Study On The Abnormality Of FHIT Gene And LOH Of Gene MS On Chromosome 3p And Expression Of FHIT, Survivin Protein In Bladder TCC

The bladder tumor, the most common cancer of urogenital system in China, affects males four times as frequently as females, and most commonly occurs in adults between the age of 50 and 70. The incidence of bladder tumor worldwide has been increasing with population senescence year after year, Threating people's life and health seriously. 95% of the bladder tumor is the epidermis tumor. The transitional cell carcinoma(TCC) accounts for 90%. Others are squamous cell carcinoma, accounting for2-3% and adenocarcinoma, accounting for 2-3%.The non-epidermis tumor has been rarely found. It shows high recurrence rate and multiple characteristic.nearly 1/3 of bladder tumors show multiple.A number of risky factors has been evalutated: (1) Contacting with some carcinogens for a long time, such as dye, spinning and weaving, leather, rubber, paint and so on; (2) smoking; (3) urinary bladder chronic infection and stimulation concern;(4) long time intake of analgesics,like phenacetin.But little is known about the etiology of bladder tumor. With the rapid development of molecular biology technology and molecular genitics deep study for tumor, early diagnosis and biology therapy to bladder tumor has became possible.Loss of heterozygosity (LOH) and genetics abnormality have frequently been detected at the region of 3 chromosome short arms for many tumors.LOH for bladder tumor shows a frequent molecular event here. Research in 1979 showed that t(3:8) breakpoint was located at 3p, then FRA3B was found to be located at 3p too.There are three tumor suppressor gene,FHIT,hMLH and VHL being located separately at 3p14-cen, 3p21-22 and 3p25-26 region.FHIT (fragile histindine triad) gene, a human gene that has been cloned by Ohta in exon trapping in 1996, located at 3p14-cen, is composed of ten exons. Abnormality of FHIT gene, as common LOH and methylation, rarely mutation ,was detected at many tumors and tumor cell lines.The function of FHIT product is associated with the following aspects:(1) AP3A hydrolase activity.(2) microtubule protein.(3) cell cycle control and apoptosis.(4) stress ability for extemal environment.Up to now, tumorigenesis mechanism of FHIT gene still is not clear. There are three hypothesises to explain it as follows: (1) PKCI (protein kinase C inhibitor) hypothesis.(2) AP3A hydrolase-cell proliferaton and ragulation hypothesis.(3) ApnA-FHIT polymers-apotosis hypothesis.Survivin gene, one of 8 inhibitor of apoptosis proteins (IAPs), cloned by Altieri in 1997, is located at 17q25, is composed of three introns and four exons with 142 coding nucleotides, Survivin protein is one of the most power founction in IAPs.The exfoliated urothelial cells can reflect genetic character of the bladder TCC. Urine detection is a simple and nonwound means. There are many urine detection methods to be used to diagnoze the bladder TCC, cancer-associated antigen, telomerase activity, plasma tumor marker,flow cytometric,and so on. But the specificity and sensitivity reported are various.Microsatellite(MA) DNA, short tandem repeat sequences, 5% in human genome,are found in all prokaryotic and eukaryotic genomes. Abnormality of microsatellite, performance as MSI and LOH, can be detected in many kinds of tumors.It can be used as tumor marker in clinical diagnosis for tumor.Method1. LOH and mutation of exon3,4,5,8 linked with FHIT gene were detected with PCR-SSCP stain method in tissue DNA from 10 cases of the normal urinary bladder tissues and 49 cases of the bladder.2.Ethylation of FHIT gene was detected by Methy-specific PCR in 10 cases of the normal urinary bladder tissues and 49 cases of the bladder TCC tissues.3. The expressions of FHIT gene and survivin gene were studied by S-P immunohistochemisty in 10 cases of the normal urinary bladder tissues and 49 cases of the bladder TCC tissues.4. Four microsatellite markers:D3S1234,D3S1300 linked with FHIT gene,D3S1561 linkedwith hMLH1 gene and D3S1038 linked with VHL gene were detected with PCR-PAGE stain method in both the urine segment DNA and tumor tissue DNA from 10 cases of the normal urinary bladder and 49 cases of the bladder TCC.Results1. 21 cases out of 49 caces of bladder TCC tissues have one or more LOH of exons in FHIT gene, The LOH frequencies of exons detected in the bladder TCC tissues from the 49 cases were 42.9% (21/49), LOH of exon 3 was 8.2% (4/49), LOH of exon 4 was10.2% (5/49), LOH of exon 5 was 32.7% (16/49), LOH of exon 8 was 0(0/49). There was not mutation of FHIT gene in the bladder TCC .The frequency of methylation detected in the bladder TCC tissues from the 49 cases was 16.3% (8/49). There were not LOH ,mutation and methylation in the normal bladder tissues.2. All the normal urinary bladder tissues showed positive staining pattern of FHIT protein, negative staining pattern of survivin protein. Positive rate of FHIT protein in 49 cases of the bladder TCC tissues is 46.9%(23/49). The difference of positive rate between Grade I and Grade II,III cases was significant. The difference of positive rate between Tis~T1 cases and T2~T4 cases showed a trend of decreasing, but there was no statistically significant difference; Positive rate of survivin in 49 cases of the bladder TCC tissues is 57.1%(28/49). The difference of positive rate between Grade I and GradeII,III cases was significant, the difference of positive rate between Tis~T1 cases and T2~T4 cases was significant, too. The expression of FHIT protein was reversely correlated to survivin protein.3. LOH frequency of D3S1234,D3S1300 that was linked with FHIT gene was found In informative 49 cases as 50%(17/34), 40%(12/30) in tissue and 47.1%(16/34), 40%(12/30) in urine segment, respectively. LOH frequency of D3S1561 that was linked with hMLH1 gene was found in informative 49 cases as 40%(8/20) in tissue and urine segment. LOH frequency of D3S1038 that was linked with VHL gene was found In informative 49 cases as 35.5%(11/31) in tissue, 32.3%(10/31) in urine segment, respectively. The LOH of D3S1234,D3S1300 were not correlated to the LOH of D3S1561 and D3S1038. The sensitivity and the specifity of the way using LOH of MS in urine segment for detection of bladder TCC were 75.5% (37/49) and 100%, positive rate of regular urine cytology is 16.3(8/49). Conclusion1. Here were frequent LOH and methylation of FHIT gene in bladder TCC. LOH was mainly alteration of FHIT gene.The LOH was mainly found at exon 5. There was not LOH of exon 8.2. FHIT protein and survivin protein may play an important role in bladder TCC. FHIT and Survivin gene may be a late molecular even to the bladder TCC. Expression of FHIT protein and survivin protein may be used as prognostic marker for the bladder TCC. Expression of FHIT protein correlated significantly to survivin protein. FHIT protein may suppress the development of the bladder TCC by the way of apoptosis.3. There were high frequent LOH of MS at FHIT gene, VHL gene and hMLH1 gene in the bladder TCC. LOH of MS in FHIT gene, VHL gene and hMLH1 gene may be independent events in the bladder TCC. The analysis of LOH of the MS linked with FHIT gene, hMLH1 gene and VHL gene in urine segment was a sensitive and specific tool in detection of bladder TCC.