ABSTRACT
BACKGROUND: Transcriptional profiling of prostate cancer (PC) has unveiled new markers of neoplasia and allowed insights into mechanisms underlying this disease. Genomewide analyses have also identified new chromosomal abnormalities associated with PC. The combination of both classes of data for the same sample cohort might provide better criteria for identifying relevant factors involved in neoplasia. Here we describe transcriptional signatures identifying distinct normal and tumoral prostate tissue compartments, and the inference and demonstration of a new, highly recurrent copy number gain on chromosome 17q25.3. METHODS: We have applied transcriptional profiling to tumoral and non-tumoral prostate samples with relatively homogeneous epithelial representations as well as pure stromal tissue from peripheral prostate and cultured cell lines, followed by quantitative RT-PCR validations and immunohistochemical analysis. In addition, we have performed in silico colocalization analysis of co-regulated genes and validation by fluorescent in situ hybridization (FISH). RESULTS: The transcriptomic analysis has allowed us to identify signatures corresponding to non-tumoral luminal and tumoral epithelium, basal epithelial cells, and prostate stromal tissue. In addition, in silico analysis of co-regulated expression of physically linked genes has allowed us to predict the occurrence of a copy number gain at chromosomal region 17q25.3. This computational inference was validated by fluorescent in situ hybridization, which showed gains in this region in over 65% of primary and metastatic tumoral samples. CONCLUSION: Our approach permits to directly link gene copy number variations with transcript co-regulation in association with neoplastic states. Therefore, transcriptomic studies of carefully selected samples can unveil new diagnostic markers and transcriptional signatures highly specific of PC, and lead to the discovery of novel genomic abnormalities that may provide additional insights into the causes and mechanisms of prostate cancer.
Subject(s)
Adenocarcinoma/genetics , Chromosome Aberrations , Chromosomes, Human, Pair 17 , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/genetics , Cell Line, Tumor , Gene Dosage , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Nucleic Acid Hybridization , Prostatic Neoplasms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, GeneticABSTRACT
The introduction of high-throughput techniques is increasingly providing abundant information on molecular alterations requiring validation at the posttranscriptional level. Protein expression is now efficiently evaluated in large series of tumors included in tissue microarrays. We propose, describe and validate a technique to elaborate paraffin-embedded cell line microarrays (PECLIMA) from fixed cell cultures, which can be processed like standard surgical pathology biopsies prior to immunophenotyping. Our results show a reliable protein immunoexpression profiling in six widely used cell lines under different fixation conditions. This technique permits the simultaneous analysis of multiple antigens in multiple cell lines under different experimental conditions. Additional features of these arrays are long-term storage, their suitability for a variety of techniques including immunocytochemistry and in situ hybridization and their low cost.
Subject(s)
Antigens, Neoplasm/analysis , Cell Line, Tumor/metabolism , Histocytological Preparation Techniques/methods , Paraffin Embedding , Protein Array Analysis/methods , Antigens, Neoplasm/immunology , Biomarkers, Tumor/metabolism , Humans , Immunohistochemistry , In Situ Hybridization , PhenotypeABSTRACT
The CHK2 gene codifies for a serine/threonine kinase that plays a central role in DNA damage response pathways. To determine the potential role of CHK2 alterations in the pathogenesis of lymphoid neoplasms we have examined the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid samples. A heterozygous Ile157Thr substitution, also present in the germ line of the patient, was detected in a blastoid mantle cell lymphoma (MCL). CHK2 protein and mRNA expression levels were similar in all types of lymphomas and reactive samples, and these levels were independent of the proliferative activity of the tumors. However, 5 tumors, one typical MCL, 2 blastoid MCLs, and 2 large cell lymphomas, showed marked loss of protein expression, including 2 samples with complete absence of CHK2 protein. These 2 lymphomas showed the highest number of chromosomal imbalances detected by comparative genomic hybridization in the whole series of cases. However, no mutations, deletions, or hypermethylation of the promoter region were identified in any of these tumors. mRNA levels were similar in cases with low and normal protein expression, suggesting a posttranscriptional regulation of the protein in these tumors. CHK2 gene and protein alterations were not related to p53 and ATM gene status. In conclusion, CHK2 alterations are uncommon in malignant lymphomas but occur in a subset of aggressive tumors independently of p53 or ATM alterations. The high number of chromosomal imbalances in tumors with complete absence of CHK2 protein suggests a role of this gene in chromosomal instability in human lymphomas.
