| BACKGOUDNasopharyngeal carcinoma (NPC) is one of the aggressive malignant head and neck squamous cell cancers (HNSC) with a high incidence in Southern China and Southeast Asia. Although Epstein-Barr virus (EBV),genetic predisposition and certain chemical carcinogens are considered to be closely associated with this cancer,relatively little is known about the molecular mechanisms involved in the intiation and progression of the disease. Noteworthily,the fact that 5-10 percent of NPC patients have a family history suggests that genetic susceptibility might play an important role in the pathogenesis of NPC. In this way,we can say NPC is a hereditary susceptible disease involving multi-genetic inheritance.Recently,cytogenetic abnormalities,especially consistent,non-random and significant loss of heterozygosity (LOH) at 3p in NPC has been detected by using comparative genomic hybridization (CGH) and genome-wide scan with microsatellite allelotyping. In particular,LOH studies have indicated multiple regions of involvement,including 3pl4-21,3p21.3-22 and 3p25-26. Functional evidence,via mono-chromosome transfer,suggested that theposition of NPC related tumor suppressor gene was contained within 18 centimorgan on the regions of chromosome 3p21.3 bounded by D3S1298 to D3S1295,and centered around D3S1568.Moreover,another functional evidence,via mono-chromosome transfer,suggested that the activity of telomerase can be strongly repressed by the experimental introduction of a normal copy of human chromosome 3p21.3-22 region bounded by D3S1298 to D3S1295,and that repression is tightly associated with the induction of permanent growth arrest. It is known that LOH on the regions of chromosome 3p21-22 is one of the most frequent genetic alterative in multiple human cancers,including renal cancer,lung cancer,head and neck squamous cell carcinoma,cervical carcinoma and breast cancer. As a result,it suggests that this region may harbor multiple tumor suppressor genes which may play a critical role in the pathogenesis of cancers.PURPOSE AND METHODSWe are focused on isolation of a new tumor suppressor gene on the regions of chromosome 3p21-22 by bioinformatic analysis and molecular biology technology) as well as,application of in silico cloning combined with positional cloning strategy. Meanwhile,the expression of two known candidate tumor suppressor genes (HASSFIA and DLECl),which were also located on chromosome 3p21-22,were detected in NPC. In a word,We may clarify the critical role of the unknown and known putative tumor suppressor genes which are located on chromosome 3p21-22 in the carcinogenesis of NPC and identify useful markers in the early diagnosis and clinical therapy of NPCRESULTSThe main results and findings are as follows:1. Analysis of differential expression of 63 ESTs located on chromosome 3p21-22We searched the Homo sapien ESTs database using D3S1609 and D3S1295 as the query and obtained 63 single copy of Unigene ESTs clusters which mapped at D3S1609-D3S1295. We assembled these ESTs clusters and obtained a long or complete cDNA consensus by in silico cloning strategy. These Unigene consensus were studied by means of semi-quantitative RT-PCR in 32 cases of low-differentiated squamous cell carcinoma from patients with primary NPC,16 cases of histologically normal nasopharyngeal epithelia,as well as,4 NPC cell lines (CNE1 > CNE2 and HNE1,HNE2). We found 49 ESTs expressed equally in both NPC and normal nasopharyngeal epithelia,8 ESTs expressed neither in NPC nor in normal nasopharyngeal epithelia,2 ESTs expressed higher in NPC than normal nasopharyngeal epithelia,4 ESTs expressed lower or undetectable in NPC than in normal nasopharyngeal epithelia. Besides,the expression of Hs.27566 gene was undetected in all NPC cell lines,and it was significantly reduced or undetected in NPC biopsies in comparison with normal nasopharyngeal mucosae fcKO.OOl).2. To study the down-regulated Hs.27566 gene by analysing the loss of heterozygosity,expression in multiple human tissues and loca...
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