[Epigenetic changes in the promoter of the fragile histidine triad (FHIT) gene in human sebocytes under the influence of in vitro culture]. / Epigenetische Veränderungen am Promotor des fragilen Histidin-Trias(FHIT)-Gens in menschlichen Sebozyten unter dem Einfluss der In-vitro-Kultur.

BACKGROUND: Due to the lack of tumor suppressor function of the fragile histidine triad (FHIT) gene product, sebaceous gland carcinomas can develop. OBJECTIVE: The model of the sebocyte cell line SZ95 was used to identify methylated CpG islands at the 5'-end of the FHIT gene and the decrease of gene expression as well as the increase of double-stranded (ds) DNA breaks were examined. MATERIAL AND METHODS: Methylation, immunofluorescence analysis, promotor sequencing and treatment of SZ95 cells with 5­azacytidine/trichostatin A (TSA). RESULTS: The cultivation was accompanied by an increasing methylation of the CpG islands, a decrease of the FHIT gene expression and an accumulation of ds-DNA breaks. Treatment with 5­azacytidine/TSA showed a decrease in DNA methylation and a re-expression of FHIT transcripts. DISCUSSION: Epigenetic changes in the cellular genome are caused by in vitro cell culture. Consequently, a positive selection of sebocytes with an epigenetically inactivated FHIT locus occurs.

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.

Interaction between fragile histidine triad methylation, protein expression and human papillomavirus 16 infection in cervical carcinogenesis / 中华流行病学杂志

Objective 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.Methods 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 softvare.Differences among groups were assessed by Chi-square and Kruskal-Wallis tests.The interaction effects were evaluated by additive model.Results 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 (x2=47.623,P＜0.001),FHIT methylation rate (x2=40.147,P＜0.001) and the rate of FHIT protein low expression (x2=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.Conclusion Hypermethylation of FHIT gene,low expression of FHIT protein and HPVI6 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.

Interaction between fragile histidine triad methylation, protein expression and human papillomavirus 16 infection in cervical carcinogenesis / 中华流行病学杂志

Objective 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.Methods 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 softvare.Differences among groups were assessed by Chi-square and Kruskal-Wallis tests.The interaction effects were evaluated by additive model.Results 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 (x2=47.623,P＜0.001),FHIT methylation rate (x2=40.147,P＜0.001) and the rate of FHIT protein low expression (x2=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.Conclusion Hypermethylation of FHIT gene,low expression of FHIT protein and HPVI6 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.

A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1.

Fragile histidine triad (FHIT) gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissues. Though FHIT has been established as an authentic tumor suppressor, the mechanism underlying tumor suppression remains opaque. Most experiments designed to clarify FHIT function have analyzed the consequence of re-expressing FHIT in FHIT-negative cells. However, carcinogenesis occurs in cells that transition from FHIT-positive to FHIT-negative. To better understand cancer development, we induced FHIT loss in human bronchial epithelial cells with RNA interference. Because FHIT is a demonstrated target of carcinogens in cigarette smoke, we combined FHIT silencing with cigarette smoke extract (CSE) exposure and measured gene expression consequences by RNA microarray. The data indicate that FHIT loss enhances the expression of a set of oxidative stress response genes after exposure to CSE, including the cytoprotective enzyme heme oxygenase 1 (HMOX1) at the RNA and protein levels. Data are consistent with a mechanism in which Fhit protein is required for accumulation of the transcriptional repressor of HMOX1, Bach1 protein. We posit that by allowing superinduction of oxidative stress response genes, loss of FHIT creates a survival advantage that promotes carcinogenesis.

Loss of fragile histidine triad gene expression in papillary thyroid carcinoma / 中华内分泌外科杂志

Objective To study fragile histidine triad gene(FHIT) gene mRNA expression in 60 samples of papillary thyroid carcinoma(PTC) and matched adjacent non-concerous tissues (NCE),and to explore the role of FHIT gene in the development in PTC.Methods Reverse transcription polymerase chain reaction(RT-PCR) was used to detect FHIT gene mRNA expression in 60 samples of PTC and matched NCE.Results In PTC tissues,the positive rate of FHIT gene mRNA expression was 45.0％ (27/60),lower than that in NCE tissues (100.0％,60/60).FHIT gene mRNA expression was correlated with pathology stage,TNM stage and lymph node metastasis (P ＜ 0.05).Conclusion FHIT loss and transcript abnormality might play important roles in proliferation and metastasis of PTC,and might be a useful marker for evaluating the biological behavior of PTC.

Influence of folate on fragile histidine triad gene expression,cell proliferation and apoptosis in cervical cancer cell lines / 中华流行病学杂志

Objective To explore the effect of folate on cell proliferation and apoptosis as well as on DNA methylation,expression of mRNA and protein of fragile histidine triad(FHIT)gene in cervical cancer cells. Methods Cervical cancer cell lines including CaSki(HPV16-positive)and C33A(HPV-negative)were cultured in vitro with different folate concentrations. Cell proliferation and apoptosis were determined by viable cell counting and flow cytometry while FHIT gene DNA methylation was used with methylation specific PCR(MSP). Both gene expression of FHIT protein and mRNA were detected by Western blot and Real-time PCR,respectively. Results Folate could inhibit the proliferation and promote the apoptosis in two kinds of cervical cancer cells. The number of viable cells decreased (C33A:r=0.98,P<0.001;CaSki:r=0.98,P<0.001) and the apoptosis rate increased(C33A:r=0.98,P<0.001;CaSki:r=0.99,P<0.001)along with the increase of folate concentration. FHIT gene DNA methylation showed all positive at the folate concentration levels of 1μg/ml and 10μg/ml,partially positive at 100μg/ml and 250μg/ml,but negative at 500μg/ml and 1 000μg/ml in both C33A and CaSki cells. Comparing with the control group,the mRNA or protein relative expression levels of FHIT gene in different folate concentrations were statistically significant in two kinds of cells,and showing that the FHIT gene mRNA expression(C33A:r=0.96,P<0.001;CaSki:r=0.94,P<0.001)and protein expression (C33A:r=0.96,P<0.001;CaSki:r=0.97,P<0.001) both increased along with the increase of folate concentration. Conclusion Our findings indicated that adequate folate seemed to be able to effectively inhibit the proliferation of cervical cancer cells and facilitate their apoptosis in vitro,so would reverse the aberration mRNA and protein expression of FHIT gene.

Influence of folate on fragile histidine triad gene expression,cell proliferation and apoptosis in cervical cancer cell lines / 中华流行病学杂志

Objective To explore the effect of folate on cell proliferation and apoptosis as well as on DNA methylation,expression of mRNA and protein of fragile histidine triad(FHIT)gene in cervical cancer cells. Methods Cervical cancer cell lines including CaSki(HPV16-positive)and C33A(HPV-negative)were cultured in vitro with different folate concentrations. Cell proliferation and apoptosis were determined by viable cell counting and flow cytometry while FHIT gene DNA methylation was used with methylation specific PCR(MSP). Both gene expression of FHIT protein and mRNA were detected by Western blot and Real-time PCR,respectively. Results Folate could inhibit the proliferation and promote the apoptosis in two kinds of cervical cancer cells. The number of viable cells decreased (C33A:r=0.98,P<0.001;CaSki:r=0.98,P<0.001) and the apoptosis rate increased(C33A:r=0.98,P<0.001;CaSki:r=0.99,P<0.001)along with the increase of folate concentration. FHIT gene DNA methylation showed all positive at the folate concentration levels of 1μg/ml and 10μg/ml,partially positive at 100μg/ml and 250μg/ml,but negative at 500μg/ml and 1 000μg/ml in both C33A and CaSki cells. Comparing with the control group,the mRNA or protein relative expression levels of FHIT gene in different folate concentrations were statistically significant in two kinds of cells,and showing that the FHIT gene mRNA expression(C33A:r=0.96,P<0.001;CaSki:r=0.94,P<0.001)and protein expression (C33A:r=0.96,P<0.001;CaSki:r=0.97,P<0.001) both increased along with the increase of folate concentration. Conclusion Our findings indicated that adequate folate seemed to be able to effectively inhibit the proliferation of cervical cancer cells and facilitate their apoptosis in vitro,so would reverse the aberration mRNA and protein expression of FHIT gene.

Reduced FHIT expression is associated with mismatch repair deficient and high CpG island methylator phenotype colorectal cancer.

Colorectal cancer (CRC) is a heterogeneous disease and a major contributor to world cancer mortality rates. Molecular subtypes of CRC have become standards for CRC classification and have established prognostic potential. Here, we attempt to corroborate and provide further insight pertinent to the fragile histidine triad (FHIT) gene in microsatellite instable (MSI), microsatellite stable (MSS), and CpG island methylator phenotype (CIMP) CRC subtypes. We employed array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA) techniques to survey genomic aberrations in FHIT gene and their effects on FHIT protein expression using immunohistochemistry (IHC) in a CRC cohort. We further studied FHIT protein expression by IHC in a larger CRC cohort defined for its mismatch repair (MMR) protein expression and genomic methylation profiles. Our results show FHIT genomic deletions centered in exons 4 and 5 in most of MSI-CRC samples. Moreover, we confirmed the significant association of FHIT protein expression diminution (p=0.035) with MSI-CRC. In the larger cohort, reduced FHIT protein expression was significantly associated with CIMP-high subtype of CRC (p=0.009) and loss of PMS2 protein expression (p=0.017). We conclude that FHIT expression may be a valuable marker for CRC subtyping, and its diagnostic, prognostic, and therapeutic potential should be perused.

Research on tumor suppressor gene FHIT / 上海第二医科大学学报

Fragile histidine triad(FHIT) gene is a tumor suppressor gene that locates on chromosome 3p14.2.FHIT can induce cell apoptosis and inhibit cell growth by activating caspase,inhibiting PI3K-Akt-survivin signal pathway and phosphorylation of I?B-?,and binding with microtube.The inactivation of FHIT is closely related with carcinogenesis.The advances in research on the structure,biological function,relationship between inactivation and carcinogenesis,and gene therapy of FHIT are reviewed in this paper.

Effects of exogenous FHIT gene on proliferation and apoptosis of human glioma cell line U87 / 上海第二医科大学学报

Objective To investigate the effects of exogenous fragile histidine triad(FHIT) gene on apoptosis of human glioma cell line U87. Methods By the method of liposome transfection,plasmids pcDNA3.1/myc-His(-)B-FHIT and pcDNA3.1/myc-His(-)B were transfected into glioma cell line U87.U87 cells were divided into three groups: U87-FHIT group,U87 cells transfected by plasmids pcDNA3.1/myc-His(-)B-FHIT;U87-vector group,U87 cells transfected by plasmids pcDNA3.1/myc-His(-)B;and blank control group,U87 cells without transfection.The expression of exogenous FHIT protein was detected by Western blot and immunofluorescence staining.The effects of FHIT on the growth characteristics of U87 were observed by MTT and flow cytometry. Results Growth inhibitory rate and apoptosis rate of the cells in U87-FHIT group were significantly higher than those in U87-vector group and blank control group(P

The expression of fragile histidine triad in primary hepatocellular carcinoma / 临床外科杂志

Objective To investigate the clinical significance of the expression of fragile histidine triad (FHIT) protein in primary hepatocellular carcinoma (HCC).Methods Immunohistochemical (S-P) method was used to detect the expression of FHIT in the 46 patients with HCC and 10 normal controls.Results In the patients with HCC,the expression rate of FHIT in the tumor tissue was 56.52%,significantly lowerthan that in adjacent non-tumor tissue and normal tissue.The absence of FHIT protein was correlated neither with the size,tumor capsule,serum AFP level nor with chronic hepatitis B virus infection and cirrhosis of liver.There was significant relationship between the expression of FHIT and differentiation and thrombus in the portal vein.Conclusion The loss of FHIT gene protein is a frequent event in HCC.Furthermore,the loss is closoly related to tumor prognosis prognosis and FHIT loss.

Change of human fragile histidine triad gene expression in BEAS-2B cells irradiated with ~(60)Co ?-ray / 第二军医大学学报

Objective:To study dynamic changes of fragile histidine triad(FHIT) gene expression in ionizing radiation injury and radiation carcinogenesis. Methods: BEAS-2B cells were divided into 0.5,1,2,4,8,16 Gy irradiation groups and control group. In 24 h, 72 h and 10 d after irradiation, the expression of FHIT gene was studied with single-cell RT-PCR and DNA sequencing separately. Results: Different types of FHIT gene mutations occured in different phases after irradiation with different doses (All mutations were exon deletion mutations by DNA sequencing), while abnormal FHIT gene was not detected in control group. The percentage of mutation in 0.5,1,2,4,8,16 Gy dose groups was 52.6%,66.7%,57.9%,76.5%,64.7% and 81.3% respectively 24 h after irradiation;17.6%,22.2%,50.0%,47.4%,47.1% and 68.4% respectively 72 h after irradiation;and 21.1%,25.0%,60.0%,57.9%,61.1% and 68.4% respectively 10 d after irradiation. Conclusion: These results suggest that ionizing radiation can cause deletion of FHIT gene.