Expression of fragile histidine triad in primary hepatocellular carcinoma and its relation with cell proliferation and apoptosis

AIM: To evaluate the expression of fragile histidine triad (FHIT) gene protein, product of a candidate tumor suppressor, and to investigate the relationship between FHIT, cell apoptosis and proliferation, and pathological features of primary hepatocellular carcinoma (HCC). METHODS: Forty-seven HCC and ten normal liver specimens were collected during surgical operation between 2001 and 2003. FHIT and proliferating cell nuclear antigen (PCNA) expression were detected by immunohistochemistry, and apoptotic level was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay on the tissue sections. RESULTS: All normal liver tissues showed a strong expression of FHIT, whereas 28 of 47 (59.6%) carcinomas showed a significant loss or absence of FHIT expression (P= 0.001). The proportion of reduced FHIT expression in those carcinomas at stages Ⅲ-Ⅳ (70.6%) and in those with extrahepatic metastasis (86.7%) showed an increasing trend compared with those at stages HI (30.8%, P= 0.013) and those without metastasis (46.9%, P = 0.010) respectively. Apoptotic incidence in advanced TNM stage carcinoma and those with positive FHIT expression was higher than that in early stage carcinoma (P=0.030) and in those with negative FHIT expression (P=0.044) respectively. The proliferating potential of hepatocellular carcinoma was associated with FHIT expression (P= 0.016) and the aggressive feature (P = 0.019). Kaplan-Meier analysis demonstrated that the survival time of these 47 patients correlated with TNM stage, FHIT expression and metastasis. CONCLUSION: There is marked loss or absence of FHIT expression, as well as abnormal apoptosis-prdiferation balance in HCC. FHIT may play an important role in carcinogenesis and development of HCC.

Decreased fragile histidine triad expression in colorectal cancer and its association with apoptosis inhibition

AIM： To detect the expression of fragile histidine triad （FHIT） in normal colorectal tissue, colorectal adenoma and colorectal cancer （CRC） tissue, and to analyze its relationship with the clinicopathological features of CRC, and apoptosis-associatecl proteins （Bcl-2, Bax, survivin） and apoptosis in colorectal cancer. METHODS： FHIT mRNA analysis was performed by nested reverse transcription-polymerase chain reaction （RT-PCR） assay. Tissue microarray （TMA） was established to detect the expression of FHIT, Bcl-2, Bax and survivin genes in 80 CRC tissue specimens, 16 colorectal adenoma tissue specimens and 16 hemorrhoid （PPH） tissue specimens during the same period of time as the control. Citrate-microwave-SP was used as immunohistochemical method. The relationship between clinicopathological factors, such as differentiation grades and 5-year survival rate was observed. TUNEL assay was used to detect the apoptosis index in 80 CRC tissue specimens. RESULTS： Ten out of 26 （38.5%） CRC tissue specimens expressed aberrant FHIT transcripts, none of the aberrant FHIT transcripts was observed in the matchednormal tissue and colorectal adenoma tissue by nested RT-PCR assay. The positive rate of FHIT gene expression in normal colorectal tissue, colorectal adenoma and carcinoma tissue was 93.75%, 68.75% and 46.25%, respectively. Clinicopathological analysis of patients showed that the decreased FHIT gene expression was not associated with age, sex, serum CEA levels, tumor site and size, histological classification. However, the expression of FHIT was correlated with differentiation grades, pathological stages, lymph node metastases and 5-year survival rate after operation. The positive rate of apoptosis-associated proteins （Bax, Bcl-2 and survivin） in CRC tissue was 72.50%, 51.25% and 77.50%, respectively. The expression of these apoptosisassociated proteins in CRC tissue was correlated with the expression of FHIT. The mean apoptosis index in FHIT negative tumors was significantly lower than that in FHIT- positive tumors （5.41 ± 0.23 vs 0.56 ± 0.10, P 〈 0.01）. CONCLUSION： The FHIT gene plays an important role in the regulation of apoptosis and decreased FHIT expression plays a key role in the initiation and progression of colorectal carcinoma.

Effect of fragile histidine triad gene transduction on proliferation and apoptosis of human hepatocellular carcinoma cells

AIM:To evaluate the inhibitory effects of human fragile histidine triad(FHIT) gene on cell proliferation and apoptosis in human hepatocellular carcinoma line Hep3B in vitro. METHODS:A recombinant pcDNA3.1(+) /FHIT including the functional region of FHIT gene was constructed and transferred into human hepatocellular carcinoma cells in vitro. mRNA and protein expression of the FHIT gene in the transfected cells was detected by RT-PCR and Western blot,respectively. The effect of FHIT on proliferation was detected by MTT assay. Changes in cell cycle and apoptosis were assayed by flow cytometry. Five mice received subcutaneous transplantation of Hep3B-FHIT;5 mice received subcutaneous transplantation of normal Hep3B and Hep3B-C as controls. The body weight of nude mice and tumor growth were measured. RESULTS:RT-PCR and Western blot analysis showed that the expression level of FHIT-mRNA and FHIT protein was higher in Hep3B cells after infection withpcDNA3.1(+) /FHIT. The growth of Hep3B cells treated with pcDNA3.1(+) /FHIT was significantly inhibited. The pcDNA3.1(+) /FHIT-transfected Hep3B cells showed a significantly higher cell rate at G0-G1 phase and increased apoptosis in comparison with controls(P < 0.05) . The growth of transplanted tumor was inhibited markedly by FHIT. Tumors arising from the Hep3B-FHIT cells occurred much later than those arising from the Hep3B and Hep3B-C cells. The growth of Hep3B-FHIT cells was slow and the tumor volume was low. CONCLUSION:Transduction of FHIT gene inhibits the growth of human hepatocellular carcinoma cells and induces cell apoptosis in vivo and in vitro.

Fragile histidine triad gene alterations are not essential for hepatocellular carcinoma development in South Korea

AIM: To establish the role of FHIT in the pathogenesis hepatocellular carcinoma (HCC). METHODS: We examined genomic alterations, as well as, mRNA and protein expression patterns from the FHIT gene, in 48 surgically resected hepatocellular carcinoma (HCC) tissues. Additionally, p53 mutations were analyzed. RESULTS: Aberrant FHIT transcripts were detected in 11 of 48 surrounding non-tumor liver tissues and 27 of 48 HCC samples (22.9% vs 56.3%, P = 0.002). No point mutations were identified within the open reading frame region of FHIT. Loss of heterozygosity (LOH) of the FHIT locus was detected in 4 of 42 informative cases for D3S1300, and 3 of 29 informative cases for D3S1313. Reduced expression of FHIT protein (Fhit) was observed in 8 (16.7%) of 48 HCC samples, with complete loss of Fhit in only 1 case. There were no associations with abnormal transcripts, LOH, and Fhit expression. p53 mutations were identified in 9 of the 48 HCC cases. However, none of the cases displayed a G to T transversion at p53 codon 249. CONCLUSION: Aberrant FHIT transcripts were more common in HCC tissues as compared to non-cancerous liver tissues. However, Fhit expression was lost or reduced in a minor fraction of HCC tissues, while it was strongly expressed in non-cancerous liver tissues.Therefore, our study suggests that FHIT plays a role in relatively few HCC cases in South Korea.

Loss of fragile histidine triad protein expression in inflammatory bowel disease

AIM： To investigate the expression of fragile histidine triad （FHIT） protein in 64 patients with ulcerative colitis （UC） and Crohn＇s disease （CD）, and its relation with clinicopathological data. METHODS： Rabbit-anti-FHIT antibody was used to detect FHIT protein expression in 64 formalin-fixed, paraffin-embedded tissue specimens of inflammatory bowel disease （IBD） by citrate-microwave-streptavidin （SP）-HRP immunohistochemical method. RESULTS： The positive FHIT protein expression was 22.79% ± 16.16%, 42.14% ± 16.82% in active and remittent phases of UC, 36.07% ± 19.23% in CD, and 57.05% ±8.86% in normal colon mucosa. Statistically significant differences in FHIT protein expression were observed between the active and remittent phases of UC, between the active phase of UC and normal colon mucosa, as well as between the remittent phase of UC and normal colon mucosa, and between CD and normal colon mucosa. CONCLUSION： Our results show that FHIT protein expression is completely absent or reduced in IBD, suggesting that the FHIT gene might be associated with the oncogenesis and progression of IBD, an early event from inflammatory conditions to carcinoma in IBD.

Loss of fragile histidine triad and amplification of 1p36.22 and 11p15.5 in primary gastric adenocarcinomas

AIM: To investigate the genomic copy number alterations that may harbor key driver genes in gastric tumorigenesis. METHODS: Using high-resolution array comparative genomic hybridization (CGH), we investigated the genomic alterations of 20 advanced primary gastric adenocarcinomas (seventeen tubular and three mucinous) of Chinese patients from the Jilin province. Ten matching adjacent normal regions from the same patients were also studied. RESULTS: The most frequent imbalances detected in these cancer samples were gains of 3q26.31-q27.2, 5p, 8q, 11p, 18p, 19q and 20q and losses of 3p, 4p,18q and 21q. The use of high-resolution array CGH increased the resolution and sensitivity of the observed genomic changes and identified focal genetic imbalances, which included 54 gains and 16 losses that were smaller than 1 Mb in size. The most interesting focal imbalances were the intergenic loss/homozygous deletion of the fragile histidine triad gene and the amplicons 11q13, 18q11.2 and 19q12, as well as the novel amplicons 1p36.22 and 11p15.5. CONCLUSION: These regions, especially the focal amplicons, may harbor key driver genes that will serve as biomarkers for either the diagnosis or the prognosis of gastric cancer, and therefore, a large-scale investigation is recommended.

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

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

Expression of Ki-67, galectin-3, fragile histidine triad, and parafibromin in malignant and benign parathyroid tumors

Background It is widely recognized that the diagnosis of parathyroid carcinoma （PC） is often difficult because of the overlap of characteristics between malignant and benign parathyroid tumors, especially at an early stage. Our study aimed to investigate the differential expression of Ki-67, galectin-3, fragile histidine triad （FHIT） gene, and parafibromin in PC, parathyroid adenoma （PA）, parathyroid hyperplasia （PH）, and normal parathyroid （NP） tissues; then to assess these expression values for use in differential diagnosis of malignant and benign parathyroid tumors. Methods Data of 15 cases with PC, 19 PAs, and 8 PHs were retrospectively analyzed for their clinical characteristics. The expression of Ki-67, galectin-3, FHIT, and parafibromin were detected via immunohistochemistry in the above-mentioned specimens and 6 NPs as control. Results Complete loss of parafibromin expression was seen in 9 of 15 （60%） carcinomas, and all normal parathyroid tissues and parathyroid benign tumors stained positive for parafibromin except for one （4%） adenoma. Galectin-3 staining was positive in 11 of 15 （73%） carcinomas, 5 of 19 （26%） adenomas, 1 of 8 （12%） hyperplasias, and 0 of 6 normal tissues. The Ki-67 proliferative index was high in 4 of 15 （27%） carcinomas, 1 of 19 （5%） adenomas, and none of the hyperplasia or normal tissues. FHIT expression did not differ appreciably among the tumor types. The combination of overexpression of galectin-3 or loss of parafibromin increased sensitivity for PC to 87%, while the specificity of both positive galectin-3 and positive Ki-67 could reach 100%. Conclusions These data suggested that loss of parafibromin and overexpression of galectin-3 and Ki-67 might help to distinguish parathyroid carcinoma from other parathyroid tumors. And the combination of two or three of these markers might produce better sensitivity and/or specificity for the diagnosis of parathyroid carcinoma.

EGFR Mutation and FHIT Methylation: Inverse Relationship in Patients with Lung Adenocarcinoma and Tuberculosis

Objective:To investigate the genetic correlations between epithelial growth factor receptor(EGFR)mutation and FHIT methylation in patients diagnosed with lung adenocarcinoma(AC)and pulmonary tuberculosis(TB).Methods:The presence of EGFR mutations and the methylation status of the FHIT gene in patients presenting with AC and TB were analyzed.The correlation between TB status and the observed genetic and epigenetic variations was also examined.Results:Among the 90 patients included in the study,38 exhibited EGFR mutations(14 among those with TB and 24 among those without TB),while 29 exhibited FHIT myelination(19 among those with TB and 10 among those without TB).Furthermore,the protein expression levels of EGFR and FHIT were significantly higher in patients diagnosed solely with AC compared to those presenting with both AC and TB.A robust inverse correlation was identified between TB status and the frequency of EGFR mutation(P<0.001).Moreover,significant associations were observed between TB status and FHIT methylation(P<0.01).Conclusion:The findings suggest a correlation between TB and the prevalence of EGFR mutation and FHIT methylation in the pathogenesis of AC.

Relationship between abnormality of FHIT gene and EBV infection in gastric cancer

AIM: To examine the aberrant expression of fragile histidine triad (FHIT) gene and protein in gastric cancer, and to evaluate the role of FHIT gene and the relationship between FHIT gene and EBV infection in gastric carcinogenesis. METHODS: FHIT transcripts were detected by nested RT-PCR in 30 cases of gastric cancer and their products were sequenced.FHIT protein was detected by Western blot. EBV infection was detected by PCR method in 50 cases of gastric cancer. RESULTS: The wild type transcripts were detected in all 30 matched normal tissues of gastric cancer.Aberrant transcripts were found in 11/30 (36.7%) gastric cancerous tissues. Sequencing analysis of the aberrant fragments found an RT-PCR product missing exons 5-7 in one case of gastric cancer, and another product missing exons 4-7. Four of ten (40.0%) cases of primary gastric cancer showed absent or decreased expression of FHIT protein as compared with their matched normal tissues.EBV was detected in 5/50 (10%) gastric cancers,among which 4/5 (80%) had aberrant transcripts of FHIT gene. CONCLUSION: Loss of FHIT gene or FHIT protein plays an important role in carcinogenesis,development and progression of gastric cancer.EBV infection might influence carcinogenesis of gastric cancer by inducing the abnormality of FHIT gene.

Relationship between Methylation of FHIT and CDH13 Gene Promoter Region and Liver Cancer

In order to demonstrate the relationship between methylation of fragile histidine triad(FHIT)and T-cadherin/H-cadherin(CDH13)genes and liver cancer,the methylation status of FHIT and CDH13 was detected in healthy individuals and in Mongolian and Han patients with liver cancer.The phenol-chloroform method was used to extract genomic DNA.The methylation specific polymerase chain reaction method was applied to detect the methylation status of FHIT and CDH13.The relationship between smoking and alcohol consumption and gene(FHIT and CDH13)methylation was analyzed.There was significant difference in methylation rate of FHIT(72.67%,34.67%)and CDH13(72.0%,28.0%)between liver cancer patients and healthy individuals of Mongolian descent(P<0.05),as well as that of FHIT(68%,30.67%)and CDH13(64%,26%)between liver cancer patients and healthy individuals of Han individuals(P<0.05).There was also a relationship between smoking and drinking and the methylation of FHIT and CDH13(P<0.05).Thus,the methylation of FHIT and CDH13 had a relationship with liver cancer incidence.Smoking and alcohol ingestion may promote the methylation of FHIT and CDH13.

Influence of DNA methyltransferase 3b on FHIT expression and DNA methylation of the FHIT promoter region in hepatoma SMMC-7721 cells

BACKGROUND: Alterations in DNA methylation occur during the pathogenesis of human tumors. In this study, we investigated the influence of DNA methyltransferase 3b (DNMT3b) on fragile histidine trial (FHIT) expression and on DNA methylation of the FHIT promoter region in the hepatoma cell line SMMC-7721. METHODS: DNMT3b siRNA was used to down-regulate DNMT3b expression. DNMT3b and FHIT proteins were determined by Western blotting. Methylation-specific PCR was used to analyze the methylation status of the FHIT gene. RESULTS: After DNMT3b siRNA transfection, the expression of DNMT3b was inhibited in SMMC-7721 cells, and the expression of FHIT was significantly higher than that in the control group. There was no significant difference in methylation status between the DNMT3b siRNA transfected cells and control cells. CONCLUSION: DNMT3b may play an important role in regulation of FHIT expression in hepatoma SMMC-7721 cells, but not through methylation of the FHIT promoter. (Hepatobiliary Pancreat Dis Int 2009; 8: 273-277)

Absence of FHIT expression is associated with apoptosis inhibition in colorectal cancer

Objective： To investigate the expression of fragile histidine triad （FHIT） protein in normal colorectal tissue, colorectal adenoma and colorectal cancer （CRC）, and to study the relationships between the expression of FHIT protein and the clinical pathology, the apoptosis-associated protein （Bcl-2, Bax, Survivin）, apoptosis in colorectal cancer. Methods： Tissue microarray （TMA） and immunohistochemistry SP were used to detect the expression of FHIT gene, Bcl-2, Bax and Survivin in 16 cases of the normal colorectal tissue, 16 cases of colorectal adenoma and 80 cases of the colorectal cancer. TUNEL was used to detect the apoptosis index （Al） in 80 cases of the colorectal cancer. Results： （1） The positive rates of FHIT gene expression in normal colorectal tissue, colorectal adenoma and adenocancer were 93.75%, 68.75% and 46.25% respectively. There were no significant differences in the relationships between the FHIT gene expression and histological types, the gender as well as the age （P〉0.05）. There were significant relationships between FHIT gene expression and lymph node metastasis, histological grades, Duke＇s system as well as the 5-year survival rate after operation. （2） The positive rates of Bax, Bcl-2 and Survivin in colorectal adenocancer were 72.50%, 51.25%, 77.50% respectively. The expression of FHIT gene was positively correlated with that of Bcl-2, Bax and Survivin. （3） The mean AI in FHIT negative tumors was significantly lower than that in FHIT positive tumors （P〈0.01）. Conclusion： FHIT gene may play a role in the oncogenesis and progression of colorectal cancer. The abnormal regulation of apoptosis may play an important role in the pathogenesis of colorectal cancer.

PROAPOPTOTIC FUNCTION OF FHIT GENE

Tumor suppressor gene plays an important role in maintaining the homeostasis between cell loss and growth. Fragile histidine triad （FHIT） gene found recently was studied in a deep going way; it becomes the focus as a result of its role of anti-tumor in human various type of tissue. Due to the high efficiency of FHIT gene benefiting the anti-tumor, it is proposed as a candidate of tumor suppressor gene though there are several opposite opinions.

Relationship between FHIT Expression and HPV Status in Vulvar Squamous Cell Carcinomas

OBJECTIVE To investigate the relationship between fragilehistidine triad (FHIT) depletion and human papillomavirus (HPV)status in the vulvar squamous cell carcinomas (VSCC).METHODS Immunohistochemical method was used to detectthe expression of FHIT protein in 42 cases of VSCC and 10 casesof normal vulvar tissues. PCR was used to detect HPV infectionstatus. We analyzed the relationship of the expression of FHITprotein between the 2 groups: HPV positive and HPV negative, aswell as the clinically pathological characteristics.RESULTS The expression of FHIT was positive in all normal vul-var tissues, and 71.4% was depletion in VSCC (P < 0.01). Abnormalexpression of FHIT was significantly correlated with pathologicalgrade (P < 0.05). There was a significant difference between FHITdepletion and HPV infection based on the statistial analysis (P <0.05).CONCLUSION The FHIT depletion is related to occurrenceand development of VSCC, and the abnormal expression of FHITsignificantly correlates with HPV infection.

MSI/LOH and extron expression of the FHIT gene in gastric carcinoma

We detected loss of heterozygosity(LOH)and microsatellite instabilities(MSI),as well as extron expression of the fragile histidine triad(FHIT)gene in gastric carcinoma(GC),in order to evaluate their association with clinicopathological processes in gastric carcinogenesis.LOH and MSI of the FHIT were detected by using PCR at 4 microsatellite loci:D3S 1300,D3S 4103,D3S 1481,D3S 1234 in cancer tissues from 50 patients with primary GC,with normal mucosa acting as matched controls.FHIT transcripts were detected by nested RT-PCR in 30 cases of GC and their products were sequenced.Results show that the average frequencies of LOH and MSI of the FHIT gene in GC were 32.4%and 26.4%,respectively.There was no correlation between LOH and MSI of the FHIT gene in GC and the histological characteristics of gastric carcinoma(Bormann’s or Lauren’s classification).LOH of the FHIT gene in GC was related to depth invasiveness,and its frequency in GC where serosa was penetrated was significantly higher than that in GC without serosa penetration(73.5%vs 37.5%,P<0.05).The frequency of MSI in GC without lymph node metastasis was significantly higher than that in GC with lymph node metastasis(66.7%vs 34.3%,P<0.05).Aberrant transcripts were found in 11/30 GC tissues.Sequencing analysis of the aberrant fragments found a RT-PCR product missing exons 5–7 in one case of GC,and another product missing exons 4–7.Four of 10(40.0%)cases of primary GC showed absent or decreased expression of the FHIT protein as compared to their matched normal tissues.The findings in this study suggest that LOH and MSI of FHIT gene may induce aberrant extron expression,which might play a role in gastric carcinogenesis.

Absence of FHIT expression is associated with apoptosis inhibition in colorectal cancer

The fragile histidine triad(FHIT)gene,a candidate tumor suppressor gene located at 3p14.2,has been shown to be involved in the carcinogenesis of many human tissues,including digestive tract tissues.However,the expression and the role of the FHIT in the initiation and the development of the colorectal cancer(CRC)are poorly understood.We have shown that the FHIT gene exhibits significantly decreased expression in human CRC compared to colorectal adenoma and normal colorectal tissue by tissue microarray(TMA).The positive rate of FHIT gene expression in normal colorectal tissue,adenoma and adenocarcinoma were 93.75%,68.75%and 46.25%,respectively.We show this decreased expression to be significantly correlated with the progression of colorectal carcinoma(P<0.05)as well as with differentiation and lymph node metastasis(P<0.05).We detected two somatic alterations in the FHIT gene in human CRC.The presence of this mutation correlated significantly with decreased FHIT expression in the human CRC.In our present study we tested the hypothesis that the decreased FHIT expression resulted in apoptosis inhibition associated with abnormal expression of apoptosis related proteins.To test this hypothesis we did a series of experiments.In the first test,we assessed apoptosis status using a standard TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)assay by comparing FHIT-positive CRC vs.FHIT-negative CRC.In the second experiment,the protein expression of the FHIT and other apoptosis related proteins(Bax,Bcl-2 and Survivin)were measured in human CRC by TMA.Our combined results demonstrate the mutation in the FHIT gene significantly reduced FHIT expression in human CRC.Both TUNEL and TMA experiments demonstrated significantly inhibited apoptosis by down-regulation of Bax and the up-regulation of Survivin and Bcl-2.Collectively,these studies identify the mechanism by which an important tumor suppressor gene,FHIT is inactivated specifically in human CRC contributing to our understanding of the mechanism of colorectal carcinogenesis.