discussion must take place so that the final specification may be achieved with the involvement of the whole community.
Assuntos
Biologia Computacional/normas , Imuno-Histoquímica/normas , Hibridização In Situ/normas , Biologia Computacional/métodos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodosRESUMO
BACKGROUND: Affymetrix GeneChip Array and Massively Parallel Signature Sequencing (MPSS) are two high throughput methodologies used to profile transcriptomes. Each method has certain strengths and weaknesses; however, no comparison has been made between the data derived from Affymetrix arrays and MPSS. In this study, two lineage-related prostate cancer cell lines, LNCaP and C4-2, were used for transcriptome analysis with the aim of identifying genes associated with prostate cancer progression. METHODS: Affymetrix GeneChip array and MPSS analyses were performed. Data was analyzed with GeneSpring 6.2 and in-house perl scripts. Expression array results were verified with RT-PCR. RESULTS: Comparison of the data revealed that both technologies detected genes the other did not. In LNCaP, 3,180 genes were only detected by Affymetrix and 1,169 genes were only detected by MPSS. Similarly, in C4-2, 4,121 genes were only detected by Affymetrix and 1,014 genes were only detected by MPSS. Analysis of the combined transcriptomes identified 66 genes unique to LNCaP cells and 33 genes unique to C4-2 cells. Expression analysis of these genes in prostate cancer specimens showed CA1 to be highly expressed in bone metastasis but not expressed in primary tumor and EPHA7 to be expressed in normal prostate and primary tumor but not bone metastasis. CONCLUSION: Our data indicates that transcriptome profiling with a single methodology will not fully assess the expression of all genes in a cell line. A combination of transcription profiling technologies such as DNA array and MPSS provides a more robust means to assess the expression profile of an RNA sample. Finally, genes that were differentially expressed in cell lines were also differentially expressed in primary prostate cancer and its metastases.
Assuntos
Regulação Neoplásica da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Biologia Computacional , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Masculino , Modelos Estatísticos , Neoplasias da Próstata/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Software , Transcrição GênicaRESUMO
Copy number variation (CNV) is one of the most profound forms of somatic DNA changes that underlie most human cancers. However, the degree of complexity within and between DNA and mRNA variations in cancer cohorts has yet to be fully characterized. Here we characterized the connectivity of CNV/CNV and its contribution to transcriptome in human cancer cell lines. Strikingly, we found there is a significant nonrandom correlation of many unlinked DNA loci and also a significant association between CNV and mRNA expression in cis and in trans (called eCNV). Both distributions of DNA/DNA and DNA/mRNA associations exhibit a scale-free structure showing that, for DNA/DNA, a few loci correlate to many other loci, whereas most loci correlate to only a few loci; and for DNA/mRNA, certain chromosomal loci associate with many mRNAs and that many mRNAs are controlled by more than one locus. This suggests that a small number of DNA loci act as hubs in a hierarchical structure that is highly nonrandom in nature, and genes linking to these hot spots tend to be involved in similar biological functions. Derivation of highly connected structures suggests a process of undirected copy number changes followed by selection of those advantageous to tumor cells during tumorigenesis. Given that the cohort includes many tissue types, our observations may identify a common and important underlying structure present in human tumors.
Assuntos
DNA/genética , RNA Mensageiro/genética , Linhagem Celular Tumoral , Dosagem de Genes , Humanos , Hibridização de Ácido NucleicoRESUMO
At least in mammals, retinoic acid is a pivotal factor in maintaining the functionality of the testis, in particular, for the progression of germ cells from mitosis to meiosis. Removal of dietary vitamin A or a targeted deletion of retinoic acid receptor alpha gene (Rara), the receptor for retinoic acid, in mice, led to testicular degeneration by a dramatic loss of germ cells and a loss of control of the spermatogenic cycle. The germ cells that remained in the vitamin A deficient (VAD) rat testis were spermatogonia and a few preleptotene spermatocytes. Spermatogenesis can be reinitiated by injection of VAD rats with retinol, the metabolic precursor of retinoic acid, but to date, the functions of retinoic acid in the testis remain elusive. We have applied DNA microarray technology to investigate the time-dependent transcriptome changes that occur 4 to 24 h after retinol replenishment in the VAD rat testis. The retinol-regulated gene expression occurred both in germ cells and Sertoli cells. Bioinformatic analyses revealed time-dependent clusters of genes and canonical pathways that may have critical functions for proper progression through spermatogenesis. In particular, gene clusters that emerged dealt with: (1) cholesterol and oxysterol homeostasis, * (2) the regulation of steroidogenesis, (3) glycerophospholipid metabolism, (4) the regulation of acute inflammation, (5) the regulation of the cell cycle including ubiquitin-mediated degradation of cell cycle proteins and control of centrosome and genome integrity, and (6) the control of membrane scaffolding proteins that can integrate multiple small GTPase signals within a cell. These results provide insights into the potential role of retinoic acid in the testis.
Assuntos
Regulação da Expressão Gênica , Espermatogênese , Testículo/metabolismo , Vitamina A/metabolismo , Animais , Análise por Conglomerados , Perfilação da Expressão Gênica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Receptores do Ácido Retinoico/metabolismoRESUMO
One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.
Assuntos
Imuno-Histoquímica/normas , Hibridização In Situ/normas , Biologia Computacional/métodos , Biologia Computacional/normas , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Imuno-Histoquímica/métodos , Hibridização In Situ/métodosRESUMO
BACKGROUND: CD10 is an approximately 100 kDa transmembrane metallo-endopeptidase. CD10 is strongly expressed by normal prostate epithelium. While only 30% of primary prostate tumors express CD10, it is strongly expressed by most lymph node metastases. The function of CD10 and the interaction between CD10 and other cellular proteins in prostate cancer (CaP) is not well defined. Cellular context may ultimately determine its biologic function in CaP. In this study, we compared CD10 mRNA and protein expression between benign and malignant prostate cells and employed proteomic analysis to identify proteins that interact with CD10 in C4-2 prostate cancer cells. METHODS: CD10 mRNA and protein expression was compared using RT-PCR and Western blotting. CD10-protein complexes were isolated by immunoprecipitation using anti-CD10 monoclonal antibodies. Eluted fractions were combined, trypsinized, and the resulting peptides analyzed by microLC-ESI-MS/MS. The parent proteins were identified by searching MS/MS spectra against a human protein database using SEQUEST. RESULTS: There were no differences in CD10 mRNA length or CD10 protein molecular weight between normal tissue and CaP. We identified 75 proteins unique to or heavily enriched in the CD10 immunoprecipitates by proteomic analysis. The 27 kDa heat shock protein (HSP27) and HSP70 were identified in three separate precipitations. Protein identification by proteomics was confirmed by Western blotting. Protein complexes immunopurified from C4-2 protein extracts using anti-HSP27 and anti-HSP70 antibodies were found to contain CD10. CONCLUSIONS: The function of CD10 in prostate cancer is largely unknown. In the C4-2 CaP cell line, CD10 was found to interact with both HSP27 and HSP70.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Neprilisina/metabolismo , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Humanos , Masculino , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Neprilisina/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Recently, we described the identification of a novel protein, nuclear receptor-associated protein 80 (RAP80), which is highly expressed in spermatocytes and appears to have a role in regulating gene expression. To identify proteins interacting with this protein, we performed yeast two-hybrid screening using full-length RAP80 as bait. This screen identified one in-frame clone encoding a novel testis-specific protein (Tsp), referred to as Tsp57. Tsp57 encodes a basic protein with a mass of 56.8 kDa. The amino acid sequence of Tsp57 is highly conserved (87%) between mouse and human. The mouse and human Tsp57 genes map to chromosomes 9A1 and 11q21, respectively. Northern blot analysis showed that the expression of Tsp57 mRNA was highly restricted to the testis and temporally regulated during testicular development. Tsp57 mRNA was greatly induced between Day 21 and Day 25 of postnatal testicular development. In situ hybridization analysis demonstrated that the hybridization signal for Tsp57 mRNA was strongest in sections of seminiferous tubules at stages VI-VIII of spermatogenesis, consistent with the conclusion that Tsp57 is most highly expressed in round spermatids. Study of Tsp57 expression in several purified subpopulations of spermatogenic cells confirmed maximum levels of expression in round spermatids. Consistently, Tsp57 expression was absent in testes from vitamin A-deficient mice, which do not have any round spermatids, and was reduced in RARalpha null mice, which have lowered numbers of round spermatids in their testes. These results indicate the possibility that Tsp57 protein plays a role in the postmeiotic phase of germ cell differentiation. Tsp57 contains two putative nuclear localization signals: NLS1 and NLS2. Examination of the cellular localization showed that the green fluorescent protein-Tsp57 fusion protein localized to both cytoplasm and nucleus. After deletion of NLS1 but not NLS2, Tsp57 localized solely to the cytoplasm, indicating a role for NLS1 in the nuclear localization of Tsp57. The localization suggests a nuclear function for Tsp57. Pull-down analysis demonstrated that Tsp57 and RAP80 form a complex in intact cells.