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六、研究計畫內容：
子計畫(I)：神經纖維瘤的基因表現和染色體丟失之分析
Neurofibromatosis type 1 and 2 (NF1 and NF2) are dominantly inherited tumor predisposition syndromes that target the peripheral and central nervous systems, respectively. Two known genes, NF1 and NF2, which respond for neurofibromatosis have been isolated a decade ago. Both NF1 and NF2 genes have been function as classic tumor suppressor genes (negative growth regulators), such that their loss of expression predisposes to tumor formation. In the case of NF1, we now appreciate the critical role of ras regulation in cell proliferation. The protein of NF2, termed merlin or schwannomin, are thought to play a role in linking cytoskeletal components to cell membrane glycoproteins. Over the past few years, we have learned tremendous amount about the function of the NF1 and NF2 gene products. However, the pathways by which an inherited NF gene lesion lead to the formation of heterogeneous neurofibromas and through which these benign tumors become malignant, are still not known.
Neurofibroma is not a homogeneous disease entity. The clinicopathological features such as size, type of growth pattern, and degree of dysplasia all have been considered to be indicative of future aggressive behaviour. Different clinical phenotypes of neurofibroma tumors may have a different genetic pathway of carcinogenesis from the benign-malignant sequence. Identification of genes differentially expressed in normal and disease tissues through the use of a large-scale, cDNA microarray technology may lead to increased understanding of pathways that lead to tumor formation. Therefore, in this study, we plan to explore our attention to identify the essential genes in different subtypes of neurofibroma cancer using a combined approach. We believe that this study will add a new dimension for the complicate cancer research and hopefully provide the basis for future development of new diagnosis and therapy.
The specific aim of this study will include (1) neurofibroma tumor tissues collection and detection of gene expression profile using cDNA array in different subtypes of colorectal tumors, (2) analyzing the difference of gene expression with clinicopathological features
Patients and tumor specimens:NF1 patients who satisfied the National Institutes of Health diagnostic criteria (Stumpf et al., 1988) and who had developed either benign or malignant tumors were recruited. The protocol will be approved by the institutional review board, and all participants will provide informed consent. We plan to set up a DNA bank and establish the basic genetic information for Taiwanese neurofibroma patients by means of collecting DNA samples from all the patients and their family members if possible from both blood and tumor samples. All tissue samples will be quickly frozen in liquid nitrogen within 1 h after surgical resection in the operation theater and stored at –80 ° C freezer before processing for mutation analysis. In addition, DNA samples from 100 healthy individuals of Chinese origin will be screened to determine whether the mutations identified in this study were present in a control population. A total of 30 neurofibroma tumors will be analyzed in this study. The detailed morphological description, histopathological data, and clinical follow-up will be recorded for each case.
Nucleic acid preparation from human subjects:Genomic DNA will be prepared from all the samples using a standard SDS-proteinase K method (Ausubel et al. 1989). For each patient, DNA will be extract from paired samples of normal tissue or blood as a control. For the RNA preparation, tissue specimens will be crushed in liquid nitrogen, and processed by the guanidinium-acid-phenol method (Chomczynski & Sacchi, 1987). Total RNA will be prepared using Trizol reagent (Life Techenologies, Inc.) or using the RNeasy total RNA purification kit (Qiagen).
Molecular cytogenetic determination of NF1 and NF2 microdeletion:Metaphase FISH will be performed using selected DNA probes as described recently (Tinschert et al., 2000). Interphase nuclei will be prepared from peripheral blood samples inEDTA stored at -80oC. Briefly, the blood will be thawed and approximately 100 μl will be washed twice in 1 ml Red Cell Lysis Buffer (10 mM Tris-HCl, 5 mM MgCl2, 10 mM NaCl). The cell pellet will be resolved in 1X PBS and an Appropriate aliquot of the solution will be subjected to cytocentrifugation. Probe preparation and labeling, as well as pretreatment, denaturation, hybridization, and washing will follow recently described protocols (Martin-Scbero et al., 2002).
Microarray experiments (Microarray preparation, probe construction, microarray hybridization):
Total RNA will be prepared from frozen specimens using Trizol reagent (Invitrogen) and further purified by RNeasy kit (Qiagen). Purity and RNA integrity will be evaluated by absorbance at 260nm and 280 nm, by agarose gel electrophoresis. High quality RNAs with A260/280 ratio over 1.9 and intact ribosomal 18S and 28S RNA bands will be utilized for the microarray experiments.
The GMRCL Human 7K set, Version 1 cDNA array slides containing 7334 genes ( 100% sequence-verified cDNA library procured from InCyte were made by Genome Medical Center at Chang Gung Memorial Hospital All the clones were spotted in duplicated onto the arrays. 10μg of total RNA from various tissues will be used for first-strand cDNA probes synthesized by incorporation of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia) during reverse transcription of purified mRNA with SuperScript II RT (Life Technologies, Inc.). RNA will be removed by heating the sample to 94 oC for 3 min followed by treatment with NaOH (0.25N final) for 10min at 37 oC. The sample will then be neutralized by addition of an equal amount of HCl, followed by 1 M Tris (pH 7.5) to 0.2 M. Unincorporated nucleotides will be removed using glass-fiber filtration plates (Millipore).
Hybridization will be done in pairs, with total RNA from tumor and normal tissue of the same patient being prepared at the same time under identical conditions. Fluorecently labeled cDNA probes will be first concentrated by drying and resuspended in 20ul of 1X hybridization solusion (5X SSC, 5X Denhardt's solution, 1% SDS, 50% formamide, 0.1ug/ml salmon sperm DNA). After denaturation at 75-80 oC, the hybridization mixture will be deposited onto an arrayed slide under a coverslip. The arrayed cDNA clones will be hybridized overnight (16-20 hr) at 65 oC in a humidified chamber. After hybridization, the slides will be scanned with a Dual-channel Fluorecent Detection System and the data analyzed using Axon GenePix computer software.
Imaging and data Analysis of cDNA expression in neurofibroma tumors:To minimize the effects of measurement variations introduced by artificial sources during experiments, we will only include the spots that had significant signals in both channels (signal: background ratio > 1.5). Spots representing housekeeping genes will be used to normalize the entire slide so that all slide could be compared directly. Before the clustering algorithm was applied, the fluorescence ratio for each spot was first log transformed, and then the data for each sample was centered to remove experimental biases. To obtain reproducible clusters, we selected only genes that passed a filter protocol designed to exclude: (a) genes where both Cy3 and Cy5 signal intensities were lower than the cutoff value; (b) genes where values were obtained in < 50% of the samples tested; or (c) genes with SDs in observed values of < 2. Excel, Cluster and TreeView software packages will be used for subsequent analysis.
Real Time quantitative RT-PCR:As a proof-of-principle and to verify our microarray data, we will select 10 genes and examine their expression levels in a panel of tumors, cell lines and normal samples by means of real-time quantitative PCR (TaqMan real-time PCR machine, ABI 7700 Sequence Detector, Applied Biosystems). Each single-stranded cDNA will be reverse-transcribed from total RNA and diluted for subsequent PCR amplification. The PCR reaction will be normalized to the ribosomal protein L11 (RPL11) level. Each PCR will be carried out in a 25ul volume reaction containing 12.5ul TaqMan Universal MasterMix (Applied Biosystems), 0.25ul of each forward and reverse primers (10 uM), 2.0ul of dual-labeled probe (2.5uM), and cDNA from 50ng total RNA. Each reaction will be amplified by 40 cycles of 95 oC for 15 sec and 60 oC for 1 min. The primers and probes of selected genes or sequences used for real-time quantitative PCR will be obtained from GeneBank and placed into primer design software (PrimerExpress version 1.5, Applied Biosystems).
Reference:
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Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thio-cyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. |
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Martin-Subero, J. I., Harder, L., Gesk, S., Schegelberger, B., Grote, W., Martinez-Climent, J. A., Dyer, M. J., Novo, F. J., Calasanz, M. J., Siebert, R. (2002) Interphase FISH assays for the detect ion of translocations with breakpoints in immunoglobulin light chain loci. Int. J. Cancer 98: 470-474. |
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Tinschert, S., Naumann, I., Stegmann, E., Buske, A., Kaufmann, D., Thiel, G., Jenne, D. E. (2000) Segmental neurofibromatosis is caused by somatic mutation of the neurofibromatosis type I (NF1) gene. Eur. J. Hum. Genet. 8: 455-459. |
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Warren, C., James, L. A., Ramsden, R. T., Wallace, A., Baser, M. E., Varley, J. M., Evans, D. G. (2003) Identification of recurrent regions of chromosome loss and gain in vestibular schwannomas using comparative genomic hybridization. J. Med. Genet. 40: 802-806. |
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