Search

Summary: Background: The role of the loss of p27 protein expression in the oncogenesis of colorectal cancer is still in debate. In this study, we prospectively examined the immunohistochemical expression of p27 in 108 consecutive colorectal cancers, and we analysed the relationship with the results, the clinicopathological data, microsatellite instability (MSI) and other genetic alterations of tumours. Methods: Unselected patients (108) who underwent curative colorectal resection for sporadic colorectal cancer in a three-year period were evaluated for MSI using 6 microsatellite markers, and for the presence of p27, p53, Fhit, Mlh1 and Msh2 proteins by means of immunostaining. The relationships between these markers were analysed. p27 protein expression was examined for association with disease recurrences and survival. Results: Lack of p27 expression was noted in 33 out of 108 (30.5%) colorectal cancer cases (P<0.05). This altered expression was significantly higher in proximal cancers (P<0.05), mucinous tumours (P<0.001), poorly differentiated histology (P<0.01), cancers with MSI (P<0.05), tumours with altered expression of Mlh1 (P<0.01), of Msh2 (P<0.05), and of Fhit (P<0.01). Overall survival was better in the patient group with altered level of phenotypic p27 expression, although the difference does not reach statistical significance (P=0.069). The analysis performed only for patients with tumour at stage II showed significantly better survival when the tumour exhibited altered p27 expression (P<0.02). Conclusions: The results of the present study support the hypothesis that altered expression of p27 may be part of the genetic pathway involving MSI, which is responsible for the development of some colorectal cancers. [Copyright &y& Elsevier]

Abstract: Fragile histidine triad (FHIT) gene is involved in the deletions at the 3p14.2 region in various cancers. We investigated the role of Fhit protein in cell growth by examining the signaling pathway affected by Fhit. We used 3 human colon cancer cell lines, SW480, DLD-1 and COLO201, in the study. SW480 cells, in which the expression of Fhit is completely absent, were transfected with pIRES1neo vector (SW/IRES cells), wild-type FHIT vector (SW/FHIT cells) or mt-FHIT (codon 96, His changed to Asn) vector (SW/mt-FHIT cells). The growth of SW/FHIT or SW/mt-FHIT cells was suppressed in comparison with that of parent or SW/IRES cells. Especially, the growth of SW/FHIT cells was considerably suppressed. On the other hand, the silencing of FHIT by an siRNA for it in SW/FHIT or DLD-1 cells harboring Fhit demonstrated that the growth of FHIT siRNA-treated cells was significantly enhanced in comparison with that of the vector control or nonspecific siRNA control. Thus, we found that Fhit negatively contributed to cell growth in the colon cancer cell lines. Moreover, SW/FHIT cells exhibited a higher sensitivity to oxidative stress evoked by inhibitors of mitochondrial electron transport or proteasomes compared with any of the control transfectants. The base line amount of phospho-IκB-α (p-IκB-α) was reduced in SW/FHIT cells compared with that in the other transfectants. On the contrary, the FHIT siRNA-treated SW/FHIT and DLD-1 cells exhibited an elevated p-IκB-α level in an RNAi experiment on FHIT. Perturbation of nuclear factor (NF)-κB signaling was strongly suggested by the fact that the wild-type Fhit expressants of SW480 cells tended to be sensitive to sulfasarazine or parthenolide, which are inhibitors of NF-κB. The time course of the level of IκB kinase (IKK) complex (IKKα/β, phospho-IKKα/β and IKKγ) after the treatment with TNF-α was similar between the transfectants. Although p-IκB-α and phospho-NF-κB p65 (p-NF-κB) in SW/FHIT cells responded to TNF-α as those in other transfectants, the increase in the levels of p-IκB-α and p-NF-κB after a 5-min treatment was less in SW/FHIT cells than in the other transfectants. These results altogether suggest that Fhit functions as an anti-oncoprotein by inhibiting the phosphorylation of IκB-α and thereby blocking NF-κB signaling. [Copyright &y& Elsevier]

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

The FHIT gene is a relatively newly discovered tumor suppressor gene, which is located on the chromosome 3p14.2. The gene encompasses the FRA3B fragile site. The gene has been implicated in the pathogenesis of various carcinomas, particularly cancers of the stomach and oral squamous cell carcinomas. The protein encoded by the gene bears a significant homology to a group of proteins that have a histidine triad motif, designated HIT proteins. The function of the protein remains essentially unknown. Loss of heterozygosity and microsatellite instability are responsible for FHIT gene alterations. A mutation in a gene will invariably give abnormal transcripts and contribute to the pathogenesis of cancer. A better understanding of FHIT mutations will also help us to understand the pathogenesis of the multistep process of carcinogenesis. Therapeutic potentials of Fhit can also be explored. Replacement of FHIT cDNA in human cancer cell lines has resulted in the suppression of tumor growth. This opens the possibility of therapeutic potentials using recombinant vectors carrying the FHIT gene. [ABSTRACT FROM AUTHOR]

The Fhit tumor suppressor binds and hydrolyses diadenosine polyphosphates and the Fhit–substrate complex has been proposed as a proapoptotic effector, as determined by infection of susceptible cancer cells with adenoviruses carrying wild-type fragile histidine triad (FHIT) or catalytic site mutants. The highly conserved Fhit tyrosine 114 (Y114), within the unstructured loop C-terminal of the catalytic site, can be phosphorylated by Src family tyrosine kinases, although endogenous phospho-Fhit is rarely detected. To explore the importance of Y114 and identify Fhit-mediated signaling events, wild-type and Y114 mutant FHIT-expressing adenoviruses were introduced into two human lung cancer cell lines. Caspase-dependent apoptosis was effectively induced only by wild-type but not Y114 mutant Fhit proteins. By expression profiling of FHIT versus mutant FHIT-infected cells, we found that survivin, an Inhibitor of Apoptosis Protein (IAP) family member, was significantly decreased by wild-type Fhit. In addition, Fhit inhibited activity of Akt, a key effector in the phosphatidylinositol 3-OH kinase (PI3K) pathway; loss of endogenous Fhit expression caused increased Akt activity in vitro and in vivo, and overexpression of constitutively active Akt inhibited Fhit-induced apoptosis. The results indicate that the Fhit Y114 residue plays a critical role in Fhit-induced apoptosis, occurring through inactivation of the PI3K-Akt-survivin signal pathway.Oncogene (2006) 25, 2860–2872. doi:10.1038/sj.onc.1209323; published online 16 January 2006 [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to understand the clinicopathological and prognostic significance of promoter methylation of Fragile Histidine Triad (FHIT) gene in esophageal cancer. FHIT methylation in 257 primary esophageal squamous cell carcinomas was retrospectively analyzed by methylation-specific polymerase chain reaction. Aberrant methylation of FHIT was found in 85 (33%) of 257 esophageal cancer patients. The FHIT methylation was found to be significantly associated with exposure to tobacco smoke (P =0.007) and with a poor prognosis in cases of stage 1–2 cancer irrespective of recurrence. The hazard of failure after esophagectomy for stage 1–2 cancers with FHIT methylation was about 5.81 (95% CI=1.15–14.07; P =0.009) times higher than in those without. Recurrence occurred in 116 (45%) of the 257 patients studied. The survival after recurrence in stage 1–2 cancers was also poorer for patients with FHIT methylation than in those without (HR=2.31; 95% CI=1.18–7.92; P =0.03). In conclusion, aberrant methylation of the FHIT promoter was found to be significantly associated with exposure to tobacco smoke and with a poor prognosis for stage 1–2 cases, but not with recurrence rate. Our study suggests that FHIT promoter methylation may be an independent prognostic biomarker in early stage esophageal squamous cell carcinoma. [Copyright &y& Elsevier]

Abstract: The fragile histidine triad (FHIT), located in chromosome region 3p14.2, had been reported to be a frequent allele with loss of heterozygosity (LOH) in smoking lung cancer and HPV-associated cervical cancer. Additionally, FHIT LOH may act as a tumor suppressor gene to involve in smoking-related lung tumorigenesis and HPV-related cervical tumorigenesis, respectively. In our previous report, a high prevalence of HPV 16/18 infection has been observed in non-smoking female lung cancer patients, and thus it was speculated that HPV 16/18 infection may increase the LOH frequency of FHIT in female cases to implicate in lung tumorigenesis. In this study, 157 lung cancer patients were enrolled and subjected to FHIT LOH analysis with three microsatellite markers. As expected, the frequency of FHIT LOH in males, smokers, and squamous cell carcinomas lung cancer patients was significantly higher than that of their corresponding counterpart (P=0.020 for gender, P<0.001 for smoking status, and P=0.038 for tumor type). Interestingly, a correlation between HPV 16 infection and FHIT LOH was observed in female lung cancer cases. To be more specifically, FHIT LOH frequency was remarkably increased from 18% (6 of 33) in HPV 16 non-infected female cases to 46% (11 of 24) in HPV 16 infected cases. The higher frequency of FHIT LOH observed in HPV 16-infected female lung tumors suggested that the involvement of HPV infection in lung tumorigenesis may, at least in part, be mediated through FHIT LOH. [Copyright &y& Elsevier]

Background. Methylation in the promoter region of genes is an important mechanism of inactivation of tumor suppressor genes. Our objective was to analyze the methylation pattern of some of the genes involved in carcinogenesis of the gallbladder, examining the immunohistochemical expression of proteins, clinical features, and patient survival time. Methods. Twenty cases of gallbladder cancer were selected from the frozen tumor bank. The DNA extracted was analyzed by means of a methylation-specific polymerase chain reaction test for the CDKN2A (p16), MLH1, APC, FHIT, and CDH1 (E-cadherin) genes. Morphological and clinical data and follow-up information were obtained. Results. All cases were in an advanced stage: histologically moderate or poorly differentiated tumors (95%). Methylation of the promoter area of genes was observed in 5%, 20%, 30%, 40%, and 65% of cases, and an altered immunohistochemical pattern (AIP) in 5%, 35%, 21%, 25%, and 66% for the MLH1, CDKN2A, FHIT, APC, and CDH1 genes, respectively. The Kappa concordance index between methylation of the promoter area and AIP for the MLH1 and CDH1 genes was very high (K > 0.75) and substantial for APC (K > 0.45). No correlation was found between survival time and the methylation of the genes studied. Conclusions. The high frequency of gene methylation (with the exception of MLH1) and the high agreement between AIP and methylation of the gene promoter area for the MLH1, APC, and CDH1 genes suggest that the inactivation of tumor suppressor genes and of the genes related to the control of cellular proliferation through this mechanism is involved in gallbladder carcinogenesis. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman scattering (SERS) is a particularly promising technique that has the potential to perform highly selective and sensitive in situ measurements of antibody-antigen reactions. This work describes the use of silver (Ag) colloids for immunoassay-based SERS detection of the fragile histidine triad (Fhit) protein. Alterations in Fhit protein expression have been associated with several human cancers, and, thus, the detection of Fhit protein is important because it can potentially be used as a cancer diagnostic biomarker, for both cancer detection and therapy. [ABSTRACT FROM AUTHOR]

The HINT1 protein, a member of the histidine triad (HIT) family, is highly conserved in diverse species and ubiquitously expressed in mammalian tissues. However, its precise function in mammalian cells is not known. As a result of its structural similarity to the tumor-suppressor protein FHIT, we used homozygous-deleted Hint1 mice to study its role in tumorigenesis. We discovered that after 2 to 3 years of age the spontaneous tumor incidence in Hint1 −/− mice was significantly greater than that in wild-type Hint1 +/+ mice (P<0.05). Using a well-established mouse model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis we found a marked and significant (P<0.05) increase in the incidence of mammary and ovarian tumors in both, Hint1 −/− and +/− mice versus +/+ mice. The Hint1 −/− and +/− mice had similar tumor incidence and similar tumor histologies. Therefore, deletion of Hint1 in mice enhances both spontaneous tumor development and susceptibility to tumor induction by DMBA. In addition, since the Hint1 +/− tumors retained expression of the unmutated wild-type allele, Hint1 is haplo-insufficient with respect to tumor suppression in this model system.Oncogene (2006) 25, 713–721. doi:10.1038/sj.onc.1209111; published online 26 September 2005 [ABSTRACT FROM AUTHOR]

MLL rearranged acute lymphoblastic leukemia (MLL) is an aggressive type of acute lymphoblastic leukemia (ALL), diagnosed predominantly in infants (<1 years of age). Since current chemotherapy fails in >50% of patients with MLL, new therapeutic strategies are desperately needed. For this, understanding the biological features characterizing MLL is necessary. Analysis of gene expression profiles revealed that the expression of the tumor suppressor gene FHIT is reduced in children with MLL rearranged ALL as compared to ALL patients carrying germ line MLL. This finding was confirmed by quantitative real-time PCR. In 100% of the infant MLL cases tested, methylation of the FHIT 5'CpG region was observed, resulting in strongly reduced mRNA and protein expression. In contrast, FHIT methylation in infant and non-infant ALL patients carrying germ line MLL was found in only approximately 60% (P< or =0.004). FHIT expression was restored upon exposing leukemic cells to the demethylating agent decitabine, which induced apoptosis. Likewise and more specifically, leukemic cell death was induced by transfecting MLL rearranged leukemic cells with expression vectors encoding wild-type FHIT, confirming tumor suppressor activity of this gene. These observations imply that suppression of FHIT may be required for the development of MLL, and provide new insights into leukemogenesis and therapeutic possibilities for MLL. [ABSTRACT FROM AUTHOR]

Abstract: It was hypothesized as early as 1986, that the recently discovered common fragile sites could facilitate recombination events, such as deletions and translocations, that result in clonally expanded cancer cell populations with specific chromosome alterations in specific cancer types. A natural extension of this hypothesis is that the clonal expansion must be driven by alteration of genes at recombination breakpoints whose altered functions actually drive clonal expansion. Nevertheless, when the FHIT gene was discovered at FRA3B, the most active common chromosome fragile region, and proposed as an example of a tumor suppressor gene altered by chromosome translocations and deletions, a wave of reports suggested that the FHIT gene was altered in cancer simply because it was in a fragile region and not because it had contributed to the clonal expansion, thus turning the original hypothesis upside down. Now, after nearly ten years and more than 500 FHIT reports, it is apparent that FHIT is an important tumor suppressor gene and that there are genes at other fragile regions that contribute significantly to development of cancer. A second fragile gene with a demonstrated role in cancer development is the WWOX gene on chromosome 16q; alterations to the WWOX gene contribute to development of hormone responsive and other cancers. Results of our recent studies of these two fragile tumor suppressor genes were summarized at the first Fragilome meeting in Heidelberg, Feb. 2005. [Copyright &y& Elsevier]

Several studies have reported conflicting results regarding correlations of the loss of Fhit expression with clinicopathological parameters in gastric cancer. We investigated the immunohistochemical expression of Fhit in 362 cases of sporadic advanced gastric adenocarcinoma. The series included 64 cases with microsatellite instability associated with defective mismatch repair genes. Fhit expression resulted absent in 72% of the tumors analyzed. Absence of Fhit expression was more frequent in cases with diffuse and mixed histotype compared to the intestinal histotype ( P=0.009). Absence of Fhit expression also correlated with tumor stage ( P<0.001), lymph node involvement ( P<0.001), presence of distant metastasis ( P=0.033), and increasing histological grade ( P=0.005). Retained Fhit expression also correlated with microsatellite instability as 61% of instable tumors had lost Fhit expression compared to 74% of microsatellite stable cancers ( P=0.050). While loss of Fhit correlates with poorer survival in univariate analysis, it is not an independent prognostic factor in multivariate analysis and is thus not of clinical utility. [ABSTRACT FROM AUTHOR]

Abstract: Mouse models of tumor suppressors are increasingly useful to investigate biomedical aspects of cancer genetics. Some tumor suppressor genes are located at common fragile sites that are specific chromosomal regions highly susceptible to DNA lesions. The tumor suppressor gene FHIT, at the fragile site FRA3B, is the first fragile gene with a developed and characterized mouse knockout model. The human gene FHIT is frequently deleted in cancers and cancer cell lines of many epithelial tissues, and Fhit protein is absent or reduced in most cancers. The mouse Fhit ortholog is also located at a common fragile site, Fra14A2 on murine chromosome 14, and sustains homozygous deletions in murine cancer cell lines. The Fhit knockout mouse is, therefore, an adequate model to study human FHIT function. To establish an animal model and to explore the role of FHIT in tumorigenesis, we have developed a mouse strain carrying one or two inactivated Fhit alleles. Insights into Fhit mouse genetics that have emerged in the last 7 years, and are reviewed in the present article, allowed for development of new tools in carcinogenesis and gene delivery studies. [Copyright &y& Elsevier]

About 10–30% of primary liver cancers represent intrahepatic cholangiocarcinomas (IHCC). Since chromosomal losses of 3p are detectable in about 40% of cholangiocarcinomas our study aimed at the identification of mechanisms leading to functional deletion of tumor suppressor genes in this region. Our efforts focussed on genomic losses and epigenetic inactivation of two tumor suppressor genes, the fragile histidine triad (FHIT) and the ras association domain family 1 (RASSF1A) genes, both located on the short arm of chromosome 3. Methods: Methylation-specific PCR (MSP) and combined bisulfite-dependent restriction analysis (COBRA) were applied to detect epigenetic silencing of gene promoters. Genomic duplex PCR was used to identify exon losses of the FHIT gene. Nineteen paraffin-embedded samples of intrahepatic cholangiocarcinomas were studied. Results: Here we report for the first time that in addition to frequent losses of the exons 5 and 6, hypermethylation of the FHIT promoter occured in a significant portion of IHCC. Methylation specific PCR (MSP) detected epigenetic inactivation of the FHIT/FRA3B locus in 8 of 19 (42%) cases. Combined bisulfite restriction analysis (COBRA) revealed that high levels of methylated FHIT promoter sequences were present in 6 of the 8 methylation positive samples. In agreement with previous reports MSP identified hypermethylation of the RASSF1A gene in 13 of 19 (68%) IHCC specimens examined. Conclusions: Epigenetic silencing of the FHIT tumor suppressor gene is a novel inactivation mechanism to be considered in the development of intrahepatic cholangiocarcinomas. However, a statistically significant inverse correlation between K-Ras activation and RASSF1A inactivation was not found. [ABSTRACT FROM AUTHOR]

Summary: Medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumors (SPNETs) are histologically alike intracranial PNETs found in different anatomical locations of the brain. Current evidence suggests that hypermethylation of promoter CpG islands is a common epigenetic event in a variety of human cancers. The aim of this study was to investigate whether promoter hypermethylation of putative tumor suppressor genes was involved in both types of intracranial PNETs. We examined the methylation status at promoter regions of RASSF1A, FHIT, and sFRP1 by methylation-specific polymerase chain reaction in a cohort of 25 primary MBs, 9 primary SPNETs, and 3 MB and 2 SPNET cell lines. Our results revealed no promoter hypermethylation of RASSF1A, FHIT, and sFRP1 in 2 normal cerebellar and 5 normal cerebral tissue specimens examined. In contrast, promoter hypermethylation of RASSF1A was detected in 100% of primary MBs, 67% (6/9) of primary SPNETs, and all PNET cell lines. The frequency of promoter hypermethylation of RASSF1A was significantly lower in SPNETs than in MBs (Fisher exact test, P = .014). Treatment of RASSF1A-deficient PNET cell lines with 5-aza-2′deoxycytidine, a demethylating agent, restored RASSF1A expression, providing evidence that promoter hypermethylation contributes to transcriptional silencing. In addition, promoter hypermethylation of FHIT and sFRP1 was detected in 22% (2/9) and 11% (1/9) of SPNETs, respectively, but not in any MBs studied. In conclusion, our study demonstrates that promoter hypermethylation of RASSF1A is a common event in intracranial PNETs, whereas FHIT and sFRP1 are epigenetically affected in a fraction of SPNETs. [Copyright &y& Elsevier]

To clarify the role of fragile histidine triad (FHIT) in hematological malignancies, we examined the methylation status and the expression level of the FHIT gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells in comparison with the methylation of the p15INK4B gene. The FHIT methylation was found in 13 of 94 (13.8%) AML and 22 of 40 (55.0%) MDS cases, but not in normal mononuclear cells (MNCs). Both the frequency and density of methylation increased in the advanced-stages MDS and the relapsed AML cases. Although FHIT and p15INK4B methylations were not correlated in MDS and AML, increased FHIT methylation at the relapse in AML was associated with p15INK4B methylation. The median expression level in AML was significantly higher than in normal MNCs, although the median expression level in those with methylation was significantly lower than in those without methylation. Furthermore, the methylation level at relapse was significantly higher than at diagnosis in AML. These results suggested that FHIT methylation was accumulated through the disease progression of MDS and AML, and the role of the FHIT gene as a tumor suppressor seemed different in AML and MDS.Leukemia (2005) 19, 1367–1375. doi:10.1038/sj.leu.2403805; published online 19 May 2005 [ABSTRACT FROM AUTHOR]

The FHIT and WWOX genes encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3, respectively. Reduced Fhit and Wwox expression has been reported in approximately two-thirds of invasive breast tumors. Expression of these fragile gene products, as well as ErbB2 and p53, were evaluated immunohistochemically in 44 pure and 31 adjacent-to-invasive ductal carcinoma in-situ (DCIS) cases. Reduced Fhit and Wwox expression were observed in (i) 70% and 68% of pure DCIS; (ii) 52% and 55% of DCIS adjacent-to-invasive tumor cases; and (iii) 20% and 50% of adjacent normal tissue in pure DCIS cases. Reduced Wwox expression in adjacent normal tissue was observed in 30% of cases in the DCIS adjacent-to-invasive group. Reduced Fhit and Wwox expression was observed in 61% of adjoining invasive tumors. In all normal, pure DCIS, and DCIS adjacent-to-invasive lesions, Fhit and Wwox expression was positively associated ( P = 0.034, P = 0.042, P = 0.004, respectively) and in the invasive component there was a positive trend toward association ( P = 0.075). Fhit and Wwox were more frequently reduced in high-grade lesions in the DCIS adjacent-to-invasive ( P = 0.025, P = 0.004, respectively). In the pure DCIS group, there was a statistically significant negative association between Fhit and ErbB2 expression in DCIS ( P = 0.035). In summary, reduced Fhit and Wwox expression in in-situ breast cancer was associated, which may contribute to the high-grade DCIS–invasive tumor pathway. [ABSTRACT FROM AUTHOR]

This study aimed to (a) determine if DNA methylation is a mechanism of WWOX (WW domain containing oxidoreductase) and FHIT (fragile histidine triad) inactivation in lung, breast and bladder cancers; (b) examine distinct methylation patterns in neoplastic and adjacent tissues and (c) seek correlation of methylation patterns with disease status. Protein expression was detected by immunohistochemistry, and methylation status by methylation-specific PCR (MSP) and sequencing, in lung squamous cell carcinomas and adjacent tissues, invasive breast carcinomas, adjacent tissues and normal mammary tissues and bladder transitional cell carcinomas. Wwox and Fhit expression was reduced in cancers in association with hypermethylation. Differential patterns of WWOX and FHIT methylation were observed in neoplastic vs adjacent non-neoplastic tissues, suggesting that targeted MSP amplification could be useful in following treatment or prevention protocols. WWOX promoter MSP differentiates DNA of lung cancer from DNA of adjacent lung tissue. WWOX and FHIT promoter methylation is detected in tissue adjacent to breast cancer and WWOX exon 1 MSP distinguishes breast cancer DNA from DNA of adjacent and normal tissue. Differential methylation in cancerous vs adjacent tissues suggests that WWOX and FHIT hypermethylation analyses could enrich a panel of DNA methylation markers.Oncogene (2005) 24, 1625-1633. doi:10.1038/sj.onc.1208398 Published online 17 January 2005 [ABSTRACT FROM AUTHOR]

The tumor suppressor gene FHIT (fragile histidine triad) at chromosomal position 3p14.2 is altered by deletions in human tumors. The frequency and specificity of its inactivation vary among carcinomas, but few articles have referred to premalignant lesions such as dysplasia. We studied the expression of FHIT in a series of squamous cell carcinomas and premalignant lesions of the larynx. We observed 36 laryngeal carcinoma biopsy specimens and 70 dysplasia biopsy specimens. We studied FHIT expression in carcinoma and dysplasia with the immunohistochemical ABC (avidin-biotinylated peroxidase complex) method. Loss of FHIT protein was observed in 42% of the squamous cell carcinomas and 23% of the premalignant lesions. There was no significant difference among the three grades of dysplasia in FHIT expression. These findings of loss of FHIT protein expression, not only in squamous cell carcinoma, but also in premalignant lesions, indicate that FHIT alterations play an important role in the early events of carcinogenesis. [ABSTRACT FROM AUTHOR]

The presence of high-risk human papillomavirus, loss of heterozygosity on chromosome 3p and fragile histidine triad gene expression were assessed as potential markers of cancer and CIN progression in 83 cervical cancers and 74 cervical intraepithelial neoplasia grade 1 lesions. Human papillomavirus type 16 was an indicator of vascular involvement in cancers. Loss of heterozygosity, especially in the fragile histidine triad gene intron 5, was an indicator of high-grade tumours, greater tumour depth and lymph node involvement. Abnormal fragile histidine triad gene expression was more frequent in cervical intraepithelial neoplasia grade 1 lesions with increased risk of disease progression. [ABSTRACT FROM AUTHOR]

Abstract: Aberrant transcription of the fragile histidine triad (FHIT) gene was investigated in intrahepatic cholangiocellular carcinomas (ICCs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) in female Syrian golden hamsters. The animals received 70mg/kg of BOP followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with dl-ethionine and then l-methionine and administration of 20mg/kg BOP. A total of 14 ICCs were obtained 10 weeks after the beginning of the experiment and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription-polymerase chain reaction analysis. Aberrant transcripts were detected in four out of 14 ICCs (28.6%), as absence in the regions of nucleotides (nt) ?75 to 279, nt ?75 to 348 and nt ?75 to 447. These results suggest that alteration of the FHIT gene may play a role in a small fraction of ICCs induced by BOP in the hamster. [Copyright &y& Elsevier]

The expression of the tumour suppressor protein fragile histidine triad (Fhit) is often impaired in many human cancers and its restoration in Fhit-negative cancer cell lines suppresses tumorigenicity and induces apoptosis. Although the proapoptotic function of Fhit is well documented, little is known about its precise mechanism of action and further studies are needed in order to elucidate the putative therapeutic properties of this protein. To this end, we have engineered the lung cancer cell line NCI-H460 in order to express different molecules involved in the control of apoptotic pathways. Infection of these cells with an adenoviral vector transducing the Fhit gene (Ad-Fhit) revealed that complete protection from apoptosis was conferred by the inhibitor of caspases Cytokine response modifier A (CrmA) and by a dominant-negative form of the adapter protein Fas-associated death domain (FADD) and partial protection by a dominant-negative form of caspase-8, while cells over expressing mitochondrial mediators of the apoptotic response such as Bcl-2 or Bcl-x(L) that are resistant to treatment with cisplatin, remained highly susceptible to cell death triggered by Fhit gene transfer. In line to what was observed in H460 cells, Ad-Fhit efficacy was not affected by Bcl-2 overexpression also in two other lung cancer cell lines (A549 and Calu-1). Analysis of cytochrome c release also confirmed that in Bcl-2- or Bcl-x(L)-expressing cells apoptosis could be detected by terminal deoxynucleotidyl-transferase mediated dUTP nick-end labelling (TUNEL) assay before any evidence of mitochondrial membrane perturbation. In conclusion, our analysis indicates that the Fhit protein exerts its oncosuppressor activity through induction of an apoptotic mechanism that seems to be FADD dependent, caspase-8 mediated and independent from mitochondrial amplification.Oncogene (2004) 23, 9102-9110. doi:10.1038/sj.onc.1208142 Published online 18 October 2004 [ABSTRACT FROM AUTHOR]

We investigated the clinicopathological significance of aberrant methylation of the retinoic acid receptor-β2 (RARβ2), RAS association domain family 1A (RASSF1A) and fragile histidine triad (FHIT) genes located on choromosome 3p in 120 patients with primary non-small cell lung cancer (NSCLC) by a methylation-specific PCR method. Aberrant methylation of these was detected in 31 (26%), 35 (29%) and 43 (36%) tumors, respectively. There was no correlation with the methylation status of any of the genes. RARβ2 methylation was more frequently observed in patients with a smoking history (19 of 61, 31%) than in patients without one (3 of 29, 10%, P = 0.0373). RARβ2 methylation was also preferentially observed in advanced stage NSCLC (12 of 71 (17%) in stage I, 5 of 15 (33%) in stage II, 11 of 24 (46%) in stage III, and 3 of 8 (38%) in stage IV, P = 0.0057 (stage I versus II, III,and IV)). FHIT methylation was predominantly detected in tumors with vascular invasion (21 of 44, 48%, P = 0.0703) or lymphatic permeation (28 of 59, 47%, P = 0.0115). RASSF1A methylation was more frequently observed in adenocarcinomas (28 of 72, 39%) than in squamous cell carcinomas (6 of 45, 13%, P = 0.0033). These results indicate that aberrant methylation of the candidate tumor suppressor genes on 3p plays a respective role in the pathogenesis of NSCLC. [Copyright &y& Elsevier]

Genetic instability is an important facet of carcinogenesis and oncogenesis, generating chromosomal variability and extensive intratumor heterogeneity. Common fragile sites are predetermined chromosomal breakage regions. Experimentally, they can be demonstrated as site-specific gaps or breaks seen on metaphase chromosomes under conditions of replicative stress. They have been known for many years as a chromosomal expression of genetic instability and have been implicated to have a causative role in cancer. However, common fragile sites still remain enigmatic intrinsic parts of human chromosomes, and the DNA sequences at most of the fragile sites have noot been identified so far. The idea of genetically tagging fragile site DNA by insertional mutagenesis through an exogenous marker gene has provided a platform for the efficient targeted cloning of a substantial number of common fragile sites. [ABSTRACT FROM AUTHOR]

Abnormalities in the expression of the tumour suppressor fragile histidine triad (FHIT) gene have been reported in a variety of human tumours, including lung cancer and restoration of its expression in cancer cell lines resulted in the inhibition of proliferation and apoptosis induction. Most of the studies that have assigned a proapoptotic role to the FHIT gene were performed in adenoviral-FHIT-transduced cancer cells expressing high levels of the Fhit protein. The present work was the first study designed to investigate the effects of FHIT gene replacement in a human FHIT-negative non-small-cell lung cancer (NSCLC) cell line (Calu-1) by using a hormone-inducible expression system that allows tight modulation of the transgene expression. Through this approach, we demonstrated that a prolonged induction was required to accumulate the Fhit protein at levels adequate to promote a significant decrease of cell proliferation. Analysis of cell-cycle phase distribution showed an accumulation of cells in the G0/G1?phase and a concomitant decrease in the S?phase. Moreover, an upregulation of p21waf1 transcript was found, which could account for the alteration of the cycling properties of the cells. The growth-inhibitory effects observed were not associated with apoptosis appearance, and although in these conditions the Fhit protein content was higher than in normal bronchial human epithelial cells (NHBE), it was still significantly lower than the level capable of inducing apoptosis in Calu-1 cells after adenoviral-mediated FHIT gene transfer. These results indicate that the tumour suppressor properties of Fhit are strictly related to its expression level and show that the Fhit protein has a dose-dependent antiproliferative effect on the Fhit-negative Calu-1 lung cancer cell line.Oncogene (2004) 23, 8439-8446. doi:10.1038/sj.onc.1207847 Published online 13 September 2004 [ABSTRACT FROM AUTHOR]

Fhit protein is the product of the putative tumor suppressor fragile histidine triad (FHIT) gene. The way by which Fhit exerts its antitumor activity remains largely unknown, although the Fhit-Ap3A complex is believed to be the native signaling form of Fhit. Here, we have shown that Fhit protein interacts with hUbc9, a recombinant human SUMO-1 conjugating enzyme, in an adenosine(5′)triphospho(5′)nucleoside (Ap3N)-dependent manner. Our experiments showed that the dinucleoside polyphosphate hydrolase activity of Fhit is suppressed by interacting with hUbc9 protein. In the presence of equimolar hUbc9 the Vmax and Km activity of Fhit was decreased by 35%. Analysis of Fhit kinetics in the presence of different fixed concentrations of Ubc9 showed that Ubc9 is an uncompetitive inhibitor. Including SUMO-1 protein in the assay neither affected the Fhit activity nor modified the effect of Ubc9 on Fhit kinetics. Our data suggest that hUbc9-induced inhibition of Fhit may result in an elongation of the Fhit-Ap3A signaling complex lifetime leading to alteration of its antitumor activity. [Copyright &y& Elsevier]

The role of Fhit protein in the oncogenesis of colorectal cancer is still in debate. Recent studies have revealed that reduced Fhit protein expression is associated with a deficiency of the mismatch repair protein. One hundred and twenty unselected patients who underwent curative resection for sporadic colorectal cancer in a three-year period were evaluated for microsatellite instability (MSI) using six microsatellite markers, and for the presence of Fhit and mismatch repair (MMR) proteins (Mlh1 and Msh2) by means of immunostaining. The relations between these markers were analysed. Reduced or absent Fhit expression was noted in 18 out of 118 patients. This altered expression was significantly higher in right-sided cancer (P=0.005), mucinous tumours (P=0.005) and in poorly differentiated histological types (P=0.0001). MSI was found in 22 out of 109 patients, more so in right-sided cancer (P=0.0001), poorly differentiated histology (P=0.0001), and mucinous tumours (P=0.0001). No association was found with TNM stage. MSI was present in 66.7% of tumours with altered Fhit expression and in only 10% of tumours with preserved or intermediate Fhit expression (P=0.0001). Of the tumours with reduced or absent Fhit expression, 72.2% had loss of nuclear Mlh1 or Msh2 expression compared with only 14% of the preserved or intermediate Fhit expression tumours (P=0.0001). These results support the hypothesis that deficiency in a MMR gene could be a cause of the high frequency of alterations in Fhit expression, and they permit the suggestion that FHIT gene alteration may be part of the genetic pathway involving MSI through which some colorectal cancers arise. [Copyright &y& Elsevier]

Objective:Loss of the fragile histidine triad (Fhit) protein has been documented in cervical cancer and dysplasia. The goal of this study was to confirm the utility of homozygous deletions, aberrant methylation, and immunohistochemical evaluations of FHIT as functionally relevant determinants of FHIT expression.Methods:We studied matched DNA, RNA, and protein from nine early-passage cervical cancer cell lines. DNA markers spanning FHIT were used to examine the extent of homozygous deletions for each cell line. 5′ CpG island methylation of FHIT was investigated by methylation-specific polymerase chain reaction (PCR) assays. FHIT transcripts were characterized by reverse transriptase (RT)-PCR. Western blot analysis and immunohistochemistry were performed to characterize Fhit protein expression.Results:Homozygous deletions were found in six of nine cervical cancer cell lines, but only had homozygous deletions involving an exon. All nine lines had both methylated and unmethylated alleles according to methylation-specific PCR. Loss of wild-type FHIT transcripts were found in five of nine lines. By western blot analysis, Fhit protein expression was lost in five of nine lines, producing an exact correlation with RT-PCR results. Immunohistochemical staining was concordant with Fhit protein expression by western blotting in eight of nine cell lines.Conclusion:A perfect correlation was found between FHIT mRNA expression and western blot analysis. Assays for Fhit protein expression and large FHIT homozygous deletions are representative biomarkers of Fhit expression. By contrast, the aberrant methylation assay is not concordant with FHIT gene expression, and we suggest caution in its use as a functionally relevant biomarker for cervical cancer. [ABSTRACT FROM PUBLISHER]

The fragile histidine triad (FHIT) gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a tumour suppressor gene involved in different tumour types including non-small-cell lung cancers (NSCLCs). In the current study, we examined for allelic deletion at the FHIT locus in 58 primary and microdissected NSCLCs, for which a clinicopathologic profile was available. We found a loss of 87.7% in heterozygosity (LOH) frequency at one or more microsatellite markers (D3S1289, D3S2408, D3S1766, D3S1312, D3S1600). Allelic deletion of D3S1766 was related to tumour histology in 10 of 11 squamous cell carcinomas (90.9%) displaying LOH compared with nine of 17 adenocarcinomas (52.9%; P=0.049). Besides, in the subset of adenocarcinomas, a higher rate of LOH at D3S1289 was observed in male (six out of eight, 75%) than in female patients (four out of 17, 23.5%; P=0.028). However, FHIT LOH was not correlated overall with a variety of clinical parameters including sex, smoking status, staging, lymph node metastasis and survival. These results indicated that the high frequency of FHIT gene disruption was important in the development of both squamous cell carcinomas and adenocarcinomas. Furthermore, there was no association between LOH at FHIT and protein expression, suggesting the presence of complex mechanisms of Fhit inactivation. On the other hand, the association between FHIT LOH and p53 protein overexpression assessment reached statistical significance (P=0.026), implying that common alterations affect the two genes in tumour progression.British Journal of Cancer (2004) 90, 2378-2383. doi:10.1038/sj.bjc.6601778 www.bjcancer.com Published online 4 May 2004 [ABSTRACT FROM AUTHOR]

The expression of two tumor suppressor genes, fragile histidine triad (FHIT) and WW domain containing oxidoreductase (WWOX), encompassing common chromosome fragile regions, FRA3B at 3p14.2 and FRA16D at 16q23, is altered in many epithelial tumors. Since DNA sequence search shows that the FHIT gene has the E2F-1 recognition site in 5′ region, which regulates cell cycle, we tested the effect of E2F-1 overexpression in tumor cells. Ectopic E2F-1 expression led to an increase of Fhit and Wwox expression in allele remaining tumor cells and resulted in induction of apoptosis. Reporter assay showed that the E2F-1 site in FHIT 5′ region was involved in the down-stream transcription after exogenous E2F-1 introduction. Chromatin immunoprecipitation detected exogenous E2F-1 binding to the recognition site in FHIT 5′ region. The data suggest that E2F-1 overexpression plays a role in suppression of tumor, at least in part trough transcriptional regulation of FHIT and relevant activation of WWOX. [Copyright &y& Elsevier]

Cancer is a genetic disease and thus is influenced by oncogenes and tumor suppressor genes. To determine whether the genetic analysis of pleural fluid can be used to diagnose malignant effusion, we investigated p53 and FHIT mutations and microsatellite alterations (MA) in the pleural fluid of 40 patients with pleural effusion associated with malignancy (ME) and in the pleural fluid of 17 patients with tuberculous pleurisy (TB) as a control group. p53 mutations were detected in five ME patients (13%) and in no TB patient, and FHIT mutations were detected in seven ME patients (18%) and two TB patients (12%). For four microsatellite markers, D3S1234, D3S1285, D9S171, and TP53, in ME patients, loss of heterozygosity (LOH) was seen in 10 (25%), 5 (13%), 10 (25%), and 6 patients (15%), respectively, and microsatellite instability (MI) in 6 (15%), 0 (0%), 1 (3%), and 3 patients (8%), respectively. Using the same markers, in TB patients, LOH was seen in three (18%), one (6%), three (18%), and one (6%), respectively, and MI in one (6%), zero (0%), zero (0%), and zero (0%), respectively. Twenty-five ME cases (63%) exhibited MA (LOH or MI) in at least one marker. Moreover, in four (80%) of five ME cases with negative cytology and no carcinoembryonic antigen increase in pleural fluid, MAs were identified. In ME, positive cytology was found in 42.5%, and positive MA, using four markers, in 63%. Although still limited in terms of sensitivity and specificity, this study shows that molecular diagnostic strategies could enhance the diagnostic yield in cases of malignant effusion. [Copyright &y& Elsevier]

The fragile histidine triad (FHIT) gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a candidate tumour suppressor gene involved in a variety of tumours, including gastric carcinomas. Recently, it has been reported that the FHIT gene may be a target of damage in some of mismatch-deficient tumours. To clarify further the role of the Fhit protein in gastric carcinogenesis, we investigated whether Fhit expression in early gastric neoplasia is associated with mismatch repair protein expression and cellular phenotype. Fhit, Mlh1 and phenotypic expression were evaluated immunohistochemically in 87 early gastric neoplasias, comprising 32 adenomas and 55 intramucosal carcinomas, resected by endoscopic mucosal resection therapy. Significant loss or reduction of Fhit expression was noted in four (12.5%) of the 32 adenomas and 21 (38.2%) of the 55 intramucosal carcinomas. The rate of abnormal Fhit expression was significantly higher in intramucosal carcinomas than in adenomas (P=0.021). Moreover, reduced Fhit expression was found to be significantly associated with loss of Mlh1 expression in early gastric neoplasia (P=0.0011). Furthermore, we also detected a significant association between reduced Fhit expression and gastric phenotype (P=0.0018). These results suggested that reduced Fhit expression occurs in the early stage of gastric carcinogenesis and could be correlated with a lack of Mlh1 expression and gastric phenotype. [ABSTRACT FROM AUTHOR]

Aberrant transcription of the FHIT gene has been observed in many solid tumors, suggesting that 5′-CpG island methylation of the FHIT gene is involved in tumor development. The authors investigated the status of the 5′-CpG island methylation of the FHIT gene and protein expression in a series of 40 cervical carcinomas and 10 normal cervical epithelial samples, and correlated the data with clinicopathological findings. Methylation-specific PCR was applied to detect the incidence of the 5′-CpG island methylation, and immunohistochemistry was used for FHIT protein staining. 5′-CpG island methylation of the FHIT gene was detected in 40% (16/40) of the cervical cancer samples and in none of normal cervical epithelial specimens. Furthermore, the 5′-CpG island methylation was positively associated with clinical stage ( p =.002). All the normal cervical epithelial samples (10/10) and 90% (36/40) of the cervical carcinomas were positive when the unmethylated primer pair was applied. Of the 16 cervical carcinomas with 5′-CpG island methylation of the FHIT gene by PCR, 15 showed reduced FHIT protein expression. This study suggests that 5′-CpG island methylation plays an important role in inactivation of the FHIT gene in cervical cancer. [ABSTRACT FROM AUTHOR]

There is limited information on the molecular changes involved in the pathogenesis of gallbladder carcinoma (GBC). The Fragile Histidine Triad (FHIT) gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene in a variety of human malignancies. Recent studies have suggested that Fhit inactivation can be a consequence of defects in mismatch repair proteins. We analyzed Fhit and Mlh1 protein expressions using immunohistochemical methods in 20 GBCs and three gallbladder adenomas (GBAs) to elucidate the role of Fhit protein in gallbladder carcinogenesis. In addition, we examined whether Fhit and Mlh1 protein expressions correlated with P53 expression and clinicopathological findings. Significant loss or reduction in Fhit expression was noted in nine (45%) of the GBCs and one of the GBAs. Loss of Mlh1 protein expression was detected in six (30%) of the GBCs and one of the GBAs. Reduced Fhit expression was significantly associated with the absence of Mlh1 protein expression in the GBCs and the GBAs (p=0.0186). P53 overexpression was present in 11 (55%) of the GBCs, but none of the GBAs. Fhit and Mlh1 protein expressions were not significantly associated with P53 expression and clinicopathological findings. These results suggested that reduced Fhit expression might be involved in the development of GBC and be correlated with Mlh1 expression. [Copyright &y& Elsevier]

: ObjectiveThe fragile histidine triad (FHIT) gene is located at chromosome 3p14.2 and encompasses the common fragile site, FRA3B, which may contribute to chromosome breakage and rearrangement of cancer cells. Aberrant protein expression and inactivation of the FHIT gene have been identified in a variety of precancerous and cancerous lesions. To identify the potential implications of the FHIT gene in the development of cervical carcinoma and explore the clinical importance of change in gene expression, we assessed the level of Fhit protein in precancerous lesions and carcinomas of the cervix.: MethodsIn our study, 15 low-grade squamous intraepithelial lesions (LSIL), 35 high-grade squamous intraepithelial lesions (HSIL), 12 microinvasive carcinomas, and 103 invasive carcinomas were evaluated. The expression of Fhit was studied by immunohistochemistry using a polyclonal antibody specific for the protein.: ResultsAll samples of normal epithelium and LSIL exhibited intermediate to strong immunostaining of Fhit. Reduced Fhit protein expression was observed in 30 of 103 (29.1%) invasive carcinomas, 1 of 12 (8.3%) microinvasive carcinomas, and 3 of 35 (8.6%) HISL. Compared with normal epithelium and dysplasia, microinvasive and invasive carcinomas showed significantly lower Fhit expression. Fhit expression was also correlated with clinicopathological status. Reduced Fhit expression was significantly associated with lymph node metastasis (P = 0.005), parametrial invasion (P = 0.023), and vaginal involvement of the tumor (P = 0.016). In univariate analysis, Fhit expression was found to be a significant predictor of survival (relative risk 2.54, P = 0.0091): the patient with reduced Fhit expression had a 154% higher risk of dying from cervical cancer than the patient with opposite values.: ConclusionsOur results indicate that immunostaining of Fhit expression has potential as a prognostic marker in the management of cervical cancer. The trend of reduced Fhit expression in microinvasive and invasive carcinomas suggests that down-regulation of Fhit is strongly linked to cancer progression. Moreover, loss of Fhit expression was related to lymph node metastasis, parametrial invasion, and vaginal involvement in cervical carcinoma. These results imply that loss of Fhit protein is associated with highly aggressive phenotypes of cervical carcinoma. [Copyright &y& Elsevier]

To investigate the etiological association of allelic loss at chromosomal regions containing tumor suppressor genes (TSGs) in non-small cell lung cancer (NSCLC) in Taiwan, we examined 48 microdissected NSCLC samples for loss of heterozygosity (LOH) at nine loci where TSGs are localized nearby. The associations of LOH at each locus with clinicoparameters and prognosis were also examined. The frequent LOH was observed using markers, D3S1285 near the FHIT gene (58.3%), D17S938 near the p53 gene (56.7%), D9S925 near the p16 gene (54.5%), and D13S153 near the RB gene (47.6%). The occurrence of LOH at each TSG locus was compared with the patients’ clinicoparameters. The incidence of LOH at D17S938 (p53 gene) and D3S4545 (VHL gene) was significantly higher in squamous carcinoma tumors than in adenocarcinoma tumors (P=0.003 and 0.024, respectively). LOH of these two loci also occurred frequently in tumors from smoker patients compared to that from nonsmoker patients (P=0.013 and 0.025, respectively). LOH at D13S153 (RB gene) was also associated with smoking (P=0.008). In addition, the prognostic analyses indicated that the patients with LOH at D18S535 (18q21, near the SMAD2/4 gene) had significantly longer post-operative survival time compared to those without LOH (P=0.03). Our results suggested that LOH at FHIT, p53, and p16 genes may occur frequently in NSCLC patients in Taiwan. In addition, LOH at p53, RB, and VHL may associate with smoking or squamous carcinoma patients and LOH at SMAD2/4 may be correlated with better prognosis. [ABSTRACT FROM AUTHOR]

The tumor suppressor gene, fragile histidine triad (FHIT), encompasses the most common human chromosomal fragile site, at 3p14.2. Detection of FHIT gene is important in cancer diagnostics since its alterations have been associated with several human cancers. A unique multi-functional biochip for simultaneous detection of FHIT DNA and FHIT protein on the same platform was applied. The design of the biochip is based on miniaturization of photodiodes, where functioning of multiple optical sensing elements, amplifiers, discriminators, and logic circuitry are integrated on a single IC board. Performance of biochip is based on biomolecular recognition processes using both DNA and protein bioreceptors, Cy5-labeled probes and laser excitation. Application of biochip for concurrent detection of various immobilized target DNA and protein molecules and multiplex of DNA and protein on the same microarray was accomplished. Linearity of biochip for quantitative measurements was demonstrated. Results demonstrated utility of this multi-functional biochip as a useful detection technology with applications in biological and clinical laboratories. [ABSTRACT FROM AUTHOR]

The FHIT gene, a member of the histidine triad family has been identified as a tumor suppressor gene. Molecular genetic approaches to determine alterations in the FHIT gene in colorectal cancers have produced varying results with reported abnormalities of the FHIT gene transcripts in 13% to 50% of cases studied. FHIT has been reported to be involved in the regulation of apoptosis and cell cycle in cell culture systems. Immunohistochemical (IHC) studies of FHIT expression in human colon cancer and its correlation to apoptosis and clinical prognosis have been sparse. We studied 100 human colorectal cancers by IHC and a computerized image analysis (CIA) method to evaluate FHIT expression and the rate of apoptosis in tumors and corresponding mucosae. Follow-up data for at least five years was available for all patients. We correlated the results with tumor grade, stage and clinical prognosis. We used commercially available polyclonal anti FHIT antibody and Apoptaq kit on paraffin-embedded tumors and their corresponding mucosae and the SAMBA 4000 CIA system to evaluate the labeling index (LI), the mean optical density (MOD) of the stain and calculate a quick score (QS). The LI is the ratio of positively stained areas to the total area of the tissues examined, the MOD represents the concentration of the positive stain as measured per positive pixels and the QS a numeric product of the LI and MOD for each microscopic area examined. Image analysis of IHC staining of the tumor sections defined three main groups based on the reactivity of the anti FHIT polyclonal antibody. Group I included 23 cases where the LI was less than 55% with a mean of 36%. Eight cases in this group showed complete loss of FHIT expression. Group II included 41 cases where the LI was between 55% and 65% with a mean of 60%. Group III was composed of 36 cases where the LI was more than 65% with a mean of 69%. Our data showed that the absence or reduction of FHIT protein in the tumors, relative to morphologically normal mucosa, plays a role in the development of a few colorectal cancers (23%). Poorly differentiated carcinomas showed absent or decreased FHIT. A reduction of FHIT was positively correlated with the rate of distant metastases and worse prognosis. Over-expression of FHIT is directly proportional to the apoptotic rate in the tumors examined. CIA provides an objective and accurate assessment of the staining patterns and generates numerical data evaluating intensity better than depending on subjective light microscopy alone. [ABSTRACT FROM AUTHOR]

The FHIT gene is a putative tumour suppressor gene. In this study, we analysed a set of 50 gastric tumours for alterations of FHIT, and found 38 of 45 tumours (84%) exhibiting loss of heterozygosity (LOH) within the FHIT gene. We used both nested Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and single step RT-PCR to analyse the FHIT transcripts and found 34 of 39 (87%) tumours and seven of the 11 (64%) corresponding non-cancerous tissues showed low or aberrant expression of FHIT mRNA and the appearance of the aberrant FHIT transcripts depended on the conditions of the RT-PCR. In these aberrant transcripts, frequent deletions and/or insertions were detected by direct sequencing. All breakpoints for deletions and insertions were at splicing sites. All insertions came from the adjacent introns, whose appearance was completely in accordance with the ‘GU-AG’ rule for pre-mRNA splicing. It may be suggested that an alternative splicing mechanism functions in the formation of these aberrant transcripts. The fragile nature of FRA3B within the FHIT gene could be responsible for the formation of the aberrant mRNA. Negative or reduced Fhit expression was detected in 39 of 50 tumours (78%). Moreover, an association was found between abnormal Fhit expression and positive node status (P=0.012). Thirteen of 48 tumours (27%) displayed microsatellite instability (MSI), among which 10 tumours also showed MSI within the FHIT gene. Furthermore, we detected an association between MSI and negative node status (P=0.02). We conclude that the abnormalities of FHIT, presumably associated with the unstable nature of FRA3B within the FHIT gene, are involved in the carcinogenesis of gastric cancer, and lack of mismatch repair (MMR) could possibly promote its alteration in a subset of gastric tumours. [Copyright &y& Elsevier]

The FHIT gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene involved in multiple tumors, including esophageal carcinoma. We analyzed Fhit expression using an immunohistochemical method in invasive carcinoma, carcinoma in situ (CIS) and dysplasia, in paraffin sections of 75 esophageal squamous cell carcinomas (ESCs) to further elucidate the role of Fhit protein in esophageal carcinogenesis. In addition, we also examined whether Fhit expression correlated with p53 expression and apoptosis. Compared to adjacent normal mucosa, significant loss or reduction of Fhit expression was noted in 67 of 75 (89.3%) invasive ESCs, in 13 of 19 (68.4%) CIS lesions, and in 10 of 23 (43.5%) dysplastic lesions. There was a progressive loss or reduction of Fhit expression with progressive increases in the severity of histopathological changes (p < 0.001). However, there was no association between Fhit expression and clinicopathological findings, including tumor stage, lymph node metastasis, or overall survival. Moreover, Fhit expression was not significantly associated with p53 expression and apoptosis. These results indicate that abnormal Fhit expression is a common event in the early stage of ESC development and may occur independently of p53 expression and apoptosis mechanisms.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]