Interspecies comparative genomic hybridization (I-CGH): a new twist to study animal tumor models.

Animal models of human diseases are widely used to address questions of tumor development. Selection of a particular animal model depends upon a variety of factors, among them: animal cost, species lifespan and hardiness; availability of biomolecular and genetic tools for that species; and evolutionary distance from humans. In spite of the growth in genomic data in the past several years, many animal models cannot yet be studied extensively due to gaps in genetic mapping, sequencing and functional analyses. Thus, alternative molecular genetic approaches are needed. We have designed an interspecies comparative genomic hybridization approach to analyze genetic changes in radiation-induced brain tumors in the non-human primate, Macaca mulatta. Using homologies between the primate and human genomes, we adapted widely-available CGH techniques to generate cytogenetic profiles of malignant gliomas in four monkey tumors. Losses and gains were projected onto the corresponding homologous chromosomal regions in the human genome, thus directly translating the status of the monkey gliomas into human gene content. This represents a novel method of comparative interspecies cytogenetic mapping that permits simultaneous analysis of genomic imbalances of unknown sequences in disparate species and correlation with potential or known human disease-related genes.

From tissue phenotype to proteotype: sensitive protein identification in microdissected tumor tissue.

Correlation of disease phenotype with protein profile (proteotype) is a significant challenge for biomedical research. The main obstacles have been the need to insure sufficient quantities of pure protein sample, the reproducibility of protein display, and rapid and accurate protein identification. We present a modified approach that combines enhanced detection sensitivity with tissue microdissection from frozen primary renal cancer tissues of different histological subtypes, followed by 2D gel analysis and protein identification with MALDI mass spectrometry. We obtained reliable and highly consistent results in phenotypically similar tumors of each individual subtype by performing strict morphological control of the analyzed tumor cells without physical or chemical alteration of the frozen tissue samples. By application of non-oxidizing silver staining, proteins were resolved and identified with high levels of specificity and sensitivity. This new combination of techniques allows not only for sensitive identification of specific protein patterns that correspond to a histological tumor phenotype, but also for identification of specific disease-associated protein targets.

Individual adult human neurons display aneuploidy: detection by fluorescence in situ hybridization and single neuron PCR.

Neurons, once committed, exit the cell cycle and undergo maturation that promote specialized activity and are believed to operate upon a stable genome. We used fluorescence in situ hybridization, selective cell microdissection, and loss of heterozygosity analysis to assess degree of aneuploidy in patients with a neurodegenerative disease and in normal controls. We found that aneuploidy occurs in approximately 40% of mature, adult human neurons in health or disease and may be a physiological mechanism that maintains neuronal fate and function; it does not appear to be an unstable state. The fact that neuronal stem cells can be identified in adult humans and that somatic mosaicism may be found in neuronal precursor cells deserves further investigation before using adult neural stem cells to treat human disease.

Coexpression of erythropoietin and its receptor in endolymphatic sac tumors.

OBJECT: Von Hippel-Lindau (VHL) disease is characterized by multiple tumors in specific organs. The cell of origin and the reason for the particular organ distribution of the tumors remains unknown. Endolymphatic sac tumor (ELST) is one of the lesions associated with VHL disease. Data from previous studies of VHL disease-associated hemangioblastomas (HBs) and renal cell carcinomas (RCCs) have indicated that VHL gene deficiency causes coexpression of erythropoietin (Epo) and its receptor (Epo-R), which facilitates tumor growth. METHODS: The authors studied ELSTs from five patients with VHL germline mutations. Analysis of the five ELST samples revealed loss of the wild-type allele, consistent with Knudson's two-hit hypothesis for tumorigenesis. All five ELST specimens were characterized microscopically and by immunohistochemical analysis. Coexpression of Epo and Epo-R was found in all five tumors on immunohistochemical studies and confirmed through reverse transcription-polymerase chain reaction and Western blot analysis. CONCLUSIONS: Expression of Epo appears to be a result of VHL gene deficiency, whereas the simultaneous coexpression of Epo-R may reflect a developmental mechanism of tumorigenesis. Coexpression of Epo and Epo-R in ELSTs together with the morphological and genetic similarities of these lesions with other VHL disease-associated tumors indicates that VHL disease-associated tumors in different organs share common pathogenetic pathways.

Proteins and protein pattern differences between glioma cell lines and glioblastoma multiforme.

INTRODUCTION: Research into the pathogenesis, molecular signaling, and treatment of glioblastoma multiforme (GBM) has traditionally been conducted using cell lines derived from malignant gliomas. We compared protein expression patterns between solid primary GBMs and GBM cell lines to identify proteins whose expression may be altered in cell culture. METHODS: We cultured cell lines U87, U118, U251, and A172 and used tissue-selective microdissection of eight primary GBMs to obtain pure populations of tumor cells, which we studied using two-dimensional gel electrophoresis (2DGE) and examined using differential expression software. Select protein targets expressed differentially between GBM tumors and GBM cell lines were sequenced using tandem mass spectrometry. RESULTS: Analysis of the primary GBM tumor samples (n = 8) and the GBM cell lines revealed reproducibly similar proteomic patterns for each group, which distinguished tumors from the cell lines. Gels contained up to 500 proteins that were consistently identified in the pH 4 to 7 range. Comparison of proteins identified in the GBM tumors and in the cell lines showed approximately 160 proteins that were gained and 60 proteins that were lost on culture. Using normalized intensity patterns from the 2DGE images, ANOVA tests were done and statistically significant spots were identified. Seven proteins found in the cell lines were significantly increased when compared with the GBM tumors (P < 0.05), whereas 10 proteins were significantly decreased from cell lines compared with the GBM tumors. Proteins identified included transcription factors, tumor suppressor genes, cytoskeletal proteins, and cellular metabolic proteins. CONCLUSION: Global protein and proteomic differences were identified between primary GBM tumor samples and GBM cell lines. The proteins identified by 2DGE analysis elucidate some of the selection pressures of in vitro culture, help accentuate the advantages and limitations of cell culture, and may aid comprehension of gliomagenesis and enhance development of new therapeutics.

Combined proteomic approach with SELDI-TOF-MS and peptide mass fingerprinting identified the rapid increase of monomeric transthyretin in rat cerebrospinal fluid after transient focal cerebral ischemia.

Several proteins are known to be markedly expressed in the brain during cerebral ischemia, however the change in protein profiles within the cerebrospinal fluid (CSF) after an ischemic insult has not been fully elucidated. We studied the changes in the CSF proteome in rat transient middle cerebral artery occlusion model. Surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) was used to detect the time-course changes in CSF protein patterns after transient focal brain ischemia. According to hierarchical cluster analysis by self-organising tree algorism (SOTA), the temporal pattern of protein peaks was divided into four groups: acute increase group, chronic increase group, gradual decrease group and unchanged group. In the acute increase group, the expression of a 13.6-kDa protein was markedly increased during the acute phase. The 13.6-kDa protein was identified as monomeric form of transthyretin using two-dimensional electrophoresis and peptide mass fingerprinting based on matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The monomeric transthyretin may represent an ischemia-specific CSF marker to indicate the sequential changes according to ischemic insults of the brain.

Identification of pleiotrophin in conditioned medium secreted from neural stem cells by SELDI-TOF and SELDI-tandem mass spectrometry.

Neural stem cells (NSCs) are multipotential progenitor cells that have self-renewal activity. Since the fates of the NSCs in situ depend on their niche containing growth factors and cytokines, we performed surface enhanced laser desorption/ionization time-of flight mass spectrometry (SELDI-TOF-MS) to screen for differentially secreted proteins in conditioned medium of neural stem cells and compared with that of NIH3T3 cells. A 15.3-kDa protein detected only in the conditioned medium of neural stem cells was determined as pleiotrophin (PTN) by SELDI-TOF-MS and ProteinChip-tandem MS systems. Identification of pleiotrophin was further confirmed by one-dimensional SDS gel electrophoresis and Edman degradation analysis. The mRNA transcripts of PTN and its receptors [receptor protein tyrosine phosphatase (RPTP) beta/zeta, N-syndecan and anaplastic lymphoma kinase (ALK)] were detected in neurosphere, suggesting that pleiotrophin signaling systems are present in the neural stem cells and are involved in the modulation of fate of neural stem cells.

Protein patterns and proteins that identify subtypes of glioblastoma multiforme.

Glioblastoma multiforme (GBM) has been subdivided into two types based on clinical and genetic findings: primary tumors, which arise de novo, and secondary tumors, which progress from lower grade gliomas to GBMs. To analyse this dichotomy at the protein level, we employed selective tissue microdissection to obtain pure populations of tumor cells, which we studied using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing of select target proteins. Protein patterns were analysed in a blinded manner from the clinical and genetic data. 2-DGE clearly identified two distinct populations of tumors. 2-DGE was reproducible and reliable, as multiple samples analysed from the same patient gave identical results. In addition, we isolated and sequenced 11 proteins that were uniquely expressed in either the primary or the secondary GBMs, but not both. We demonstrate that specific proteomic patterns can be reproducibly identified by two-dimensional gel electrophoresis from limited numbers of selectively procured, microdissected tumor cells and that two patterns of GBMs, primary versus secondary, previously distinguished by clinical and genetic differences, can be recognized at the protein level. Proteins that are expressed distinctively may have important implications for the diagnosis, prognosis, and treatment of patients with GBM.

Biologic characterization of a secondary glioblastoma with extracranial progression and systemic metastasis.

Glioblastomas rarely metastasize outside the CNS. We biologically characterized a case of secondary glioblastoma associated with extracranial progression and distant metastasis. A 42-year-old male patient was subjected to craniotomy for a left temporal tumor (astrocytoma grade II) and subsequently underwent another 3 craniotomies due to tumor recurrences. At the third craniotomy, extracranial progression was noted, and the tumor was classified as a glioblastoma. In order to pinpoint the genes expressed differentially in the intracranial primary tumor and the metastatic tumors, we used cDNA microarray. The patterns of gene expression in these 2 samples were highly similar, suggesting that the mechanism of metastasis was direct infiltration of tumor cells into extracranial blood vessels. Insulin-like growth factor binding protein-2 was overexpressed in both primary and metastatic tumors. Immunohistochemical studies of DNA-dependent protein kinase, which participates in the repair of DNA, was strongly positive in the samples obtained at the first and second operations, but the positive rates were markedly reduced in the specimens obtained at the third and fourth operations. These results suggest that insulin-like growth factor binding protein-2 and deficiency of DNA-dependent protein kinase proteins promoted tumor progression in the present case.