| Laryngeal squamous cell carcinoma (LSCC) is one of common malignant neoplasms in head and neck cancer, accounting for 1%-8.4% of all malignancies. Recently, the morbidity and mortality of LSCC are increasing. Although a lot of studies have been done on etiology, pathology, diagnosis and treatment of LSCC, the understanding of the molecular changes of this disease is still poor. The inactivation of tumor suppressor genes and the amplification or overexpression of oncogenes have been implicated in the multistep progression of LSCC carcinogenesis. Frequent allelic loss was observed on chromosome 3p in laryngeal carcinoma and its premalignant lesion, indicating that inactivation of putative tumor suppressor gene on 3p may be involved in early steps of laryngeal carcinogenesis.In 1996, Ohta et al identified the FHIT (fragile histidine triad) gene by exon trapping experiment on 3p14.2, spanning the FRA3B common fragile site and the t (3;8) break point associated with hereditary renal cellcarcinoma. The 1.1 Kb FHIT cDNA is encoded by 10 small exons, and open reading frame (ORF) is from exon 5 to exon 9. The FHIT protein has been characterized as adenosine 5', 5"-P1, P4-triphosphate asymmetrical (Ap3A) hydrolase, which cleaves the Ap4A substrate, a molecule that might be involved in control of DNA replication and cell cycle, into ATP and AMP. It has been postulated that accumulation of Ap4A due to loss of FHIT function could lead to cell proliferation and subsequent tumor development. A lot of studies have indicated a role for the FHIT gene as a tumor suppressor. Introduction of the wild-type FHIT gene into cancer cells lacking endogenous FHIT protein suppressed tumorigenicity in nude mice. Highly frequent abnormal transcripts were found in a variety of human cancers including digestive tract, lung, breast, head and neck cancers and so on.At present, however, a few reports have examined abnormalities on FHIT gene in LSCC, and most of them have focused on microsatellite analyses to detect LOH (loss of heterozygosity) at 3pl4.2; Studies on FHIT gene structural alterations in LSCC were even seldom seen; There was one report which observed aberrant FHIT RT-PCR transcripts on LSCC, but no date was available on relationship between FHIT gene and clinicopathological features in LSCC; So far, there has been no report about homozygous deletion and methylation at DNA level and protein expression of FHIT gene in LSCC. In order to further investigate the role of FHIT gene in carcinogenesis and development of LSCC, in this study, we obtained 41 cases of LSCC and their matched normal squamous epithelium (NSE) and used many kinds of molecular biology techniques. Our study would offer a theoretical base to elucidate the significance ofFHIT gene in the progression of LSCC and offer the theoretical basis to the research on molecular mechanism of LSCC. This study was divided into two parts listed below:First part: Homozygous deletion, methylation, expression of FHIT gene and its clinical significance in laryngeal squamous cell carcinomaMethods:1. Exon-specific PCR amplification was used to observe the homozygous deletion of entire coding region of FHIT gene in 41 cases of LSCC and their matched NSE.2. PCR-based restriction enzyme assay technique was used to value the promoter methylation status of FHIT gene in tissues mentioned above.3. Expression of FHIT protein and PCNA (proliferating cell nuclear antigen, PCNA) in tissues mentioned above was examined by using immunohistochemical SP technique.4. The relationship between those aberrant and clinicopathological features of LSCC was analyzed at the same time.5. Statistical analysis: The SPSS Statistical Package program 10.0 was used for all analyses. Associations between the variables were tested by x2 test, Fisher's exact probability test, and Student's test. Value of less than 0.05 was deemed significant.Results:1. In NSE, entire coding exons of FHIT gene were all amplified successfully and there was no homozygous deletion. In LSCC...
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