Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas.

BACKGROUND: Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes. Loss of FHIT function may be important in the development and/or progression of various types of cancer. MATERIALS AND METHODS: We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC). Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, [3H]-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections. RESULTS: The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation. Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues. The observed changes increased with progression of these lesions. We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci. We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC). The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months. FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant. Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05). CONCLUSIONS: Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation. Aberrant FHIT gene could be a prognostic marker in lung cancer.     

人类抑癌基因beclin 1在胃癌和直结肠癌中表达下调的研究

人类抑癌基因beclin 1通过自噬作用调节细胞生长,但在胃癌和直结肠癌中其表达水平和调控机制仍不清楚．通过检测胃癌和直结肠肿瘤组织中beclin 1基因的表达水平,及DNA异常甲基化和杂合子缺失对其表达的影响,发现与癌旁组织相比,35％的胃癌标本和30％的直结肠癌标本中beclin 1基因表达显著下调．同时发现,beclin 1基因5’端存在一高密度CpG岛,在胃癌和直结肠癌中beclin 1的启动子区域和第二个内含子区域存在甲基化,而杂合子缺失仅在胃癌中发生．这些发现表明beclin 1基因的异常甲基化和杂合子缺失对其在胃癌和直结肠癌中的表达起调控作用．     

细胞角蛋白基因13在喉鳞状细胞癌中缺失和表达的研究

为了探讨细胞角蛋白基因13（Cytokeratin13,CK13)在喉癌发生中的作用,在CK13基因内部及附近选择5个微卫星引物进行杂合性丢失（loss of heterozygosity,LOH)分析,于DNA水平间接检测72例喉鳞状细胞癌患者中该基因的缺失,应用Northern Blot检测16例喉鳞癌患者的配对肿瘤及癌旁正常组织中CK13基因的表达差异;同时应用CK13蛋白单克隆抗体对不同分化程度的喉鳞癌组织进行免疫组化染色。结果表明：5个STR位点均存在LOH,其中D17S1964E、D17S2092、D17S791、D17S1665及D17S808位点的LOH频率分别为18．03％、28．13％、27．42％、39．68％和34．85％,其中D17S1665位点的LOH频率最高,至少一个位点出现LOH的病例高达77．78％（56／72）,杂合性丢失与临床分期、淋巴结转移无显著相关,但与肿瘤分化程度高低相关（P＜0．05）。Northern blot结果表明：16例喉鳞癌患者C13基因在正常组织中的表达比肿瘤组织显著增强,免疫组化结果也显示CK13蛋白在正常组织或高分化肿瘤中的表达明显高于低分化者,且存在显著差异（P＜0．01）。证实CK13基因可能在喉鳞状细胞癌的发生中具有重要作用,可能是一个新的抑癌基因。     

Does loss of heterozygosity in critical genome regions predict a local relapse in patients after laryngectomy?

BACKGROUND: Patients, who had an upper aerodigestive tract malignancy, have a high incidence of succeeding tumor development. This has been attributed to the role of "field cancerization" in carcinogenesis. The aim of this study was analysis of loss of heterozygosity (LOH) in the regions frequently lost during the course of head and neck squamous cell carcinomas (HNSCC), especially at early stages, which could answer the clinicians' question, if LOH analysis has any "predictive" value in relation to tumor occurrence. MATERIAL AND METHODS: Sixty-five larynx cancer patients were examined for loss of heterozygosity on 3p, 7q, 8p, 9p and 18q chromosomal arms with the use of 12 microsatellite markers. The material from a single patient consisted of blood, tumor, safe margin and one or two clinically unchanged mucosal samples. During follow up, the material from brush specimens (14 patients) as well as laryngeal swabs (4 patients) was also examined. RESULTS: The highest frequency of LOH was detected for marker D3S1234 in tumor tissues (29%). Analysis of margin samples (b) revealed low LOH frequencies (2-5%) and complete retention of heterozygosity for markers: D3S1234, D7S486, D8S261, D8S264, D9S171 and D18S46. Similarly, for normal appearing mucosa from upper part of larynx (c) frequencies of LOH were low (2-6%), with the complete retention of heterozygosity for markers: D3S1284, D3S1304, D3S1234, D8S264 and D9S1870. We did not detect any LOH in the material of normal appearing mucosa from tracheostoma region (d). During follow up, LOH was detected for eight markers, with the highest incidence for markers D18S46 (six cases), D7S486 (four cases) and D3S1300 (three cases). CONCLUSIONS: The data, obtained during this investigation, did not reveal the predictive value of LOH with respect to local relapse occurrence in laryngeal cancer patients. However, time of follow up did not reach 5 years, so that further clinical monitoring should be conducted.     

Molecular genetic alterations of FHIT and p53 genes in benign and malignant thyroid gland lesions

Several oncogenes and tumor-suppressor genes are involved either as early or late event in thyroid gland carcinogenesis. Human FHIT (fragile histidine triad) gene is highly conserved gene whose loss of function may be important in the development and/or progression of various types of cancer. We undertook this study to analyze FHIT and p53 gene status in different benignant and malignant thyroid tumors. Status of these genes as well as intensity of apoptosis was analyzed in tumor tissues by molecular genetic methods, immunohistochemistry, and FACS-scan analysis. The majority of the malignant thyroid cancers displayed aberrant expression of FHIT gene, concominant with p53 gene inactivation. This is followed by low rate of apoptosis, which may be important in the development and/or progression of thyroid cancer. We found higher incidence of p53 mutation and aberrant processing of FHIT mRNA in malignant tumors (papillary, follicular, medullary and anaplastic carcinomas) and in those tumors with distant metastasis. The growth of p53(-)/FHIT(-) follicular carcinoma of human origin was much faster in nude mice than p53(+)/FHIT(+) follicular carcinoma, and mice had shorter survival rate. Our results show a correlation between aberrant FHIT and p53 expression, low rate of apoptosis, and malignancy. Concomitant aberration of FHIT gene and p53 could be responsible for development of highly malignant types of thyroid cancer and may be considered as a prognostic marker for these tumors.     

SEMA3B基因在鼻咽癌组织中的表达、杂合性丢失和甲基化分析

SEMA3B基因定位于鼻咽癌高频缺失区域3p21.3上,最近被证明具有抑瘤基因的功能.分析了鼻咽癌组织中SEMA3B基因的表达、杂合性丢失(LOH)和甲基化情况.首先应用逆转录-聚合酶链式反应(RT-PCR)方法检测了33例鼻咽癌组织和15例慢性鼻咽炎组织中SEMA3B基因的表达,结果显示75.8%(25/33)鼻咽癌组织中SEMA3B基因表达缺失或下调,显著低于慢性鼻咽炎组织中的表达(P=0.001).进一步选取3个微卫星位点D3S1568、D3S1621和D3S4597分析了20例鼻咽癌组织中SEMA3B基因LOH的情况,结果表明3个位点的丢失率分别为10%、20%和15%,总的丢失率为45%,统计分析发现LOH与基因表达之间存在明显相关(P=0.023).最后,采用甲基化特异性PCR方法分析了SEMA3B基因启动子区甲基化,结果发现在100%的鼻咽癌组织和73.3%的慢性鼻咽炎组织中检测到SEMA3B基因启动子区高甲基化.由此得出结论,SEMA3B基因在鼻咽癌组织中表达缺失或下调,LOH是引起其表达异常的原因之一.     

中国人胃癌染色体17q21区域基因遗传不稳定性研究

To investigate microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396, D17S579 and D17S855, and their effect on the expression of nm23H1 and BRCA1 of gastric cancer, which would provide experimental basis for clinical treatment and prognosis analysis of gastric cancer. DNA was extracted from paraffin-embedded materials. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analyze MSI and LOH. Expression of nm23H, and BRCA1 was detected by Envision immuno-histochemistry and Leica-Qwin computer imaging techniques. In the forty cases of gastric cancer, the frequency of MSI, LOH and nm23H1 protein were 20.00%, 17.50% and 55.00% respectively at locus D17S396, while at locus D17S579, the frequency of MSI, LOH and BRCA1 protein were 22.50%, 15.00% and 37.50% respectively; at locus D17S855, the frequency of MSI, LOH and BRCA1 of thirty-seven cases were 18.92%, 18.92%, 37.84% respectively. In tumor node metastasis (TNM) staging, at locus D17S396, D17S579 and D17S855, MSI in stages I + II appeared more frequently than that in stages III + IV, while LOH appeared the contrary tendency. In the group of metastasis of gastric cancer, MSI had a less frequency (5.00%) than that with no metastasis (35.00%, P < 0.05) at locus D17S396, but LOH appeared more frequently (30.00%) than that with no metastasis (5.00%, P < 0.05). At locus D17S579, MSI had an increasing tendency with the degree of tumor differentiation (50.00% in high differentiation cases, 20.00% in middle differentiation cases, and 0% in low differentiation cases, P < 0.05). The frequency of nm23H1 and BRCA1 protein in stages TNM I + II was higher than that in stages TNM III + IV; and that in higher differentiation cases was higher than in poor differentiation cases. The frequency of nm23H1 protein in the group of metastasis (30.00%) was less than that with no metastasis significantly (80.00%, P<0.01). The frequency of nm23H1 protein in the group positive to MSI (87.50%) was higher than that in the group negative to MSI (46.88%, P < 0.05). However, nm23H1 protein in group positive to LOH (14.29%) was lower than that in the group negative to LOH (63.64%, P < 0.05). The frequency of BRCA1 protein in the group positive to MSI (66.67%) was more than that in the group negative to MSI (29.03%, P < 0.05). The results of experiments indicate that MSI and LOH may separately control the development of sporadic colon cancer with different pathways. MSI may be an early period molecule marker for sporadic colon cancer, enhanced expression of nm23H1 protein can effectively inhibit colon cancer metastasis and improve prognosis of sporadic colon cancer patients. By comparison, LOH mostly arises in the late period of sporadic colon cancer and endows a high aggressive and poor prognostic phenotype. nm23H1 protein could effectively restrain gastric cancer metastasis and development; and BRCA1 protein could restain tumor from becoming lower differentiation.     

HPV感染、FHIT蛋白表达与非小细胞肺癌的相关性研究

目的探讨人乳头瘤病毒感染在NSCLC发生中的病因学意义,同时分析FHIT蛋白表达与NSCLC发生及HPV感染的关系.方法采用PCR方法选用通用型引物和HPV16、18型特异性引物分别对42例NSCLC及14例肺良性病变组织进行检测.免疫组化SP法检测FHIT蛋白的表达水平.结果 HPV DNA检出率肺癌组为42.9%,肺良性病变组为7.1%,二者有显著性差异(P<0.05).HPV感染率在肺鳞癌(53.6%)显著高于腺癌(21.4%),且随着分化程度降低,其感染率增高,在吸烟患者(57.7%)显著高于不吸烟患者(18.8%);FHIT蛋白异常表达率肺癌组为61.9%,与肺良性病变组(28.6%)比较有显著性差异(P<0.05).肺癌中HPV阳性组FHIT蛋白异常表达率为83.3%,明显高于HPV阴性组(45.8%,P<0.05).结论 HPV感染与NSCLC组织学类型、分化程度及吸烟有关,它可能是导致NSCLC发生的重要病因学因素之一;FHIT蛋白异常表达与NSCLC发生及HPV感染有关,HPV可能通过诱导FHIT表达异常参与NSCLC的发生发展过程.     

Helicobacter pylori Infection and Family History of Gastric Cancer Decrease Expression of FHIT Tumor Suppressor Gene in Gastric Mucosa of Dyspeptic Patients

Background:  The expression of a fragile histidine triad (FHIT) protein is lost in stomach tumors. The study aimed at determining whether FHIT expression is affected by Helicobacter pylori infection, strain virulence ( vacA and cagA genes) and histopathological changes in the gastric mucosa of patients with functional dyspepsia having first-degree relatives with gastric cancer.
Materials and Methods:  Eighty-eight never-smoking patients with functional dyspepsia were selected for the study, and 48 of them had first-degree relatives with gastric cancer. Bacterial DNA amplification was used to identify H. pylori colonization. The level of FHIT gene expression was determined by qRT-PCR (mRNA) and Western blot (FHIT protein) analyses.
Results:  For patients having first-degree relatives with gastric cancer FHIT expression was lower (mRNA by ca. 40–45% and protein by 30%) compared with the control patients ( p  < .05). H. pylori infection decreased the FHIT mRNA level by 10–35% and the protein level by 10–20%. Bacterial strain vacA (+) cagA (+) lowered FHIT mRNA by ca. 30–35% in the antrum samples of both groups and in corpus samples of patients with first-degree relatives with gastric cancer ( p  < .05). The FHIT mRNA level was twice as high in control H. pylori- negative patients with intestinal metaplasia, compared with those with non-atrophic gastritis.
Conclusions:  The decreased FHIT gene expression associated with hereditary factors and with H. pylori infection, especially with vacA (+) cagA (+)-positive strains, may be related to gastric carcinoma development.     

A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production.

We have isolated a naturally arising human immunodeficiency type 1 (HIV-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominant production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced messages contributed to reduced virus replication as a result of a reduction in wild-type Rev protein. The point mutation within exon 6D activated exon 6D inclusion when the exon and its flanking splice sites were transferred to a heterologous minigene. Introduction of the point mutation into an otherwise wild-type HIV-1 proviral clone resulted in virus that was severely inhibited for replication in T cells and displayed elevated usage of exon 6D. Exon 6D contains a bipartite element similar to that seen in tat exon 3 of HIV-1, consisting of a potential exon splicing silencer (ESS) juxtaposed to a purine-rich sequence similar to known exon splicing enhancers. In the absence of a flanking 5' splice site, the point mutation within the exon 6D ESS-like element strongly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing. We propose, therefore, that exon silencers may be a common element in the HIV-1 genome used to create balanced splicing of multiple products from a single precursor RNA.     

Isolation and characterization of new microsatellites at the nm23-H1 and nm23-H2 gene loci and application for loss of heterozygosity (LOH) analysis

 The nm23-H1 gene has been suggested to be a metastasis suppressor gene. Studies about the events of loss of heterozygosity (LOH) at the nm23 locus and its correlation to metastasis are controversially discussed. To optimize detection of LOH at the nm23 locus, we screened two P1 clones for additional microsatellites. Tumor and normal DNA from 37 colorectal, 16 gastric, and 8 germ cancer patients were examined for LOH. We found two new CA repeats, one 5′ to nm23-H1 and another 3′ to nm23-H2. Using these nm23 locus-specific CA repeats and five other chromosome 17 loci (D17S1522, D17S1566, D17S855, D17S515, and TP53), allele loss was observed in 4/32 (12.5%) patients with colon cancer, 2/14 (14.3%) with gastric cancer, and 1/7 (14%) with germ cancer. No isolated LOH of the nm23 region was observed. Received: 5 May 1997 / Accepted: 2 June 1997     

Comparative genomic hybridization study of nasal-type NK/T-cell lymphoma

BACKGROUND: Nasal-type NK/T-cell lymphoma is a rare type of non-Hodgkin's lymphoma. The genetic changes associated with pathogenesis have not been well defined. This study investigates the nonrandom genetic alteration of nasal-type NK/T-cell lymphoma. METHODS: Nine cases were studied. Comparative genomic hybridization (CGH) was carried out using fresh tumor tissues of seven nasal-type NK/T-cell lymphomas. To complement the data by CGH, loss of heterozygosity (LOH) of chromosomes 6q, 1p, and 17p using polymorphic markers and p53 gene mutation by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) were analyzed. RESULTS: The DNA copy number changes of seven nasal-type NK/T-cell lymphomas were gains on chromosomes 2q(5), 13q(4), 10q(3), 21q(2), 3q(2), 5q(2), and 17q(2), and losses involving chromosomes 1p(4), 17p(4), 12q(3), 13q(2), and 6q(1). One of six cases informative for at least two markers for chromosome 6q showed LOH at D6S300, D6S1639, D6S261, D6S407, and D6S292. Two cases showing loss of 1p and 17q by CGH revealed LOH at D1S214, D1S503, and D17S559. P53 mutation was detected in exon 8 in one of nine cases. CONCLUSIONS: Frequent DNA losses at 1p, 17p, and 12q and gains at 2q, 13q, and 10q suggested that these regions could be targets for further molecular genetic analysis to investigate tumor suppressor genes or oncogenes associated with tumorigenesis of NK/T-cell lymphoma. Infrequent alteration of 6q contrary to previous studies raises doubt about an implication of 6q loss in the pathogenesis of early-stage NK/T-cell lymphoma. Further studies on more defined cases are required to verify their association.     

Expression of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 in gastric mucosa, intestinal metaplasia, and gastric carcinoma

Aberrant mucin O-glycosylation is often observed in cancer and is characterized by the expression of immature simple mucin-type carbohydrate antigens. UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6) is one of the enzymes responsible for the initial step in O-glycosylation. This study evaluated the expression of ppGalNAc-T6 in human gastric mucosa, intestinal metaplasia, and gastric carcinomas. Our results showed that ppGalNAc-T6 is expressed in normal gastric mucosa and in intestinal metaplasia. A heterogeneous expression and staining pattern for this enzyme was observed in gastric carcinomas. ppGalNAc-T6 was expressed in 79% of the cases, and its expression level was associated with the presence of venous invasion. Our results provide evidence that ppGalNAc-T6 is an IHC marker associated with venous invasion in gastric carcinoma and may contribute to the understanding of the molecular mechanisms that underlie aberrant glycosylation in gastric carcinogenesis and in gastric carcinoma.     

宫颈癌p16基因甲基化及表达的研究

为了探讨p16基因甲基化及异常表达在宫颈癌中的意义,分别采用甲基化特异性PCR（MSP）方法检测不同病理类型和临床分期的60例宫颈癌组织中p16基因启动子区域5´CpG岛甲基化状态;采用PCR方法检测p16基因外显子1（E1)和外显子2(E2)纯合缺失情况;采用免疫组化的方法分析p16蛋白的表达缺失和减弱情况。结果显示正常对照组织及癌旁p16基因无甲基化,且无E1和E2缺失和p16蛋白表达异常。60例宫颈癌标本的甲基化率为21.67%（13/60）;p16 基因缺失率为15.00%（9/60）;p16蛋白表达下降或无表达为51.67%（31/60）。可见p16基因蛋白的阳性表达率随着临床分期升高呈明显下降趋势。结果提示p16基因失活在宫颈癌中多见且与病理分级密切相关。p16 基因甲基化在宫颈癌发生中起着一定作用。Abstract： To detect hypermethylation and aberrant expression of the p16 gene in cervical carcinoma (CC), methylation-specific PCR (MSP) was used to determine the methylation status of 5´CpG islands of the p16 gene, loss or decrease of p16 expression was analyzed by immunohistochemistry (IHC), and homozygous deletion of exon 1 (E1) and/or exon 2 (E2) was determined by PCR. in 60 cases of CC at different pathological grades and clinical stages. The results showed absence of methylation and presence of normal expression of the p16 gene in the control and adjacent tissues of CC. Hypermethylation, loss or decrease of expression and deletion of the p16 gene were detected in 21.67%(13/60), 51.67%(31/60) and 15.00%(9/60) of the tumor tissues, respectively. The rate of p16 expression markedly reduced with the increase of clinical stages. Our data suggested that inactivation of the p16 gene was a frequent event and positively correlated with pathological grades in CC, and that methylation of the p16 gene was an important event in carcinogenesis of CC.