Locus-specific transcription silencing at the FHIT gene suppresses replication stress-induced copy number variant formation and associated replication delay.

Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome instability reside in late-replicating domains associated with large transcribed genes and provided indirect evidence that transcription is a factor in their instability. Here, we compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type and isogenic cells in which FHIT gene transcription was ablated by promoter deletion. Two promoter-deletion cell lines showed reduced or absent CNV formation and CFS expression at FHIT despite continued instability at the NLGN1 control locus. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was reversed at FHIT by the promoter deletion. Altering RNase H1 expression did not change CNV induction frequency and DRIP-seq showed a paucity of R-loop formation in the central regions of large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results demonstrate that large gene transcription is a determining factor in replication stress-induced genomic instability and support models that CNV hotspots mainly result from the transcription-dependent passage of unreplicated DNA into mitosis.

FHIT and C-MYC expression in cervical histology and cytology as biomarkers for detecting high-grade intraepithelial neoplasia in human papillomavirus-positive women.

BACKGROUND: The current cervical cancer screening strategies based on Papanicolaou (Pap) and Human papillomavirus (HPV) tests receive great achievement but still exhibit many limitations in clinical practice. Exploring new biomarkers as stratified management method in HPV primary screening is becoming the tendency of current research. METHODS: Immunocytochemistry (ICC) of FHIT and C-MYC were performed on exfoliated cervical cells from 197 eligible high-risk HPV positive women. Mann-Whitney U test, Pearson Chi-Square test, logistic regression analysis and receiver operating characteristic (ROC) curves were used to assess the diagnostic efficiency. RESULTS: ICC staining intensity of FHIT and C-MYC in high-grade cervical intraepithelial neoplasia (CIN) specimens was significantly different from low-grade CIN and normal specimens. Compared with Pap test, ROC analysis of ICC in detecting high-grade CIN resulted in a larger area under the curve (AUC) (0.805 and 0.814 vs 0.723, p< 0.001). FHIT achieved higher sensitivity than Pap test (79.41% vs 66.67%, p= 0.04). Logistic regression analysis of the combination of two biomarkers led to higher AUC value, specificity and PPV than any single biomarker. CONCLUSIONS: The utility of FHIT and C-MYC ICC analysis in cervical exfoliated cells of HPV-positive women displayed superior diagnostic potential and may improve clinical performance of cervical cancer screening.

Brachypodium distachyon triphosphate tunnel metalloenzyme 3 is both a triphosphatase and an adenylyl cyclase upregulated by mechanical wounding.

Proteins with a CyaB, thiamine triphosphatase domain (CYTH domain) may play a central role at the interface between nucleotide and polyphosphate metabolism. One of the plant CYTH domain-containing proteins from Brachypodium distachyon, BdTTM3, is annotated in NCBI databases as an 'adenylyl cyclase (AC)' or a 'triphosphate tunnel metalloenzyme'. The divergent nomenclature and the search for plant ACs induced us to experimentally confirm the enzymatic activity of BdTTM3. Based on in vitro analysis, we have shown that the recombinant form of BdTTM3 is a protein with high triphosphatase activity (binding both tripolyphosphate and ATP) and low AC activity. Furthermore, the analysis of BdTTM3 transcriptional activity indicates its involvement in the mechanism underlying responses to wounding stress in B. distachyon leaves.

Oxadiazon affects the expression and activity of aldehyde dehydrogenase and acylphosphatase in human striatal precursor cells: A possible role in neurotoxicity.

Exposure to herbicides can induce long-term chronic adverse effects such as respiratory diseases, malignancies and neurodegenerative diseases. Oxadiazon, a pre-emergence or early post-emergence herbicide, despite its low acute toxicity, may induce liver cancer and may exert adverse effects on reproductive and on endocrine functions. Unlike other herbicides, there are no indications on neurotoxicity associated with long-term exposure to oxadiazon. Therefore, we have analyzed in primary neuronal precursor cells isolated from human striatal primordium the effects of non-cytotoxic doses of oxadiazon on neuronal cell differentiation and migration, and on the expression and activity of the mitochondrial aldehyde dehydrogenase 2 (ALDH2) and of the acylphosphatase (ACYP). ALDH2 activity protects neurons against neurotoxicity induced by toxic aldehydes during oxidative stress and plays a role in neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. ACYP is involved in ion transport, cell differentiation, programmed cell death and cancer, and increased levels of ACYP have been revealed in fibroblasts from patients affected by Alzheimer's disease. In this study we demonstrated that non-cytotoxic doses of oxadiazon were able to inhibit neuronal striatal cell migration and FGF2- and BDNF-dependent differentiation towards neuronal phenotype, and to inhibit the expression and activity of ALDH2 and to increase the expression and activity of ACYP2. In addition, we have provided evidence that in human primary neuronal precursor striatal cells the inhibitory effects of oxadiazon on cell migration and differentiation towards neuronal phenotype were achieved through modulation of ACYP2. Taken together, our findings reveal for the first time that oxadiazon could exert neurotoxic effects by impairing differentiative capabilities of primary neuronal cells and indicate that ALDH2 and ACYP2 are relevant molecular targets for the neurotoxic effects of oxadiazon, suggesting a potential role of this herbicide in the onset of neurodegenerative diseases.

Fhit down-regulation is an early event in pancreatic carcinogenesis.

Aberrant Fhit expression characterizes a large proportion of primary pancreatic ductal adenocarcinomas (PDACs), but fragmentary information is available on Fhit expression during the phenotypic changes of pancreatic ductal epithelium during multistep transformation. We assessed Fhit expression by immunohistochemistry in two different multistep pancreatic carcinogenic processes: pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). We considered 105 surgically treated PDACs/IPMNs and selected 30 samples of non-neoplastic pancreatic parenchyma, 50 PanIN lesions, 30 IPMNs, 15 IPMNs with associated invasive carcinoma, and 60 adenocarcinomas. Normal pancreatic ducts and surrounding acinar cells consistently showed moderate to strong Fhit immunoreactivity. Significant down-regulation of Fhit expression was observed in association with increasing severity of dysplastia/neoplastia in both carcinogenic processes. This was further confirmed by studying multiple lesions obtained from the same surgical specimen. Of 60 PDACs, only 14 showed Fhit expression comparable to normal pancreatic ductal epithelium, while the remainder (77%) showed clearly negative or reduced Fhit expression. This study demonstrates that Fhit down-regulation is an early event in both multistep carcinogenic processes leading to PDAC.

[Research of Metachromatic Reaction of Saccharomyces cerevisiae].

This work is a continuation of research of the Chizhevsky-Velhover's bio-astronomic effect. Monitoring of volutin granule metachromatic staining of Saccharomyces yeasts (S. cerevisiae UCM Y-517, S. cerevisiae CRY, S. cerevisiae CNX, S. bayanus UCM Y-493, S. unisporus UCM Y-2065, S. rosinii UCM Y-2614, S. exiguus UCM Y-649) under conditions of different space weather was carried out. S. cerevisiae UCM Y-517, which displayed the metachromatic reaction in 96.7 % cases, showed the biggest sensitivity to the space weather changes. The changes in phosphoric metabolism of S. cerevisiae CNX cells, which can not synthesize exopolyphosphatases PPX1 and PPN1 (CF 3.6.1.11), did not influence the metachromatic reaction. Yeast cells grown on wort-agar displayed more intensive metachromatic reaction compared to those grown on YEPD-agar. However, increasing concentration of phosphorus in YEPD-agar improved visualization of the metachromatic staining. The strain S. cerevisiae UCM Y-517 is recommended as a model for monitoring of volutin granules metachromatic reaction in the research project "Heliomed" because of its high sensitivity to the space weather changes.

The Obesity-Linked Gene Nudt3 Drosophila Homolog Aps Is Associated With Insulin Signaling.

Several genome-wide association studies have linked the Nudix hydrolase family member nucleoside diphosphate-linked moiety X motif 3 (NUDT3) to obesity. However, the manner of NUDT3 involvement in obesity is unknown, and NUDT3 expression, regulation, and signaling in the central nervous system has not been studied. We performed an extensive expression analysis in mice, as well as knocked down the Drosophila NUDT3 homolog Aps in the nervous system, to determine its effect on metabolism. Detailed in situ hybridization studies in the mouse brain revealed abundant Nudt3 mRNA and protein expression throughout the brain, including reward- and feeding-related regions of the hypothalamus and amygdala, whereas Nudt3 mRNA expression was significantly up-regulated in the hypothalamus and brainstem of food-deprived mice. Knocking down Aps in the Drosophila central nervous system, or a subset of median neurosecretory cells, known as the insulin-producing cells (IPCs), induces hyperinsulinemia-like phenotypes, including a decrease in circulating trehalose levels as well as significantly decreasing all carbohydrate levels under starvation conditions. Moreover, lowering Aps IPC expression leads to a decreased ability to recruit these lipids during starvation. Also, loss of neuronal Aps expression caused a starvation susceptibility phenotype while inducing hyperphagia. Finally, the loss of IPC Aps lowered the expression of Akh, Ilp6, and Ilp3, genes known to be inhibited by insulin signaling. These results point toward a role for this gene in the regulation of insulin signaling, which could explain the robust association with obesity in humans.

Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of Breast Cancer Cells.

The tumor-suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho-MAPK, and phospho-STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation.

Relationship between expression of P27, Fragile Histidine Triad (FHT), phosphatase and tensin homolog deleted on chromosome ten (PTEN), P73, and prognosis in esophageal squamous cell carcinoma.

To investigate the significance between the expression of P27, Fragile Histidine Triad (FHIT), phosphatase and tensin homolog deleted on chromosome ten (PTEN), and P73 in esophageal squamous cell carcinoma (ESCC), paraffin-embedded tissue blocks of 200 cases were obtained from the Fourth Hospital of Hebei Medical University. The sections were used for (HE) and immunohistochemical staining streptavidin-perosidase (SP). The immunologic reagents used included antibodies against P27, FHIT, PTEN, and P73. From I- to II- and III-graded ESCC, the positive expression of P27 was decreased, but the P73 was increased, showing a ladded change (P < .05). The oncogene and tumor suppressor gene protein expression were related to the differentiation and can be one of the factors of influencing prognosis. The oncogene and tumor suppressor gene protein expression was related to the prognostic factor, and thus, it is valuable for clinical treatment and judging prognosis to detect the expression of P27, FHIT, PTEN, and P73 in ESCC.

c-Myc suppresses microRNA-29b to promote tumor aggressiveness and poor outcomes in non-small cell lung cancer by targeting FHIT.

The dual role of the microRNA-29 (miR-29) family in tumor progression and metastasis in solid tumors has been reported. Evidence for the role of miR-29 in tumor malignancy and its prognostic value in overall survival (OS) and relapse-free survival (RFS) in non-small cell lung cancer (NSCLC) remains conflicting. Mechanistic studies presented herein demonstrated that c-Myc suppressed the expression of miR-29b, promoting soft agar growth and invasion capability in lung cancer cells. Interestingly, the decrease in the expression of miR-29b by c-Myc is responsible for soft agar growth and invasiveness mediated by FHIT loss due to promoter methylation. Among patients, low expression of miR-29b and FHIT was more common in tumors with high c-Myc expression than in tumors with low c-Myc expression. Kaplan-Meier and Cox regression analysis showed that tumors with high c-Myc, low miR-29b and low FHIT expression had shorter OS and RFS periods than their counterparts. In conclusion, the decrease in the expression of miR-29b by c-Myc may be responsible for FHIT loss-mediated tumor aggressiveness and for poor outcome in NSCLC. Therefore, we suggest that restoration of the miR-29b expression using the c-Myc inhibitor might be helpful in suppressing tumor aggressiveness mediated by FHIT loss and consequently improving outcomes in NSCLC patients with tumors with low expression of FHIT.

FHIT suppresses epithelial-mesenchymal transition (EMT) and metastasis in lung cancer through modulation of microRNAs.

Metastasis is the principal cause of cancer death and occurs through multiple, complex processes that involve the concerted action of many genes. A number of studies have indicated that the Fragile Histidine Triad (FHIT) gene product, FHIT, functions as a tumor suppressor in a variety of common human cancers. Although there are suggestions of a role for FHIT loss in progression of various cancers, a role for such loss in metastasis has not been defined. Here, via in vivo and in vitro assays, we reveal that the enforced expression of FHIT significantly suppresses metastasis, accompanied by inhibition of the epithelial-mesenchymal transition (EMT), a process involved in metastasis through coordinate modulation of EMT-related genes. Specifically, miR-30c, a FHIT-upregulated microRNA, contributes to FHIT function in suppression of EMT and metastasis by directly targeting metastasis genes Metadherin (MTDH), High-mobility group AT-hook 2 (HMGA2), and the mesenchymal markers, Vimentin (VIM) and Fibronectin (FN1), in human lung cancer. Finally, we demonstrate that the expression pattern of FHIT and miR-30c is inversely correlated with that of MTDH and HMGA2 in normal tissue, non-metastatic and metastatic tumors, serving as a potential biomarker for metastasis in lung cancer.

Assessment of the prognostic value of methylation status and expression levels of FHIT, GSTP1 and p16 in non-small cell lung cancer in Egyptian patients.

BACKGROUND: Methylation of tumor suppressor genes has been investigated in all kinds of cancer. Tumor specific epigenetic alterations can be used as a molecular markers of malignancy, which can lead to better diagnosis, prognosis and therapy. Therefore, the aim of this study was to evaluate the association between gene hypermethylation and expression of fragile histidine triad (FHIT), glutathione S-transferase P1 (GSTP1) and p16 genes and various clinicopathologic characteristics in primary non-small cell lung carcinomas (NSCLC). MATERIALS AND METHODS: The study included 28 primary non-small cell lung carcinomas, where an additional 28 tissue samples taken from apparently normal safety margin surrounding the tumors served as controls. Methylation-specific polymerase chain reaction (MSP) was performed to analyze the methylation status of FHIT, GSTP1 and p16 while their mRNA expression levels were measured using a real-time PCR assay with SYBR Green I. RESULTS: The methylation frequencies of the genes tested in NSCLC specimens were 53.6% for FHIT, 25% for GSTP1, and 0% for p16, and the risk of FHIT hypermethylation increased among patients with NSCLC by 2.88, while the risk of GSTP1 hypermethylation increased by 2.33. Hypermethylation of FHIT gene showed a highly significant correlation with pathologic stage (p<0.01) and a significant correlation with smoking habit and FHIT mRNA expression level (p<0.05). In contrast, no correlation was observed between the methylation of GSTP1 or p16 and smoking habit or any other parameter investigated (p>0.05). CONCLUSIONS: RESULTS of the present study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC. FHIT methylation may play a role in lung cancer later metastatic stages while GSTP1 methylation may rather play a role in the early pathogenesis.

Fragile histidine triad (FHIT) suppresses proliferation and promotes apoptosis in cholangiocarcinoma cells by blocking PI3K-Akt pathway.

Fragile histidine triad (FHIT) is a tumor suppressor protein that regulates cancer cell proliferation and apoptosis. However, its exact mechanism of action is poorly understood. Phosphatidylinositol 3-OH kinase (PI3K)-Akt-survivin is an important signaling pathway that was regulated by FHIT in lung cancer cells. To determine whether FHIT can regulate this pathway in cholangiocarcinoma QBC939 cells, we constructed an FHIT expression plasmid and used it to transfect QBC939 cells. Protein and mRNA expression were measured by western blotting and qRT-PCR, respectively. The viability and apoptosis of QBC939 cells were then assessed using MTT assays and flow cytometry. Our results revealed that the expression of survivin and Bcl-2 was downregulated, and caspase 3 was upregulated, in cells overexpressing FHIT. In addition, FHIT suppressed the phosphorylation of Akt. The changes in cell proliferation and apoptosis were obvious in cells overexpressing FHIT which parallels that of treatment with LY294002, a potent inhibitor of phosphoinositide 3-kinases. Treatment with LY294002 further decreased the expression of survivin and Bcl-2 and increased caspase-3 levels. These results suggest that FHIT can block the PI3K-Akt-survivin pathway by suppressing the phosphorylation of Akt and the expression of survivin and Bcl-2 and upregulating caspase 3.

Abnormal FHIT protein expression may be correlated with poor prognosis in gastric cancer: a meta-analysis.

Our current meta-analysis is aimed to investigate the relationships between fragile histidine triad (FHIT) protein expression and prognosis in gastric cancer patients. We searched MEDLINE (1966 ~ 2013), the Cochrane Library Database (Issue 12, 2013), EMBASE (1980 ~ 2013), CINAHL (1982 ~ 2013), Web of Science (1945 ~ 2013), and the Chinese Biomedical Database (CBM) (1982 ~ 2013) without any language restrictions. The meta-analysis was conducted using the STATA 12.0 software. Crude hazard ratios (HR) with its 95 % confidence interval (95 % CI) were calculated. Eight clinical cohort studies with a total of 1,361 gastric cancer patients were involved in our meta-analysis. Our results revealed that FHIT-negative patients exhibited a shorter overall survival (OS) time than FHIT-positive patients (HR = 1.23, 95 % CI = 1.01 ~ 1.44, P < 0.001). Ethnicity-stratified analysis demonstrated that FHIT-negative patients have significantly poorer prognosis than FHIT-positive patients among both Caucasians and Asians (all P < 0.05). In conclusion, our meta-analysis provides evidences that negative expression of FHIT protein may be correlated with poor prognosis in patients with gastric cancer. Thus, FHIT expression level may be utilized as an independent prognostic marker for gastric cancer.

Promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A genes in cervical carcinoma.

BACKGROUND AND OBJECTIVES: Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of human cancer. This study explored the relationship between promoter methylation and inactivation of the DAPK1, FHIT, MGMT, and CDKN2A genes in cervical cancer. METHODS: The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A was investigated by using a methylation-specific polymerase chain reaction in 53 specimens of cervical cancer (42 squamous cell carcinoma, 11 adenocarcinoma), 22 specimens of intraepithelial neoplasia tissues, and 24 control normal cervical tissue specimens. The correlation of promoter methylation with the clinicopathological features of cervical cancer was analyzed. The expressions of DAPK1, FHIT, MGMT, and CDKN2A were detected by measuring relative mRNA levels. RESULTS: The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A in cervical cancer vs. intraepithelial neoplasia vs. normal cervical tissue was 75.5 vs. 31.8 vs. 4.2 % (p < 0.0001), 66.0 vs. 59.1 vs. 25.0 % (p = 0.0033), 34.0 vs. 27.3 vs. 20.8 % (p = 0.76), and 17.0 vs. 31.8 vs. 8.3 % (p = 0.11), respectively. The methylation of the promoter region significantly decreased the expression of only DAPK1 (p = 0.03). The methylation rate of the DAPK1 gene promoter was significantly higher in cervical cancer tissues than in cervical intraepithelial neoplasia and normal cervical tissues. CONCLUSION: Promoter methylation may therefore lead to the inactivation of the DAPK1 gene, and may be related to the progression of cervical oncogenesis.

Expression of WWOX and FHIT is downregulated by exposure to arsenite in human uroepithelial cells.

Ecological studies in Taiwan, Chile, Argentina, Bangladesh, and Mexico have confirmed significant dose-dependent associations between ingestion of arsenic-contaminated drinking water and the risk of various human malignancies. The FHIT and WWOX genes are active in common fragile sites FRA3B and FRA16D, respectively. Reduced expression of FHIT or WWOX is known to be an early indicator of carcinogen-induced cancers. However, the effect of arsenite on the expressions and molecular mechanisms of these markers is still unclear. The aims of this study were (i) to observe the expression of ATR, WWOX and FHIT proteins in urothelial carcinoma (UC) between endemic and non-endemic areas of blackfoot disease (BFD) by immunohistochemical analyses; (ii) to compare expression of these genes between arsenite-treated SV-HUC-1 human epithelial cells and rat uroepithelial cells; and (iii) to determine the role of DNMT and MEK inhibitors on expressions of WWOX and FHIT in response to arsenite in SV-HUC-1. The experiments revealed that expressions of ATR, WWOX and FHIT in UC significantly differed between BFD areas and non-BFD areas (p=0.003, 0.009 and 0.021, respectively). In fact, the results for the arsenite-treated groups showed that ATR, WWOX and FHIT are downregulated by arsenite in SV-HUC-1. However, the inhibitors suppressed the effects of arsenite on WWOX and FHIT proteins and mRNA expression. In conclusion, arsenite decreased expressions of ATR, WWOX and FHIT via ERK1/2 activation in SV-HUC-1 cells. These findings confirm that dysregulations of these markers may contribute to arsenite-induced carcinogenesis.

HIV-TAT-fused FHIT protein functions as a potential pro-apoptotic molecule in hepatocellular carcinoma cells.

Accumulating evidence has demonstrated that FHIT (fragile histidine triad) is a bona fide tumour suppressor gene in a large fraction of human tumours, including hepatocellular cancer. A virus-based delivery system has been developed to transfer the FHIT gene into many types of cancer cells to inhibit growth or even induce apoptosis. However, a protein-based replacement strategy for FHIT has not been performed in cancer cells. Here, we used HIV-TAT (transactivator of transcription)-derived peptide to transfer the purified FHIT protein into HCC (hepatocellular carcinoma) cells and determine the biological effect of this fusion protein in inducing apoptosis. Affinity chromatography was used to purify TAT peptide-fused human FHIT (TAT-FHIT) protein from BL21 Escherichia coli. Immunofluorescence staining and Western blot analysis were performed to identify the expression and internalization of TAT-FHIT in HCC cells compared with the purified FHIT protein. Our study showed that TAT-FHIT protein can translocate into cancer cells in 1 h after incubation at 37°C. Furthermore, the results of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, Annexin-V staining and Western blotting demonstrated that TAT-FHIT can robustly inhibit growth and induce apoptosis of HCC cells in vitro. In addition, a mechanistic study showed that both exogenous and intrinsic apoptotic pathways were involved in TAT-FHIT-mediated apoptosis and this effect could be attenuated partially by a mitochondrial protector TAT-BH4, indicating that mitochondrion plays a critical role in TAT-FHIT-mediated pro-apoptotic effect in cancer cells. Taken together, our study suggests that TAT-FHIT is a potential pro-apoptotic molecule in HCC cells and strengthen the hypothesis of its therapeutic application against HCC.

Association of the promoter methylation and protein expression of Fragile Histidine Triad (FHIT) gene with the progression of differentiated thyroid carcinoma.

The role of aberrant methylation of fragile histidine triad (FHIT) promoters in the differentiated thyroid carcinoma (DTC) is not yet clear. Therefore, we investigated the association of the status of FHIT promoter methylation and FHIT protein expression with the clinicopathological progression of DTC, using PCR-based methylation assay and immunohistochemical technique. While no FHIT gene promoter methylation was observed in the matched non-cancerous epithelium (NCE) specimens, 24.6% of DTC samples demonstrated methylation in the FHIT promoter region. The protein expression of FHIT in NCE and DTC was 100.0% and 41.5% (P<0.01), respectively. There was a negative correlation between promoter methylation and protein expression of FHIT gene (P<0.05). Additionally, the methylation status appeared to be significantly associated with the pathological grade, tumor TNM stage, and lymph node metastasis (P<0.05), and FHIT proteins were weakly expressed in only about 20% of DTC with grade II pathological changes, TNM stage III/IV, or lymph node metastasis. Finally, the gender and tumor classification but not age marginally affected the promoter methylation and protein expression of FHIT. Our results suggest that methylation of the promoter region may play a key role in inactivation of FHIT - possibly leading to subsequent carcinogenesis and progression of DTC.

Immunohistochemical FHIT expression still exists in early lesions of basal cell carcinoma.

In this study, we evaluated the expression of Fragile Histidine Triad (FHIT) in basal cell carcinoma (BCC). The FHIT locus was found to be altered in numerous types of cancer [6,7,18,20,22,25,26]. However, we found only one study dealing with FHIT expression in BCC [11]. In our study, we used immunohistochemical methods for the evaluation of FHIT expression in tissue samples of 42 BCC cases. The control group was formed by intradermal melanocytic nevi (IMN). Ki-67 labeling index was used to compare cellular proliferation of BCC with internal and external controls. The study group was further separated into two subgroups, according to the intensity of FHIT staining. The Ki-67 indexes of these subgroups were also compared with each other. As a primary result, there was no significant decrease in FHIT expression in early lesions of BCC. As a second finding, there was no correlation between the intensity of FHIT staining and Ki-67 labeling index. As a third finding, there was no difference in Ki-67 labeling index between early lesions of BCC and non-neoplastic epidermis. The results were unexpected, since FHIT expression has been reported to be lost in an above mentioned study [11]. We concluded that FHIT expression remains to be positive, at least in early lesions of BCC.

Aberrations of the FHIT gene and Fhit protein in canine lymphoma cell lines.

The fragile histidine triad (FHIT) gene is a tumor-associated gene, and aberrant FHIT gene and protein expression have been described in many types of human tumors. Furthermore, it has been reported that FHIT gene inactivation is induced by hypermethylation of 5' CpG islands in the gene or by genomic deletion around the open reading frame (ORF). In this study, we explored the aberrations in the canine FHIT gene and Fhit protein expression and assessed the methylation status and genomic deletions by using 5 canine lymphoma cell lines. We found that the decrease in the expression of the Fhit protein in canine lymphoma cell lines was similar to that in human tumors. The expression of the wild-type FHIT transcript was reduced in all 5 cell lines. However, we could not confirm the involvement of aberrant methylation events in the 5' CpG islands of the canine FHIT gene. We were able to identify homozygous or heterozygous deletions in the canine FHIT genes in all 5 cell lines. Moreover, a widespread genomic deletion of the FHIT gene, which included the ORF region, was detected in 1 cell line. In the present study, we detected aberrations in the FHIT gene and Fhit protein expression in all 5 canine lymphoma cell lines, and this phenomenon might be an important factor in promoting canine lymphoma.