RESUMO
Analysis of single-cell gene expression promises a more precise understanding of human disease pathogenesis and important diagnostic applications. Here, we review the rationale for the study of gene expression at the single-cell level, practical methods to isolate homogeneous or single-cell samples, and current approaches to the analysis of single-cell gene expression. Finally, we highlight applications of laser microdissection-based gene expression analysis to the study of human disease and clinical diagnosis.
Assuntos
Células/metabolismo , Perfilação da Expressão Gênica/métodos , Animais , Separação Celular/métodos , Dissecação , Humanos , Lasers , Técnicas de Diagnóstico Molecular , Sensibilidade e EspecificidadeRESUMO
Human disease is governed by a complex array of cellular populations and sub-population. Gene expression profiling is proving an important means of understanding and classifying pathophysiologic processes by identifying genes, gene pathways and pathway networks not previously known to be associated with particular diseases. However, disease-associated gene expression can be obscured by surrounding 'normal' tissue. Laser capture microdissection allows gene expression analysis of pooled single cells, cell subpopulations and cell populations. Analysis of laser capture microdissection-procured cells will allow a better understanding of the cellular components of disease.
Assuntos
Expressão Gênica , Lasers , Técnicas de Diagnóstico Molecular , Análise de Sequência com Séries de Oligonucleotídeos , DNA Complementar/metabolismo , Dissecação , Biblioteca Gênica , Marcação In Situ das Extremidades Cortadas , Modelos Genéticos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA/metabolismoRESUMO
Intraosseous squamous cell carcinomas of the mandible arise de novo or secondary to a tumor or transformed cyst epithelium. Current diagnostic tests frequently fail to distinguish between these tumors, leading to confusing classification schemes. We report the functional genomic analysis of a mandibular odontogenic carcinoma. Malignant keratinocytes from the lesion were isolated using laser capture microdissection. Target sample generated from the total RNA of the LCM-procured cells was used to hybridize high-density oligonucleotide arrays. Functional genomic analysis of the odontogenic carcinoma database compared with four oral mucosal squamous cell carcinoma gene expression databases was performed. Preliminary results suggest a small subset of genes distinguish this odontogenic carcinoma from oral mucosal epidermoid carcinomas.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Mandibulares/genética , Tumores Odontogênicos/genética , Carcinoma de Células Escamosas/diagnóstico , Bases de Dados Genéticas , Diagnóstico Diferencial , Regulação Neoplásica da Expressão Gênica , Genoma , Humanos , Neoplasias Mandibulares/diagnóstico , Tumores Odontogênicos/diagnóstico , RNA Neoplásico/genéticaRESUMO
Disruption of the homeostatic balance between proliferation and apoptosis is widely believed to contribute to human oral carcinogenesis. Using the Syrian hamster oral cancer model, we examined normal, hyperplastic, dysplastic and malignant oral epithelium for the fraction of apoptotic, proliferating and p12(doc-1) expressing keratinocytes using the TUNEL assay, as well as PCNA and p12(doc-1) immunostaining, respectively. The percentage of TUNEL positive cells progressively increased from normal to dysplastic epithelium (P<0.0019), but returned to normal keratinocyte levels in the malignant epithelium (P<0.20). However, PCNA positive cells increased progressively through hamster oral malignant progression (P<0.0012). The overall ratio of apoptotic to proliferating keratinocytes remains similar until the transition between dysplastic and malignant epithelium, where the ratio is markedly reduced (P<0.05). p12(doc-1) labeling demonstrated a similar expression pattern (P<0.008). This study demonstrates that apoptosis, proliferation and the expression of p12(doc-1) reflects alterations reported during human oral carcinogenesis and supports the use of the Syrian hamster model for the further examination of these pathways.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/patologia , Animais , Apoptose , Carcinoma de Células Escamosas/metabolismo , Bochecha , Cricetinae , Epitélio/metabolismo , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas , Mesocricetus , Índice Mitótico , Modelos Animais , Neoplasias Bucais/metabolismo , Lesões Pré-Cancerosas/metabolismo , Proteínas Supressoras de Tumor/análiseRESUMO
Genome-wide and high-throughput functional genomic tools offer the potential of identifying disease-associated genes and dissecting disease regulatory patterns. There is a need for a set of systematic bioinformatic tools that handles efficiently a large number of variables for extracting biological meaning from experimental outputs. We present well-characterized statistical tools to discover genes that are differentially expressed between malignant oral epithelial and normal tissues in microarray experiments and to construct a robust classifier using the identified discriminatory genes. Those tools include Wilks' lambda score, error rate estimated from leave-one out cross-validation (LOOCV) and Fisher Discriminant Analysis (FDA). High Density DNA microarrays and Real Time Quantitative PCR were employed for the generation and validation of the transcription profile of the oral cancer and normal samples. We identified 45 genes that are strongly correlated with malignancy. Of the 45 genes identified, six have been previously implicated in the disease, and two are uncharacterized clones.
Assuntos
Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença , Neoplasias Bucais/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regulação Neoplásica da Expressão Gênica , Genoma , Humanos , Mucosa Bucal/patologia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatística como Assunto , Transcrição GênicaRESUMO
BACKGROUND: The Human Genome Project, or HGP, has inspired a great deal of exciting biology recently by enabling the development of new technologies that will be essential for understanding the different types of abnormalities in diseases related to the oral cavity. LITERATURE REVIEWED: The authors review current literature pertaining to the advanced microarray technologies arising from the HGP and how they can contribute to dentistry. This technology has become a standard tool for monitoring activities of genes at both academic and pharmaceutical research institutions. RESULTS: With the availability of the DNA sequences for the entire human genome, attention now is focused on understanding various diseases at the genome level. Deciphering the molecular behavior of genetically encoded proteins is crucial to obtaining a more comprehensive picture of disease processes. Important progress has been made using microarrays, which have been shown to be effective in identifying gene expression patterns and variations that correlate with cellular development, physiology and function. Arrays can be used to classify tissue samples accurately based on molecular profiles and to select candidate genes related to a number of cancers, including oral cancer. This type of oral genetic approach will aid in the understanding of disease progression, thus improving diagnosis and treatment for patients. CLINICAL IMPLICATIONS: Microarrays hold much promise for the analysis of diseases in the oral cavity. As the technology evolves, dentists may see these tools as screening tests for better managing patients' dental care.
Assuntos
Doenças da Boca/genética , Análise de Sequência com Séries de Oligonucleotídeos , Doenças Dentárias/genética , Antibacterianos/uso terapêutico , Progressão da Doença , Perfilação da Expressão Gênica , Variação Genética/genética , Genótipo , Projeto Genoma Humano , Humanos , Neoplasias Bucais/genética , Análise de Sequência com Séries de Oligonucleotídeos/classificação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Lesões Pré-Cancerosas/genética , Proteínas/genética , RNA Mensageiro/genéticaAssuntos
Micromanipulação/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Perfilação da Expressão Gênica/métodos , Humanos , Lasers , Neoplasias/genética , Neoplasias/patologia , RNA Complementar/genética , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Information determining cellular structure and function is contained in chromosomal DNA. Genes, regions of DNA encoding this information, are composed of specific sequences of nucleotides. DNA sequencing methods have been developed to identify these sequences. Even subtle alteration (or mutation) of these sequences can lead to many human syndromes and diseases. This article reviews 1) the structure of the nucleotide, 2) the methods of DNA sequencing, and 3) its recent clinical application in analysis of the nevoid basal cell carcinoma syndrome.
Assuntos
Análise de Sequência de DNA , Síndrome do Nevo Basocelular/genética , Composição de Bases/genética , Pareamento de Bases/genética , Sequência de Bases/genética , Códon/genética , DNA/genética , Doença , Neoplasias de Cabeça e Pescoço/genética , Humanos , Proteínas de Membrana/genética , Mutação/genética , Cistos Odontogênicos/genética , Receptores Patched , Receptores de Superfície Celular , SíndromeRESUMO
Chromosomal DNA transfers and stores information regarding the structure and function of the cell. Genetic information, encoded within sequences of nucleotides that compose DNA, is grouped into functional units called genes. Genetic diseases are caused by changes in the chromosomal DNA, leading to a change in the quantity or function of the protein gene product. In the past, genetic diagnosis was limited by the availability of sufficient quantity and quality of DNA and the absence of an efficient amplification procedure. The polymerase chain reaction (PCR), an inexpensive, rapid, and accurate means of amplifying DNA, is already making a major contribution to the diagnostic sciences. PCR techniques have been widely used in diverse applications, including molecular analysis of microbial pathogens, inheritable diseases and syndromes, and neoplasms. The purpose of this article is to 1) Review gene structure and function, 2) review principles of PCR technology and its applications in molecular biology, and 3) discuss an experimental clinical application of PCR to identify novel infectious agents responsible for odontogenic infections.