Identification of unstable sequences within the common fragile site at 3p14.2: implications for the mechanism of deletions within fragile histidine triad gene/common fragile site at 3p14.2 in tumors

The FRA3B, at 3p14.2, lies within the fragile histidine triad (FHIT) gene and is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin. Common fragile sites are highly unstable regions of the genome. Large intragenic deletions within FHIT, localized within the FRA3B sequences, have been identified in a variety of tumor cells. To characterize the FRA3B deletions in tumor cells and identify FRA3B sequences that are required for fragile site induction, we used microcell-mediated chromosome transfer to isolate hybrid cell clones that retain chromosome 3 homologues with various deletions within FRA3B. Detailed molecular mapping of the FHIT/FRA3B locus in the resultant hybrid cells revealed a complex pattern of instability within FRA3B. Each tumor cell line contained multiple chromosome 3 homologues with variable deletion patterns, often with discontinuous deletions, suggesting that the process of breakage and repair within FRA3B is an ongoing one. By comparing the approximate location of the breakpoints in the hybrid clones, we identified 11 recurring breakpoint/repair regions within the FRA3B. A comparison of the frequency of breaks/gaps within FRA3B in the hybrid clones with various deletions of FRA3B sequences revealed that the loss of FRA3B sequences does not reduce the overall rate of breakage and instability within the remaining FRA3B sequences. The majority of breaks occurred in the proximal portion of the FRA3B, in a 300-kb interval between exon 4 and the proximal 50 kb of intron 5. Our observations suggest that there is no single sequence within the FRA3B that influences breakage or recombination within this region; however, we cannot rule out the presence of multiple "hot spots" within the FHIT/FRA3B locus. Together, the results suggest that factors other than the DNA sequence per se are responsible for the formation of DNA breaks/gaps.     

Peutz-Jeghers综合征脆性组氨酸三连体基因突变与癌变的关系

背景与目的：Peutz-Jeghers综合征（Peutz-Jeghers syndrome,PJS）是一种常染色体显性遗传性疾病。脆性组氨酸三连体(fragile histidine triad,FHIT)基因是定位于3p14.2脆性位点区域的重要抑癌基因。我们以往的研究结果认为PJS患者在3p14.2区域可能存在易感基因。本文旨在明确PJS患者FHIT基因突变与癌变的关系和FHIT基因的改变及规律。方法：应用变性高效液相色谱（DHPLC）分析、聚合酶链反应-单链构象多肽分析技术（PCR-SSCP）和DNA测序方法,对6个PJS家系中的15个患者及其20例正常家族成员的FHIT基因进行了研究。结果：在6个家系中,有1位患者的FHIT基因第159位核苷酸由G→T,相应的第54位密码子编码的氨基酸由谷氨酸变成终止密码子;在第62位密码子处存在碱基G插入,使读码框架发生移位,在第111位密码子处提前出现终止密码子;在2例癌变患者的息肉标本DNA和癌标本DNA中检测到FHIT基因第8外显子的纯合性缺失;在3个散发的PJS患者中发现,患者与其母亲在第8外显子,有相同的SSCP带型和DHPLC峰型,DNA测序结果显示,在第98位密码子处发生同义突变,由CAT→CAC,相应编码的氨基酸未发生改变,均为苏氨酸。另外,有7例患者和2例正常人在FHIT基因的第6内含子5′端第42位核苷酸位点发生由A→G的点突变。结论：FHIT基因在PJS患者中存在点突变,突变频率较低,在癌组织中存在缺失,这提示FHIT基因突变可能参与了PJS发病过程,而FHIT基因缺失可能与癌变有关。     

乳腺癌中Fhit蛋白的低表达与转移的关系

背景与目的：脆性三联组氨酸（Fragile histidine triad,FHIT)基因为一种候选的肿瘤抑制基因,横跨大多数人类基因组的共同脆性位点FRA3B,FHIT基因的改变见于多种类型特别是上皮起源的人类类型肿瘤,并与已知的致癌物质作用有关,本研究目的为探讨乳腺癌中脆性三联组氨酸FHIT基因蛋白Fhit表达状况及其与临床病理指标的关系。方法：采用兔抗人Fhit蛋白抗体和枸椽酸－微波－链酶卵白素－过氧化物酶（Streptavidin-HRP,SP）免疫组化方法检测66例福尔马林固定,石蜡包埋的乳腺癌,其中6例为导管内癌,60例为浸润性导管癌的标本中Fhit表达状况并分析其与癌组织侵犯及淋巴结转移的关系。结果：癌组织Fhit表达强度较癌旁非癌乳腺组织低者为38例（57．6％）,其中22例为阴性（22．0％）,较癌旁非癌乳腺组织高者为18例（27．3％）,与癌旁非癌乳腺相等者为10例（15．2％）,伴局部或腋下淋巴结转移的乳腺浸润性导管癌Fhit蛋白表达低者阳性率为83．3％（20／24）,无淋巴结转移的浸润性导管癌为50．0％（18／36）,导管内癌为0／6,已伴淋巴结转移癌组83．3％（20／24）与未转移癌组42．9％（18／42）比较,差异有显著性（P＜0．01）,浸润性导管癌中已伴淋巴结转移癌组与未转移癌组比较,差异有非常显著性（P＜0．01）,结论：乳腺癌Fhit表达可能与癌组织侵犯及淋巴结转移相关,提示Fhit低表达可能对乳腺癌的演化和进展具有重要作用并可能成为一个新的预后指标。     

FHIT基因在乳腺癌中的表达及意义

目的 探讨FHIT基因在乳腺癌中的表达与临床病理分期的关系。方法 应用免疫组织化学S．P法对99例乳腺癌组织及其癌旁组织标本（其中临床病理分期：0期15例,Ⅰ期9例,Ⅱ期52例,Ⅲ期15例,Ⅳ期3例）对FHIT蛋白表达水平检测,观察FHIT基因在癌旁组织中的低水平表达或缺失情况的意义。结果 0期乳腺癌FHIT蛋白低表达率26．67％（4／15）,与Ⅰ期乳腺癌FHIT蛋白的低表达率55．56％（5／9）比较,差异无显著性（四格表精确检验法P=0．132〈1．963,P〉0．05）;Ⅰ期乳腺癌FHIT蛋白的低表达率55．56％（5／9）与Ⅱ期乳腺癌FHIT蛋白的低表达率66．67％（38／57）比较,差异无显著性（x^2=0．075〈x^2 0．05（1）=3．841,P〉0．05）;Ⅱ期乳腺癌FHIT蛋白的低表达率66．67％（38／57）与0期乳腺癌FHIT蛋白的低表达率26．67％（4／15）比较,有非常显著性差异（x^2=7．817〉x^2 0．01（1）=6．635,P〈0．01）。Ⅱ期乳腺癌FHIT蛋白的低表达率66．67％（38／57）与Ⅲ-Ⅳ期乳腺癌FHIT蛋白的低表达率94．44％（17／18）比较,差异有显著性（x^2=4．071〉x^2 0．05（1）=3．841,P〉0．05））;0-Ⅰ期乳腺癌FHIT蛋白的低表达率37．5％（9／24）与Ⅱ期以上乳腺癌FHIT蛋白的低表达率73．33％（55／75）比较,有非常显著性差异（x^2=10．215〉x^2 0．01（1）=6．635,P〈0．01）。结论 FHIT抑癌基因表达强度的下降与乳腺癌的临床病理分期有关。     

Loss of fragile histidine triad expression in colorectal carcinomas and premalignant lesions

Abnormal expression of the fragile histidine triad (FHIT) candidate tumor suppressor gene has been observed in a variety of human tumors, but little is known about its expression during colorectal tumorigenesis. Sections of 70 aberrant crypt foci (ACF), 55 adenomas, 84 primary colorectal carcinomas, and 13 metastatic lesions were evaluated immunohistochemically for Fhit expression. All normal colonic epithelium showed a strong expression of Fhit; 44% of carcinomas showed a marked loss or absence of Fhit expression. The proportion of carcinomas with reduced expression showed an increasing trend (a) with decreasing differentiation and (b) in tumors with metastases (62%) compared with tumors without metastases (38%). The proportion of metastatic lesions (12 of 13) with reduced expression of Fhit was even greater. Although only a small proportion of ACF and adenomas showed a reduction of Fhit expression, the reduced expression of Fhit was strongly associated with the degree of dysplasia in both ACF (P = 0.0002) and adenomas (P = 0.0085). The findings of reduced expression of Fhit in a small proportion of colonic precancerous lesions and in increased proportions of primary and metastatic colorectal cancers suggest that Fhit plays a role in the development and progression of some colon carcinomas.     

Aberrant crypt focus and fragile histidine triad protein in sporadic colorectal carcinoma

AIM:To characterize aberrant crypt focus (ACF) in adjoining mucosa in sporadic colorectal carcinoma and to evaluate fragile histidine triad (Fhit) protein and Ki67. METHODS:ACF was identified grossly and classified histologically in 75 resected specimens. ACF was typed into hyperplastic ACF (HACF) and dysplastic ACF (DACF). Sections of ACF, carcinoma and normal colonic mucosa as control were studied for Fhit and Ki67 expressions by immunohistochemistry and were grouped according to staining intensity and the number of positive stained cells observed in different histological groups. Comparison was done between the different groups by Pearson’s χ 2 test and γ test for the ordinal data. P value < 0.05 was considered as significant.RESULTS:Age range was 40 to 86 years in males (mean = 43.36) and 45 to 70 years in females (mean = 56). HACF was identified in all cases studied in the non-tumorous colonic mucosa; ACF was observed as non-contiguous scattered foci, which supports the hypothesis of acquisition of single focus monoclonality by colonic epithelial cells in tumor generation. Twenty-four (32%) had DACF and were observed as closure to carcinoma foci. Intensity of Fhit expression:(1) HACF 40% exhibited strong intensity, similar to normal, moderate in 36% and weak in 24%; (2) DACF strong in 25%, moderate in 37.5% and weak in 37.5%; and (3) carcinoma negative in 16%, strong in 43% and moderate and weak in 28.5% each. Significant difference was observed in intensity of the Fhit protein expressions by HACF and DACF (P < 0.05). Tumor in older patients showed a stronger Fhit intensity compared to younger patients (P = 0.036). Vegetarian diet intake and nonsmokers showed stronger Fhit intensities. Advanced stage tumor, non-vegetarian diet and younger age was associated with loss of Fhit protein. Ki67 positivity was an extended crypt pattern in HACF and DACF showed extension up to the neck region of the crypts and surface epithelium. Carcinomas showed a marked increase in Ki67 expression (P < 0.05). Fhit protein had an inverse association with Ki67 expression. CONCLUSION:Weaker Fhit intensity was associated with smoking, non-vegetarian diet intake and increasing Ki67 expression. Loss of Fhit protein expression is possibly influenced by environmental factors like smoking and non-vegetarian diet intake.     

前列腺癌组织FHIT和PSA表达的初步研究

目的:探讨脆性组氨酸三联体(fragile histidine triad, FHIT)与前列腺特异性抗原(prostate specific antigen, PSA)在前列腺癌组织中的表达及意义.方法:选取不同分级的前列腺癌组织(64例)和前列腺增生组织(32例),采用免疫组织化学SP法染色,检测FHIT及PSA的表达情况.结果:64例前列腺癌组织中病理分级(按Gleason分级系统)分化良好癌、中等分化癌和分化不良癌组织中FHIT的蛋白表达率分别为84.2%(16/19)、45.4%(10/22)和17.4%(4/23).随着Gleason评分增加,FHIT蛋白表达率显著下降,P<0.05.病理分级中分化良好癌、中等分化癌和分化不良癌PSA的蛋白表达率分别为100%(19/19)、68.2%(15/22)和34.8%(8/23),随着Gleason评分增加,PSA蛋白表达率显著下降,P<0.05.结论:FHIT的表达与前列腺癌的Gleason评分负相关;PSA的表达与Gleason评分负相关;FHIT和PSA的表达呈正相关.     

EXPRESSION OF FRAGILE HISTIDINE TRIAD AND P53 IN NON-SMALL CELL LUNG CANCER

Objective： To investigate the expression offragile histidine triad （FHIT） and p53 protein in non-small cell lung cancer （NSCLC） and explore the relationship between their expressions and the clinicopathological features. Methods： FHIT protein and p53 protein were detected by immunohistochemistry in 76 cases of NSCLCs and matched normal lung tissues. Results： Fifty-one cases （67.1%） showed negative expression of FHIT （apparent reduction or loss） and thirty-seven cases （48.7%） showed p53 positive expression （overexpression）. The difference was significant （P=0.04）. However, there was no significant difference in FHIT expression between the p53-positive group and the p53-negative group （64.9% versus 69.2%, P=0.686）. The negative rate of FHIT protein expression was higher in squamous cell carcinoma than in adenocarcinoma, in moderately and poorly differentiated carcinoma than in well-differentiated carcinoma, and in cases with smoking history than in cases without smoking history （P〈0.05）. There was no relationship between FHIT expression and clinical stage or lymph node metastasis. The negative FHIT expression was not an independent predictor of overall survival （P=0.338）. Conclusion： The frequency of negative expression of FHIT protein is higher than that of positive expression of p53 in NSCLCs. The negative expression of FHIT is independent of the expression of p53. The change of expression of FHIT may play a role in the smoking related lung tumorigenesis while it may have no relationship with the progress of NSCLC or prognosis of the patients.     

The fragile histidine triad gene: a molecular link between cigarette smoking and cervical cancer.

PURPOSE: Smoking is an epidemiologic risk factor for cervical cancer. The fragile histidine triad (FHIT) gene is a tumor suppressor gene that is altered in 80% of tobacco-associated lung cancers. We hypothesized that reduced FHIT protein expression, homozygous deletions (HD) or hemizygous deletions (HemiD) and microsatellite alterations (MA) at the FHIT/FRA3B locus occur more commonly in cervical cancers of smokers than nonsmokers. EXPERIMENTAL DESIGN: Archival tissues of 58 patients with stage IA1 to IB2 squamous cell carcinoma of the cervix were identified. FHIT protein expression was studied with immunohistochemistry. Laser capture microdissection was used to isolate tumor and normal DNA. HD/HemiD of FHIT exons 4 and 5 were analyzed by monoplex real-time PCR. MA at FHIT/FRA3B were studied with multiplex nested PCR with three fluorescently labeled microsatellite markers (D3S1300, D3S1312, and D3S1480). RESULTS: Eighteen of 26 tumors from smokers (69%) and 13 of 32 nonsmokers (41%; P < 0.05) showed loss of FHIT protein expression. Thirty-seven stage IB tumors yielded sufficient DNA for analyses. HD or HemiD of both exons tested occurred in 8 of 17 smokers (47%) and 2 of 20 nonsmokers (10%; P < 0.05). MA at more than two sites were found in 11 of 17 tumors of smokers (65%) and 6 of 20 nonsmokers (30%; P < 0.05). Mean composite genomic FHIT alteration scores were significantly higher for tumors of smokers versus nonsmokers (0.67 versus 0.40; P < 0.02). CONCLUSION: Loss of FHIT expression, HD, HemiD, and MA at the FHIT/FRA3B locus occur significantly more commonly in cervical cancers of smokers. These findings suggest that the tumor suppressor gene FHIT may represent a molecular target in cigarette smoking-associated cervical carcinogenesis.     

Cancer and the FRA3B/FHIT fragile locus: it's a HIT

The FHIT gene encompassing the most active common human chromosomal fragile region, FRA3B, was discovered in 1996 and proposed as a tumour suppressor gene for important human cancers. Seven years and more than 350 reports later, early questions concerning its tumour suppressor role have been answered. Recent studies on the role of Fhit loss in major types of human cancers report association with high proliferative and low apoptotic indices, node positivity, loss of mismatch repair protein, likelihood of progression and reduced survival.     

Large common fragile site genes and cancer

The common fragile sites are large regions of genomic instability that are found in all individuals and are hot spots for chromosomal rearrangements and deletions. A number of the common fragile sites have been found to span genes that are encoded by very large genomic regions. Two of these genes, FHIT and WWOX, have already been demonstrated to function as tumor suppressors. In this review we will discuss the large common fragile site genes that have been identified to date, and the role that these genes appear to play both in cellular responses to stress and in the development of cancer.     

结直肠癌中FHIT蛋白的异常表达及其临床意义

目的探讨结直肠癌中脆性组氨酸三联体(FH IT)基因蛋白表达状况与临床病理指标的关系。方法采用半定量免疫印迹(W estern b lot)检测30例结直肠癌及其癌旁正常组织(≥5 cm)FH IT蛋白表达状况。结果30例癌组织有56.7%的FH IT蛋白表达异常,FH IT蛋白表达量与患者的年龄、性别、肿瘤部位、肿瘤大小及组织学类型无关(P>0.05)而与肿瘤的组织分化程度、浸润深度、Dukes分期和淋巴结转移有关(P<0.05)。在浸润深度越深、分化程度越低、Dukes分期越晚和有淋巴结转移的癌组织中,FH IT蛋白低表达越明显。结论FH IT蛋白表达状况可能与结直肠癌组织学分级、浸润程度、Dukes分期及淋巴结转移相关,提示FH IT蛋白表达降低可能对结直肠癌发生、发展具有重要作用。     

FHIT基因在肺癌中的缺失与HPV感染的研究

目的　探讨肺癌发生的分子生物学机制。方法　采用逆转录 -巢式聚合酶链反应 (reverse tapepolymerasechainreaction)的方法对 42例肺癌及 10例正常肺组织中的FHIT(Fragilehistidinetriad)基因的缺失情况进行检测 ,并用PCR技术检测了肺癌组织中人乳头状瘤病毒 (humanpapillomavirus ,HPV)的DNA片段。 结果　 66.7% (2 8/4 2 )肺癌组织中检测到FHIT基因的缺失 ,而正常组织中未检测到FHIT基因的缺失 ,二者差别有显著意义 (P <0 .0 1)。 42例肺癌组织中有 8例检测到HPV的片段 ,阳性率为 19% (8/4 2 ) ,正常组织中未检测到HPV的片段 ,且在 8例HPV阳性的标本中均有FHIT基因缺失。结论　 (1)FHIT基因的缺失在肺癌的发生中起一定的作用。 (2 )FHIT基因的缺失与肺癌的不同病理分型、分化程度、临床分期无关。与吸烟有一定的相关性。 (3 )HPV的感染与肺癌的发生有一定关系 ,且与FHIT基因缺失有正相关关系。     

FRA3B and other common fragile sites: the weakest links

In 1979, the first chromosome alteration associated with familial cancer was reported. Five years later, a fragile site was observed in the same chromosome region. The product of the fragile histidine triad (FHIT) gene, which encompasses this fragile site, is partially or entirely lost in most human cancers, indicating that it has a tumour-suppressor function. Inactivation of only one FHIT allele compromises this suppressor function, indicating that a 'one-hit' mechanism of tumorigenesis is operative. Are genes disrupted at other fragile sites? And, are these genes also tumour suppressors?