Fragile Histidine Triad Protein Research Articles

Overcome Chemoresistance: Biophysical and Structural Analysis of Synthetic FHIT-Derived Peptides.

The fragile histidine triad (FHIT) protein is a member of the large and ubiquitous histidine triad (HIT) family of proteins. On the basis of genetic evidence, it has been postulated that the FHIT protein may function as tumor suppressor, implying a role for the FHIT protein in carcinogenesis. Recently, Gaudio et al. reported that FHIT binds and delocalizes annexin A4 (ANXA4) from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. They also identified the smallest protein sequence of the FHIT still interacting with ANXA4, ranging from position 7 to 13: QHLIKPS. This short sequence of FHIT protein was not only able to bind ANXA4 but also to hold its target in the cytosol during paclitaxel treatment, thus avoiding ANXA4 translocation to the inner side of the cell membrane. Starting from these results, to obtain much information about structure requirements involved in the interaction of the peptide mentioned above, we synthetized a panel of seven peptides through an Ala-scan approach. In detail, to study the binding of FHIT derived peptides with ANXA4, we applied a combination of different biophysical techniques such as differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and microscale thermophoresis (MST). Circular dichroism (CD) and nuclear magnetic resonance (NMR) were used to determine the conformational structure of the lead peptide (7–13) and peptides generated from ala-scan technique. The application of different biophysical and structural techniques, integrated by a preliminary biological evaluation, allowed us to build a solid structure activity relationship on the synthesized peptides.

Tanshinones induce tumor cell apoptosis via directly targeting FHIT

The liposoluble tanshinones are bioactive components in Salvia miltiorrhiza and are widely investigated as anti-cancer agents, while the molecular mechanism is to be clarified. In the present study, we identified that the human fragile histidine triad (FHIT) protein is a direct binding protein of sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of Tanshinone IIA (TSA), with a Kd value of 268.4 ± 42.59 nM. We also found that STS inhibited the diadenosine triphosphate (Ap3A) hydrolase activity of FHIT through competing for the substrate-binding site with an IC50 value of 2.2 ± 0.05 µM. Notably, near 100 times lower binding affinities were determined between STS and other HIT proteins, including GALT, DCPS, and phosphodiesterase ENPP1, while no direct binding was detected with HINT1. Moreover, TSA, Tanshinone I (TanI), and Cryptotanshinone (CST) exhibited similar inhibitory activity as STS. Finally, we demonstrated that depletion of FHIT significantly blocked TSA’s pro-apoptotic function in colorectal cancer HCT116 cells. Taken together, our study sheds new light on the molecular basis of the anti-cancer effects of the tanshinone compounds.

P828 DNA methylation profile defines epigenetic characteristics and molecular targets of Crohn’s disease

Abstract Background Inflammatory bowel disease(IBD) is known to be caused by a genetic predisposition involving multiple genes; however, there is growing evidence that abnormal interaction with environmental, particularly epigenetic, factors can have a significant contribution during the development of IBD. Although many studies, particularly genome-wide association studies (GWAS), have been performed to identify the genetic changes underlying the pathogenesis of Crohn’s disease (CD), the role of epigenetic changes in the development of complications arising from CD is poorly understood. Methods Here, we employed an unbiased approach to define DNA methylation alteration in CD patients using the Human Methylation 450K Bead Chip platform. Compared to normal controls, the majority of differential DNA methylation in CD patient samples was in the promoter, intergenic, and gene body regions. Results The DNA methylation profile in CD revealed 134 probes (23 hypermethylated and 111 hypomethylated probes) that were differentially methylated. We validated the methylation levels of 19 genes that showed hypermethylation in CD patients compared with normal control. Technical validation was performed using quantitative MSP analysis and we finally identified that the Fragile Histidine Triad (FHIT) genes were hypermethylated in a disease-specific manner. Using a large cohort for CD patients samples (n = 207), we found that FHIT is frequently methylated in CD patients (71%) by MSP and significantly increasing methylation level in CD patient samples. In addition, we confirmed the methylation level of FHIT gene between normal colon and CD patients. Due to hypermethylation of FHIT gene promoter in CD patients, we observed that the level of FHIT protein is downregulated in CD patient samples compared with normal by IHC analysis. Gene network analysis by GO and metascape for hypermethylated genes in CD patients suggested putative cellular and molecular interactions relevant to IBD pathology. Conclusion Overall, our DNA methylation profile identifies newly hypermethylated genes in CD, as well as paves the way to a better understanding of the role of epigenetics in the pathogenesis of CD, and provides direction for future research in the diagnosis/prognosis or therapeutic treatments for CD.

The fission yeast FHIT homolog affects checkpoint control of proliferation and is regulated by mitochondrial electron transport

Genetic analysis has strongly implicated human FHIT (Fragile Histidine Triad) as a tumor suppressor gene, being mutated in a large proportion of early‐stage cancers. The functions of the FHIT protein have, however, remained elusive. Here, we investigated aph1 +, the fission yeast homolog of FHIT, for functions related to checkpoint control and oxidative metabolism. In sublethal concentrations of DNA damaging agents, aph1Δ mutants grew with a substantially shorter lag phase. In aph1Δ mutants carrying a hypomorphic allele of cds1 (the fission yeast homolog of Chk2), in addition, increased chromosome fragmentation and missegregation were found. We also found that under hypoxia or impaired electron transport function, the Aph1 protein level was strongly depressed. Previously, FHIT has been linked to regulation of the human 9‐1‐1 checkpoint complex constituted by Hus1, Rad1, and Rad9. In Schizosaccharomyces pombe, the levels of all three 9‐1‐1 proteins are all downregulated by hypoxia in similarity with Aph1. Moreover, deletion of the aph1 + gene reduced the Rad1 protein level, indicating a direct relationship between these two proteins. We conclude that the fission yeast FHIT homolog has a role in modulating DNA damage checkpoint function, possibly through an effect on the 9‐1‐1 complex, and that this effect may be critical under conditions of limiting oxidative metabolism and reoxygenation.

Expressions of FHIT, BCRP and Bcl-2 Proteins in Breast Invasive Ductal Carcinoma

Objective: To explore the expressions of FHIT (Human fragile histidine triad), BCRP (breast cancer resistance protein) and Bcl-2 proteins in breast invasive ductal carcinoma (IDC) and its relationship with clinical pathological factors. Methods: The expressions of FHIT, BCRP and Bcl-2 proteins in 84 cases of intraoperative resection of primary breast IDC and 36 cases of normal breast tissue about 3 cm away from the neoplastci foci were detected with the method of immunohistochemical streptavidin-peroxidase (SP) to analyze the relationship between the three proteins and the pathological features of breast IDC. Results: The positive expression rates of FHIT, of BCRP and Bcl-2 were 42.86%, 72.62% and 61.90% respectively in the breast IDC group and 72.22%, 36.11% and 38.89 % respectively in the normal breast tissue control group, between which the differences were statistically significant (P <0.05). In the IDC group, the expression of the three proteins was related to the lymph nodes metastasis and tumor recurrence of breast IDC, but was not significantly correlated with the expression of estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene human epidermal growth factor receptor 2 (Her-2). The expression of FHIT was negatively correlated with the expression of and Bcl-2 (r = -0.322, -0.389), but BCRP and Bcl-2 expression were positively correlated (r = 0.276). Conclusion: The abnormal expressions of FHIT, BCRP and Bcl-2 proteins in breast IDC may play an important role in its onset and development.

Effects of FHIT gene on proliferation and apoptosis of osteosarcoma cells.

Regulatory effects of fragile histidine triad (FHIT) gene on proliferation and apoptosis of osteosarcoma cells were studied. The hFOB1.19 and Saos2 cells were routinely cultured, pcDNA3.1-FHIT overexpression vectors carrying FHIT gene fragments and blank pcDNA3.1 vectors were transfected into Saos2 cells, respectively, and the cells were divided into hFOB, Saos2, transfection and no-load transfection groups. After transfection for 48 h, the cells were collected and analyzed. The expression of FHIT messenger ribonucleic acid (mRNA) was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of FHIT protein was detected by western blot analysis. Cell Counting Kit 8 (CCK8) was used to detect cell proliferation, and flow cytometry was used to detect apoptosis. The expression of FHIT mRNA was significantly decreased in Saos2 group compared with that in hFOB group, and the difference was statistically significant (P<0.05). The expression of FHIT mRNA was significantly increased in transfection group compared with that in Saos2 group, and the difference was statistically significant (P<0.05). The expression of FHIT protein was obviously decreased in Saos2 group compared with that in hFOB group, and there was a statistically significant difference (P<0.05). The expression of FHIT protein was obviously increased in transfection group compared with that in Saos2 group, and the difference was statistically significant (P<0.05). Compared with that in the hFOB group, the cell proliferation rate was remarkably increased in Saos2 group, while the apoptosis rate was remarkably decreased, showing statistically significant differences (P<0.05). Compared with those in Saos2 group, the cell proliferation rate was significantly decreased in transfection group, while the apoptosis rate was significantly increased, and the differences were statistically significant (P<0.05). In conclusion, FHIT gene regulates the proliferation and apoptosis of Saos2 osteosarcoma cells, inhibits the proliferation and promotes apoptosis of Saos2 osteosarcoma cells.

Effect of interventional embolotherapy on FHIT and p16 expression in hepatocellular carcinoma patients.

Effects of interventional embolotherapy on the expression of fragile histidine triad (FHIT) and p16 in hepatocellular carcinoma (HCC) patients were investigated. Patients with primary HCC who were definitely diagnosed and treated in the Department of Gastroenterology in Qingdao Central Hospital from March 2014 to March 2016 were selected, and they underwent interventional embolotherapy. HCC and cancer-adjacent tissues of the patients were harvested for immunohistochemical staining. The correlation between the expression levels of FHIT and p16 was analyzed at the gene and protein level. Clinical data were collected, and whether they were correlated with the expression of FHIT and p16 was investigated. The expression levels of FHIT and p16 in primary HCC tissues were remarkably lower than that in cancer-adjacent tissues (P<0.05). In HCC tissues, FHIT expression was obviously positively correlated with p16 expression (Spearman's correlation coefficient, r=0.308; P=0.025). FHIT was related to HCC tumor-node-metastasis (TNM) staging, the differentiation degree in Edmondson-Steiner grading, lymph node metastasis and portal vein thrombosis (P<0.05 in all comparisons), whereas, p16 was associated with tumor size and the differentiation degree in Edmondson-Steiner grading (P<0.05 in all comparisons). The expression of FHIT and p16 genes and proteins in HCC tissues were obviously lower than those in cancer-adjacent tissues (P<0.05 in all comparisons). FHIT and p16 genes, as tumor suppressor genes, inhibit the proliferation of HCC, and there is a positive correlation between them. The proteins of the FHIT and p16 can be used as new indicators for clinical detection, thus providing a new method for clinical diagnosis.

The Clinical Relevance of Fragile Histidine Triad Protein (FHIT) in Patients with Bladder Cancer.

BackgroundThe present study aimed to investigate the clinical relevance of fragile histidine triad protein (FHIT) in patients with bladder cancer (BC).Material/MethodsThree independent BC microarray studies were collected and reanalyzed. The expression of FHIT was evaluated between BC samples and normal bladder tissues. The correlation between the expression of FHIT and clinicopathological features was analyzed using the chi-square test. Log-rank based survival analysis was conducted to detect the survival significance of FHIT in patients with BC. Gene set enrichment analysis (GSEA) was performed to identify the mechanisms.ResultsFHIT was significantly downregulated in BC cells (p=0.0044). BC patients in the FHIT high expression group had better clinical characteristics (including invasiveness, tumor grade, disease progression, and T staging) than those in the FHIT low expression group (p<0.0001, p<0.0001, p=0.031, p<0.0001, and p=0.056, respectively). Patients in the FHIT high expression group had better cancer-specific survival (p<0.0001) and overall survival (p=0.0008) than those in the FHIT low expression. GSEA results indicated that BC samples in the FHIT low expression group were enriched in interferon alpha response, apoptosis, androgen response, interferon gamma response, heme metabolism, and transforming growth factor (TGF) beta signaling.ConclusionsFHIT predicts better clinical relevance for patients with BC, which may be a promising therapeutic target.

Interaction between abnormal expression of fragile histidine triad and methyl-CpG-binding protein 2 on cervical cancerization

Objective: To explore the relationship between abnormal expression of fragile histidine triad (FHIT) gene and methyl-CpG-binding protein 2 (MeCP2) as well as their interaction on cervical cancerization. Methods: A total of 73 patients with cervical squamous cell carcinoma (SCC), 113 patients with cervical intraepithelial neoplasia (CIN Ⅰ, n=45; CINⅡ/Ⅲ, n=68) and 60 women with normal cervix (NC) were included in the study. Real time PCR and Western blot were performed to detect the expression levels of mRNA and protein about FHIT and MeCP2, respectively. The methylation status of FHIT gene CpG island was tested by methylation-specifc PCR (MSP). Kruskal-Wallis H test, χ(2) test, trend χ(2) test and Spearman correlation analysis were conducted with software SPSS 20.0. The interaction was evaluated by generalized multifactor dimensionality reduction (GMDR) model. Results: With the deterioration of cervical lesion, the methylation rates of FHIT gene CpG island (χ(2)=18.64, P<0.001; trend χ(2)=18.08, P<0.001) increased gradually, while the expression levels of FHIT mRNA (H=27.32, P<0.001; trend χ(2)=12.65, P<0.001) and protein (H=47.10, P<0.001; trend χ(2)=29.79, P<0.001) decreased gradually. There was a negative correlation between the methylation rates of FHIT gene CpG island and the expression level of FHIT protein (r=-0.226, P<0.001). The levels of MeCP2 mRNA (H=26.19, P<0.001; trend χ(2)=11.81, P=0.001) and protein (H=69.02, P<0.001; trend χ(2)=47.44, P<0.001) increased gradually with the aggravation of cervical lesions. There was a positive correlation between the expression level of MeCP2 protein and the FHIT mRNA Ct ratio (r=0.254, P<0.001). Expression of proteins were negatively correlated between MeCP2 and FHIT (r=-0.213, P=0.001). The results analyzed by GMDR model showed that there were interactions among high MeCP2 protein expression, the CpG island methylation of FHIT and mRNA and protein expression in CINⅡ/Ⅲ group, and among high MeCP2 mRNA and protein expression, the CpG island methylation of FHIT and low mRNA and protein expression in SCC group. Conclusion: High expression of MeCP2 mRNA and protein, the CpG island methylation and low mRNA and protein expression of FHIT could increase the risk of cervical carcinogenesis, and there might be a synergistic effect on cervical carcinogenesis.

Folate deficiency and aberrant DNA methylation and expression of FHIT gene were associated with cervical pathogenesis.

Aberrant DNA methylation is a recognized feature in various types of human cancer, and folate has a vital role in the epigenetics of mammalian cells by supplying methyl groups for DNA methylation reactions. Fragile histidine triad (FHIT) is a tumor suppressor gene that is frequently silenced in cervical cancer (CC) and preneoplastic lesions. Promoter hypermethylation was previously observed in CC, and its epigenetic silencing has been observed at mRNA or protein levels. Changes in folate intake to modulate DNA methylation may be a mechanistic link to cancer, but this remains to be elucidated. The aim of the present study was to evaluate the influences of folate on FHIT gene methylation and expression in the progression of cervical cancerization. In the present study, red blood cell (RBC) folate levels, FHIT gene methylation status, and mRNA and protein expression levels were detected in 254 women, including normal cervix (NC, n=80), cervical intraepithelial neoplasm grade 1 (CIN1, n=55; CIN2/3, n=55) and cervical squamous cell carcinoma (SCC, n=64) samples. The methylation status of FHIT gene and its mRNA and protein expression levels were measured in CaSki (HPV16 positive) and C33A (HPV16 negative) CC cells treated with different concentrations of folate. The results indicated that FHIT gene methylation rate increased with the severity of cervix lesions, however, RBC folate levels, FHIT mRNA and protein expression levels were reduced. The proliferation inhibition rate, apoptosis rate, and FHIT protein and mRNA expression levels increased along with rising concentrations of folate, whereas the degree of FHIT gene methylation gradually weakened in CaSki or C33A cell lines. The present findings indicated that folate deficiency, FHIT gene promoter hypermethylation and reduced expression were significantly associated with cervical carcinogenesis. The results indicated that folate was able to enhance apoptosis and inhibit the cervical cell proliferation while regulating FHIT gene methylation and expression. Adequate intake of folate to maintain normal DNA methylation status is an effective way for cervical lesions prevention, and demethylation treatment may offer a new strategy for therapy of CC.

Fragile histidine triad protein expression in colorectal adenoma and carcinoma

Fragile histidine triad protein expression in colorectal adenoma and carcinoma

Association of FHIT expression and FHIT gene hypermethylation with liver cancer risk: a PRISMA-compliant meta-analysis.

BackgroundThere have been suggestions that fragile histidine triad protein (FHIT) expression and FHIT gene hypermethylation were crucial to the pathogenesis of liver cancer. However, the conclusions remained unclear because of small sample size, disease subtype, and different detection techniques. Therefore, we performed a meta-analysis to estimate the associations of FHIT expression and FHIT gene hypermethylation with liver cancer pathogenesis.MethodsStudies that were published in electronic databases, such as PubMed, Web of Knowledge, China National Knowledge Infrastructure (CNKI), VIP, and WanFang, were retrieved and selected for the meta-analysis. Relative risk (RR) and 95% confidence interval (CI) were calculated to determine the correlations of FHIT expression and FHIT gene hyper-methylation with liver cancer pathogenesis with Stata 12.0 software.ResultsA total of 17 papers that evaluated the associations of FHIT expression (14 articles) and FHIT gene methylation (3 articles) with liver cancer pathogenesis were included in this meta-analysis. In the overall analysis, the pooled relative risk was 1.93 (95% CI =1.72–2.17), which indicated a significant association between FHIT low expression and liver cancer risk. According to the results of clinical information, there were significant associations of FHIT expression with TNM-stage (RR =2.13, 95% CI =1.72–2.64), tumor size (RR =1.67, 95% CI =1.36–2.05), and merger of cirrhosis (RR =1.34, 95% CI =1.06–1.69) of liver cancer in the Chinese population. In addition, the FHIT gene hypermethylation was significantly associated with the risk of liver cancer (RR =1.45, 95% CI =1.08–1.93).ConclusionThe FHIT expression and hypermethylation of FHIT gene were significantly associated with the risk of liver cancer, especially in the Chinese population. Furthermore, the results indicated significant associations between FHIT low expression and TNM-stage, tumor size, and merging of cirrhosis of liver cancer in the Chinese population.

Reduction in the copy number and expression level of the recurrent human papillomavirus integration gene fragile histidine triad (FHIT) predicts the transition of cervical lesions.

Cervical cancer is the second most common cancer and the third leading cause of cancer death in females worldwide, especially in developing countries. High risk human papillomavirus (HR-HPV) infection causes cervical cancer and precancerous cervical intraepithelial neoplasia (CIN). Integration of the HR-HPV genome into the host chromatin is an important step in cervical carcinogenesis. The detection of integrated papillomavirus sequences-PCR (DIPS-PCR) allowed us to explore HPV integration in the human genome and to determine the pattern of this integration. We performed DIPS-PCR for 4 cell lines including 3 cervical cancer cell lines and 40 tissue samples. Overall, 32 HR-HPV integration loci were detected in the clinical samples and the HeLa and SiHa cell lines. Among all the integration loci, we identified three recurrent integration loci: 3p14.2 (3 samples), 13q22.1 (2 samples and a SiHa cell line) and 8q24 (1 sample and a HeLa cell line). To further explore the effect of HR-HPV integration in the 3p14.2 locus, we used fluorescence in situ hybridization (FISH) to determine the copy number of the 3p14.2 locus and immunohistochemistry (IHC) to determine the protein expression levels of the related FHIT gene in the clinical samples. Both the 3p14.2 locus copy number and FHIT protein expression levels showed significant decreases when CIN transitioned to cervical cancer. HPV copy number was also evaluated in these clinical samples, and the copy number of HPV increased significantly between CIN and cervical cancer samples. Finally, we employed receiver operating characteristic curve (ROC curve) analysis to evaluate the potential of all these indexes in distinguishing CIN and cervical cancer, and the HPV copy number, FHIT copy number and FHIT protein expression levels have good diagnostic efficiencies.

Prognostic value of EUS combined with MSCT in predicting the recurrence and metastasis of patients with gastric cancer.

This study aims to explore the prognostic value of endoscopic ultrasonography combined with multi-slice spiral computed tomography in predicting the recurrence and metastasis of gastric cancer, as well as investigate the correlation of fragile histidine triad protein expression with the tumor-node-metastasis stage of gastric cancer patients. A total of 81 gastric cancer patients were selected in our study. All patients were examined by endoscopic ultrasonography and multi-slice spiral computed tomography before operation, and gastric cancer tissues and adjacent normal tissues were obtained after operation. Immunohistochemistry was performed to detect fragile histidine triad expression. All patients were followed up for 3 years after operation. Univariate and multivariate analysis of risk factors were conducted for the prognosis of gastric cancer patients. Endoscopic ultrasonography combined with multi-slice spiral computed tomography could increase the accuracy of preoperative tumor-node-metastasis stage of gastric cancer patients. In gastric cancer tissues, fragile histidine triad expression was mostly weakly positive with a positive rate of 60.5%. In gastric cancer adjacent normal tissues, the positive fragile histidine triad expression was mostly moderate with a positive rate of 79.0%. The fragile histidine triad expression was negatively correlated with tumor-node-metastasis stage of gastric cancer patients. The fragile histidine triad expression decreased along with the increase of T-stage, N-stage and M-stage of gastric cancer patients. Univariate and multivariate analysis showed that T-stage and N-stage were risk factors for the recurrence/metastasis and 3-year mortality of gastric cancer patients, while fragile histidine triad expression was a protective factor. Our study demonstrated that endoscopic ultrasonography combined with multi-slice spiral computed tomography may be more accurate in assessing the preoperative tumor-node-metastasis stage of gastric cancer patients.

The clinical significance of loss of FHIT and PTEN expression in 289 patients with non-small-cell lung cancer

Background: The Fragile histidine triad (FHIT) gene is a nucleotide metabolism associated with the Ap3A hydrolase, which may regulate cell cycle and induce cell apoptosis. Phosphate and tensin homolog deleted on chromosome ten (PTEN) had been found to be a dual specificity phosphatase activity (DSP) of the tumor suppressor gene. However, the roles of FHIT and PTEN in patients with non-small-cell lung cancer (NSCLC) is not well established so far. Methods: Immunohistochemistry staining was used to determine the expression of FHIT and PTEN in 76 cases of normal lung tissue and benign pulmonary lesion tissues and 289 cases of NSCLC. Results: The negative rate of FHIT and PTEN expression in NSCLC was significantly higher than that in normal lung tissues and benign pulmonary lesion tissues (P<0.001). The negative expression of FHIT and PTEN was closely associated with cell differentiation, TNM stages and lymph node metastasis and smoking history in NSCLC (P<0.001). Furthermore, the expressing level of FHIT and PTEN protein in NSCLC was associated with a poor survival of patients (P<0.001). The result of multivariate Cox analysis showed that smoking, TNM stage and loss of FHIT and PTEN expression were independent prognosticators. Conclusions: Loss of FHIT and PTEN expression in clinical specimens could be related to invasion and metastasis of NSCLC. FHIT and PTEN expression are independent prognostic factor for patients with NSCLC. Restoring the imbalance of FHIT and PTEN expression may become a new target to treat NSCLC. In addition, tobacco can induce gene deletion mutations and result in the loss of FHIT expression.

Interaction between fragile histidine triad methylation, protein expression and human papillomavirus 16 infection in cervical carcinogenesis

To investigate the association between fragile histidine triad (FHIT) gene methylation, abnormal protein expression and HPV16 infection as well as their interactions in cervical carcinogenesis. A total of 108 patients with normal cervical (NC), 142 cases of cervical intraepithelial neoplasia (CIN1, n=72; CIN2+, n=70), and 100 new cases of cervical squamous cell carcinoma (SCC) were chosen from the Shanxi Tumor Hospital, Second Hospital of Shanxi Medical University, Maternal and Child Health Center in Taiyuan and Jiexiu during September 2009 and March 2011. HPV16 was detected by multiple PCR. FHIT methylation and protein expression levels were detected by methylation specific PCR (MSP) and Western Blot, respectively. All the data were performed with SPSS 20.0 statistical software. Differences among groups were assessed by Chi-square and Kruskal-Wallis tests. The interaction effects were evaluated by additive model. The prevalence rates of HPV16 infection in CIN1 (45.8%), CIN2+(68.6%) and SCC (73.0%) were significantly higher than that in NC (28.7%, P<0.001). In NC, CIN1, CIN2+ and SCC groups, the FHIT gene methylation rates were 3.7%, 13.9%, 21.4% and 38.0% while the protein expression levels were 1.255±0.130, 1.184±0.172, 1.133±0.126 and 1.099±0.148, respectively. Differences among the groups were statistical significant (P<0.001). With increasing degrees of cervical lesions, the HPV16 infection rate (χ(2)=47.623, P<0.001), FHIT methylation rate (χ(2)=40.147, P<0.001) and the rate of FHIT protein low expression (χ(2)=65.098, P<0.001) were all gradually increasing. There appeared positive additive interaction between FHIT methylation, FHIT protein low expression and infection of HPV16. Hypermethylation of FHIT gene, low expression of FHIT protein and HPV16 infection could increase the risk of cervical cancer and precancerous lesions. These results suggested that there might be synergistic action between FHIT gene hypermethylation and HPV16 infection in the progression of cervical cancer and the same was true between the low expression of FHIT protein and HPV 16 infection.

Immunohistochemical characterization of FHIT expression in normal human tissues.

Fragile histidine triad (FHIT) is a tumor suppressor gene that is commonly inactivated in human tumors. Interestingly, the normal pattern of FHIT expression is largely unknown. This study is aimed to characterize the expression of FHIT protein in normal human tissues. A total of 119 normal human tissue specimens were analyzed for the FHIT expression using immunohistochemistry technique. The inclusion criteria included: normal/inflammatory tissue with no evidence of cellular atypia. All studied specimens were stained positively with FHIT and showed either nuclear or cytoplasmic expression. Interestingly, the pattern of FHIT staining was similar among different specimens from each organ. FHIT is located predominantly in the nucleus, although cytoplasmic staining is also present in some cell types. Oral squamous epithelium, breast ductal epithelium, squamous and tubal metaplastic epithelium of the uterine cervix, esophageal squamous epithelium, salivary glands, and bronchial epithelia all strongly expressed the nuclear protein. In connective tissue, FHIT has shown strong cytoplasmic expression in histocytes including macrophages and dendritic cells, fibroblasts, and myofibroblasts. Documentation of the pattern of FHIT expression in normal tissues will contribute to our understanding of the normal function of this protein and to interpretation of potentially altered FHIT expression in human tumors.

Crystal structure of HINT from Helicobacter pylori.

Proteins belonging to the histidine triad (HIT) superfamily bind nucleotides and use the histidine triad motif to carry out dinucleotidyl hydrolase, nucleotidyltransferase and phosphoramidite hydrolase activities. Five different branches of this superfamily are known to exist. Defects in these proteins in humans are linked to many diseases such as ataxia, diseases of RNA metabolism and cell-cycle regulation, and various types of cancer. The histidine triad nucleotide protein (HINT) is nearly identical to proteins that have been classified as protein kinase C-interacting proteins (PKCIs), which also have the ability to bind and inhibit protein kinase C. The structure of HINT, which exists as a homodimer, is highly conserved from humans to bacteria and shares homology with the product of fragile histidine triad protein (FHit), a tumour suppressor gene of this superfamily. Here, the structure of HINT from Helicobacter pylori (HpHINT) in complex with AMP is reported at a resolution of 3 Å. The final model has R and Rfree values of 26 and 28%, respectively, with good electron density. Structural comparison with previously reported homologues and phylogenetic analysis shows H. pylori HINT to be the smallest among them, and suggests that it branched out separately during the course of evolution. Overall, this structure has contributed to a better understanding of this protein across the animal kingdom.

Adenylylsulfate-ammonia adenylyltransferase activity is another inherent property of Fhit proteins.

Fhits (fragile histidine triad proteins) occur in eukaryotes but their function is largely unknown, although human Fhit is believed to act as a tumour suppressor. Fhits also exhibit dinucleoside triphosphatase, adenylylsulfatase and nucleoside phosphoramidase activities that in each case yield nucleoside 5'-monophosphate as a product. Due to the dinucleoside triphosphatase activity, Fhits may also be involved in mRNA decapping. In the present study, we demonstrate Fhit-catalysed ammonolysis of adenosine 5'-phosphosulfate, which results in the formation of adenosine 5'-phosphoramidate. This reaction has previously been associated with adenylylsulfate-ammonia adenylyltransferase (EC 2.7.7.51). Our finding shows that the capacity to catalyse ammonolysis is another inherent property of Fhits. Basic kinetic parameters and substrate specificity of this reaction catalysed by human Fhit are presented.

Novel reactivity of Fhit proteins: catalysts for fluorolysis of nucleoside 5'-phosphoramidates and nucleoside 5'-phosphosulfates to generate nucleoside 5'-phosphorofluoridates.

Fragile histidine triad (HIT) proteins (Fhits) occur in all eukaryotes but their function is largely unknown. Human Fhit is presumed to function as a tumour suppressor. Previously, we demonstrated that Fhits catalyse hydrolysis of not only dinucleoside triphosphates but also natural adenosine 5'-phosphoramidate (NH2-pA) and adenosine 5'-phosphosulfate (SO4-pA) as well as synthetic adenosine 5'-phosphorofluoridate (F-pA). In the present study, we describe an Fhit-catalysed displacement of the amino group of nucleoside 5'-phosphoramidates (NH2-pNs) or the sulfate moiety of nucleoside 5'-phosphosulfates (SO4-pNs) by fluoride anion. This results in transient accumulation of the corresponding nucleoside 5'-phosphorofluoridates (F-pNs). Substrate specificity and kinetic characterization of the fluorolytic reactions catalysed by the human Fhit and other examples of involvement of fluoride in the biochemistry of nucleotides are described. Among other HIT proteins, human histidine triad nucleotide-binding protein (Hint1) catalysed fluorolysis of NH2-pA 20times and human Hint2 40times more slowly than human Fhit.