Identification of the overexpressed genes in 12q13.3 Amplicon of Nasopharyngeal Carcinoma

Autor: Kai-Hsi Lu, 盧凱熙
Rok vydání: 2009
Druh dokumentu: 學位論文 ; thesis
Popis: 98
Nasopharyngeal carcinoma (NPC) is an endemic cancer with a distinct racial and geographical distribution. It is particularly prevalent in southern China and southeastern Asia. Like other cancers, nasopharyngeal cancer show several genomic changes involving chromosomal gain and loss. Most of the results on genomic changes in NPC cells have been obtained from traditional methods such as G-banding, microsatellite marker analysis, and comparative genomic hybridization (CGH). Because of limitations of detecting resolution, these methods cannot provide enough information for further analysis. Traditional methods often involve large DNA fragments, which usually contain more than one gene. To solve this problem, we adopted the following strategy to delineate the minimal regions of deletion or amplification in NPC. We first used high-resolution array-based CGH (array CGH) to investigate the genomic abnormalities in NPC. We then combined real-time quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry (IHC) to evaluate the expression of individual genes. Previous literature has shown that high-level amplification of genomic fragments points toward the existence of one or more relevant, dosage-dependent proto-oncogene genes. By performing amplicon mapping and parallel analyses of DNA copy number, mRNA, and protein expression levels, we can identify candidate genes that may be important mediators in tumor formation or progression. Taken together, the combination of copy number analysis and gene expression profiling provides an improved strategy for oncogene discovery in NPC. Clinical samples of primary NPC at different clinical stages and 6 NPC cell lines, namely, CNE1, CNE2, HK1, HONE1, TW01, and TW06, were used as the study materials. To elucidate the regions of gain or amplification, array CGH was applied to characterize the common amplicons in NPC cell lines and clinical samples. In 10 clinical samples and 5 NPC cell lines, except HK1, we found that frequent gains were on chromosomes 12q13 (80%), 22q11.2 (70%), 11ptel (50%), 11q13 (50%), 2p12 (40%), 7q11.23 (40%), 8q24 (40%), 11p11.2 (40%), 17q23.2-25 (40%), 19p13.2 (40%), 20q13.2 (40%), and 22q11.23 (40%). The chromosomes frequently lost were 3p14.2 (70%), 9p21.3 (90%), 9p21 (60%), 5q21-22 (50%), 3q26.3 (50%), 3p24.3 (40%), 8p21.3-22 (40%), 15q11-13 (40%), 18q21.3 (40%), 22qtel (40%), and Xp22.3 (40%). These results were similar to our previous CGH findings. High incidences of gains were identified on chromosome 12q and 22q. On the basis of these results, the amplicon regions were further confirmed by fluorescence in situ hybridization (FISH) analysis in all 6 NPC cell lines. The smallest regions of gain at 12q13 were further confirmed in 6 NPC cell lines by using FISH with 3 BAC clones, namely, RP11-479A23, RP11-91I15, and RP11-165F12. The results of FISH analysis indicated that the copy number of 12q13 was higher than that of 12q14, by about 1–2 copies in all NPC cell lines, except HK1. We further used Q-PCR to detect the change in copy numbers of individual genes located at the 12q13 to 12q14 adjacent regions. The results showed that the CDK2 gene, located at the boundary of the amplicon, had high copy number changes. Q-PCR also revealed high expression level of the CDK2 gene in 5 NPC cell lines. However, other genes near the CDK2 gene did not show the same expression pattern. Finally, we determined the CDK2 protein expression level in another 22 NPC samples of different clinical stages by IHC. The results showed that the CDK2 expression level was higher in early stages than in late stages. CDK2 expression levels at the T1 and N0 stages were also higher than those at the T2–T4 and N1–N3 stages. Our results provided a picture of multiple genetic lesions in NPC and gave hints for further identification of critical tumor oncogenes involved in NPC tumorigenesis. We can speculate that the expression pattern of genes located in the amplicon may offer possible tumor markers for diagnosis or prognosis of primary NPC. Furthermore, we characterized the most commonly detected amplicon in NPC and found that CDK2 might be a putative oncogene involved in the tumorigenesis of NPC.
Databáze: Networked Digital Library of Theses & Dissertations
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