Fhit Expression Research Articles

The expression of FHIT and the relativity with cigarette in NSCLC

目的 研究脆弱性组氨酸三联体(FHIT)的表达与非小细胞肺癌(NSCLC)的发生发展及与吸烟的关系.方法采用免疫组化(S-P)法检测7例正常支气管黏膜和81例NSCLC患者中FHIT的表达.结果 FHIT在正常支气管黏膜及NSCLC中的表达差异有显著意义(P＜0.05),其表达与分化程度、吸烟指数及吸烟率差异有显著意义(P＜0.01,P＜0.05,P＜0.05),与性别、年龄、TNM分期及分型无关.吸烟与鳞癌呈正相关.结论 FHIT是NSCLC发生发展分子机制之一;FHIT可能与烟草致癌有相关性。

Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential

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.

Increased Sensitivity to Cisplatin in Non-Small Cell Lung Cancer Cell Lines after FHIT Gene Transfer

To evaluate the relevance of fragile histidine triad (FHIT) status in relation to drug treatment, we analyzed the sensitivity of the Fhit-negative non-small cell lung cancer (NSCLC) cell line NCI-H460 to different drugs, after treatment with an adenoviral vector expressing the FHITtransgene. Expression of Fhit resulted in reduced sensitivity to etoposide, doxorubicin, and topotecan. This feature was associated with Fhit-induced downregulation of DNA topoisomerases I and II. In contrast, expression of Fhit did not modulate sensitivity to Taxol, but produced a slight increase in sensitivity to cisplatin, as shown by colony-forming assays. Analysis of apoptosis revealed that, after cisplatin exposure, the number of apoptotic cells was two-fold higher in Fhitexpressing H460 cells. Moreover, it appeared that wildtype p53 was required for sensitization to cisplatin because the effect was marginal in A549 and Calu-1 cells, where the p53 pathway is altered and simultaneous restoration of p53 and Fhit in Calu-1 cells increased cisplatin sensitivity. Fhit could also partially restore sensitivity to cisplatin in Bcl-2- and BCl-xL-overexpressing H460 cells that are normally resistant to this drug. Our results support the possible relevance of FHIT in cisplatin-based chemotherapy as well as in the reversal of drug resistance in NSCLC.

Silencing of the tumor suppressor gene FHIT is highly characteristic for MLL gene rearranged infant acute lymphoblastic leukemia

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.

FHIT expression in neoplastic, hyperplastic, and normal endometrium

FHIT expression in neoplastic, hyperplastic, and normal endometrium

Loss of Fhit expression is associated with poorer survival in gastric cancer but is not an independent prognostic marker

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.

Fhit and CHK1 Have Opposing Effects on Homologous Recombination Repair

Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in approximately 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit-/- cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage-induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI-induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation-induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation-induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.

Concordant loss of fragile gene expression early in breast cancer development

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.

P-943 Effect of inducible Fhit and p53 expression in Calu-1 lung cancercell line

P-943 Effect of inducible Fhit and p53 expression in Calu-1 lung cancercell line

Hypermethylation patterns in the Fhit regulatory region are tissue specific

DNA hypermethylation is associated with decreased expression of tumor suppressor genes. We previously observed decreased Fhit expression and Fhit promoter region hypermethylation in rodent tumors induced by various carcinogens, and noted that the 5' regulatory regions in the promoter, exon 1, and intron 1 were differentially methylated, depending on the tissue of origin. Because different carcinogens were used for induction of tumors of the different organs, we could not conclude that the methylation patterns were tissue-specific. To determine if in rat tissues: (1) Fhit methylation status is related to expression levels and (2) Fhit methylation patterns were tissue- or carcinogen-specific, we examined Fhit methylation status and expression levels in DMBA- and MNU-induced benign and malignant mammary tumors. Fhit intron 1 was methylated in 3/9 DMBA and all of MNU-induced benign mammary tumors, in association with reduced Fhit expression levels; Fhit promoter and intron 1 were methylated in all DMBA and MNU-induced carcinomas in association with highly reduced Fhit expression levels. Treatment of rat cancer cells in vitro with the DNA methyltransferase inhibitor, 5'-Aza-2'deoxycytidine, for 4 d, increased Fhit expression and altered the methylation status. Before treatment, both promoter and intron 1 regions were methylated; after treatment, only intron 1 remained methylated. Thus, in carcinogen-exposed rat tissues there is an overall association of Fhit expression with regulatory region methylation, and hypermethylation patterns did not vary with carcinogen. The specific patterns of hypermethylated CpGs in the Fhit regulatory regions thus appear to be tissue-specific.

Components of DNA Damage Checkpoint Pathway Regulate UV Exposure–Dependent Alterations of Gene Expression of FHIT and WWOX at Chromosome Fragile Sites

Common chromosome fragile sites are highly recombinogenic and susceptible to deletions during the development of environmental carcinogen-induced epithelial tumors. Previous studies showed that not only genetic but also epigenetic alterations in cancerous cells are involved in inactivation of the genes FHIT and WWOX at chromosome fragile sites, reported to be potential tumor suppressor genes. Here we investigated the effect of UV light on the gene expression. After exposure to UV, the mRNA and protein of the two genes in murine embryonic fibroblasts (MEF) were unstable, apparently at the G1-S phase of the cell cycle, which was consistent with nuclear run-on assay. A study of MEFs synchronized via a double thymidine block indicated that, after the exposure, the expression of Fhit and Wwox was reduced in E2f-1-deficient cells and markedly in wild-type cells, whereas the reduction was partially inhibited in Trp53-deficient cells; cells at the S phase seemed to be sensitive to exogenous FHIT, suggesting a role of the checkpoint at the G1-S phase in the stability of gene expression and a possible involvement of FHIT function at the S phase. The transfection experiment showed that the UV-induced decrease in expression was partially inhibited by transfection of kinase-dead Atr (ataxia telangiectasia mutated and Rad3 related), which is a sensor of UV-induced damage. Taken together, the present study showed that UV-induced alterations of the fragile site gene expression are involved at least partially in the checkpoint function, suggesting the role in the process of carcinogenesis after exposure to UV.

Loss of Fhit Expression in Squamous Cell Carcinoma and Premalignant Lesions of the Larynx

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.

Fragile genes as biomarkers: epigenetic control of WWOX and FHIT in lung, breast and bladder cancer

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.

Loss ofFHITexpression in gastric mucosa of patients with family histories of gastric cancer andHelicobacter pyloriinfection

To answer the question whether FHIT gene expression is affected by the family history of gastric carcinoma and the presence of Helicobacter pylori (H pylori) in the gastric mucosa of patients with dyspepsia. FHIT gene expression in two different topographic sites of the gastric mucosa of twenty-one patients with dyspepsia and with or without familial gastric carcinoma, infected or not infected with H pylori, was evaluated by reverse transcription-PCR (RT-PCR) and IMAGE QUANT methods. A rapid urease test and histopathological examination were used to determine H pylori colonization. In the gastric mucosa of patients with family histories of gastric carcinoma, the amount of FHIT protein mRNA was reduced down to 32%, and for patients with H pylori colonization, to 24% in comparison to controls with dyspepsia and without cancer in the family. FHIT expression was independent of the topography of specimens (corpus vs antrum), and for the control patients it was less sensitive to infection with H pylori. A considerable statistical difference in FHIT levels was observed in the gastric mucosa from the corpus of patients with family histories of gastric carcinoma in respect to H pylori colonization (P = 0.06). Macroscopic evaluation of the gastric mucosa demonstrated that pathologic changes classified according to the Sydney system had no significant influence on FHIT expression within each tested group of patients. Loss of FHIT expression was observed in patients with dyspepsia and family histories of gastric carcinoma, especially those infected with H pylori. Such results may constitute an early indication of the development of gastric carcinoma, which is associated with family factors including heredity and H pylori infection. The loss of the FHIT gene may serve as a marker for early diagnosis and prevention of gastric carcinoma, especially in context of early monitoring of H pylori infection in individuals with a record of familial stomach cancer.

Expression and significance of Ki67 and FHIT In adrenocortical tumor tissue

[Objective] To study the relationship between Ki67, P16 and adrenocortical tumors. [Methods] The Labeling Index (LI) of Ki67 and P16 was detected by immunohistochemical staining in 68 cases of adrenocortical carcinoma(8 cases), adrenocortical adenoma (60 cases) and normal adrenocortical tissue beside tumor (9 cases). [Results] There is the highest expression of Ki67 and the lowest expression of P16 in adrenocortical carcinoma. There exist negative correlation between the expression of Ki67 and P16 (r=-0.7136,P 0.05). There is significant difference in the LI of Ki67 and that of P16 between adrenocortical carcinoma and adrenocortical adenoma (P 0.001 ). The cutpoint of LI of Ki67 was 4% and the cutpoint of LI of P16 was 12% in adrenocortical carcinoma. [Conclusions] The LI of Ki67 above 4% and LI of P16 below 12%,is a sensitive indicator of adrenocortical carcinoma. It is suggested that combine detecting the expression of Ki67 and P16 may be taken as one of biomarkers for differentiation of adrencortical adenomas from adrencortical carcinomas.

4-Hydroxybutyl(butyl)nitrosamine-induced urinary bladder cancers in mice: characterization of FHIT and survivin expression and chemopreventive effects of indomethacin

The administration of 4-hydroxybutyl(butyl)nitrosamine (OH-BBN) to male B6D2F1 mice yielded a high incidence of large palpable urinary bladder cancers. Since prior studies demonstrated chemopreventive effects of non-steroidal anti-inflammatory drugs (NSAIDs), we further explored the efficacy of the NSAID indomethacin using different treatment regimens. OH-BBN was administered twice per week for 12 weeks (the first week of treatment was designated week 1). In Experiment I continual indomethacin treatment (20 mg/kg diet) was initiated either prior to (week -1) or following (week 13) OH-BBN dosing. Palpable bladder masses (subsequently diagnosed as cancers) developed in 32% of carcinogen-treated only mice by 32 weeks, while mice administered indomethacin either prior to or after OH-BBN developed palpable masses in 3 and 6% of the animals, respectively. In Experiment II mice were treated with indomethacin beginning 1 week after OH-BBN for either 12 weeks (limited treatment, weeks 13-24) or for 30 weeks (weeks 13-42). Continual treatment resulted in a 77% decrease in palpable bladder masses and an 82% decrease in all cancers (palpable and microscopic), while limited treatment decreased palpable masses by 48% but failed to decrease the number of bladder cancers (palpable plus microscopic). In Experiment III OH-BBN-treated mice were followed for 61 weeks. Palpable masses developed in 66% of control mice, while 26% of mice treated with indomethacin continually from 1 week after OH-BBN (weeks 13-61) developed palpable masses. A separate group in this study treated with indomethacin beginning when 5% of the mice had palpable bladder masses continued to develop new masses for an additional 4 weeks. By 6 weeks after beginning indomethacin treatment, however, these animals showed a profound decrease in the development of additional cancers. The expressions of FHIT and survivin in normal urinary bladder epithelium and in bladder cancers were determined by immunohistochemical analysis. FHIT was expressed at high levels in normal epithelium, but was minimally expressed in cancers, and even showed decreased expression in papillomas. The anti-apoptotic protein survivin was not expressed in normal bladder epithelium, but was variably expressed in cancers. FHIT and survivin expressions were similar in cancers from indomethacin-treated and non-treated mice.

5′CpG island hypermethylation and aberrant transcript splicing both contribute to the inactivation of the FHIT gene in resected non-small cell lung cancer

The relative contribution of promoter hypermethylation and aberrant splicing to the inactivation of the fragile histidine triad ( FHIT) gene is unclear. Using genetic and epigenetic analyses, the current investigation examines the loss of protein and mRNA expression, and 5′CpG hypermethylation and allelic imbalance of the FHIT gene in a series of 129 non-small cell lung cancer (NSCLC) samples, in parallel with clinicopathological analyses. We found that 50% of NSCLC patients had aberrant protein expression, which was more frequent in squamous cell carcinomas (SQ) (69%) than in adenocarcinomas (AD) (28%) ( P < 0.0001). 5′CpG hypermethylation of FHIT was identified in 31% of patients. Abnormally-sized FHIT transcripts were also observed in 24% of patients and were attributed to various exonic deletions, mainly in the region of exons 4–8. Allelic imbalance of the FHIT locus and its correlation with the status of Fhit expression, 5′CpG hypermethylation, and aberrant splicing, indicated that biallelic inactivation of Fhit expression could be induced by 5′CpG hypermethylation of one allele and alternative splicing in the other allele. Moreover, an 83% concordance in the methylation status of FHIT was demonstrated between 12 samples of bronchial precancerous lesions taken before surgery and their matched resected tumours. Our data suggest that FHIT 5′CpG hypermethylation and splicing alterations are both predominant mechanisms involved in the aberrant expression of the FHIT gene, and that FHIT 5′CpG methylation may be potentially used as a supplemental detection marker for NSCLC.

Immunochemoconjugate of anti-DNMT1/HDAC2 bispecific F(ab)2-bsAb linked with cleavable disulfide to vinorelbine induces apoptosis in chemoresistant infiltrating ductal carcinoma (IDC) characterised by HDAC2 overexpression and 5'CpG island hypermethylation of the FHIT, RAR-β2, BRCA-1, APC, p16(CDKN2A), RASSF1A, CDH1(E-cadherin), 14–3-3-σ (stratifin), HIC1 and MDG1 tumor suppressor genes

2088 One of the reasons for chemoresistance to IDC is deacetylation and DNA methylation which causes silencing of tumor suppressor genes(TSGs).DNMT1 interacts with HDAC2 to repress transcription of TSGs. IDC cells obtained by surgical excision from a patient were analysed by IHC,PCR-based LOH, ChIP, MSP,RT-PCR and NB.There was loss of FHIT expression,LOH at FHIT and 5' CpG island methylation of the FHIT .There was transcriptional silencing of the following TSGs: BRCA1,RAR-β2,APC,p16 (CDKN2A), RASSF1A,CDH1(E-cadherin),14–3-3-σ (stratifin),HIC1 and MDG1.There was overexpression of DNMT1 and HDAC2 suggesting a link between histone deacetylation,cytosine methylation,local chromatin condensation and subsequent transcriptional repression.This chemoresistant IDC was defined as CIMP+.We treated IDC cells with anti-DNMT1/HDAC2 bispecific F(ab)2-bsAb linked with cleavable disulfide to VRL . Post-treatment, there was inhibition of HDAC2 and DNMT1 blocking the 5'CpG island methylation of the tumour suppressor genes resulting to transcriptional activation by upregulation of their mRNA. There was histone hyperacetylation which opens chromatin structure in which the DNA is more loosely wrapped around the histones making it more receptive to interaction with transcription factors.Overexpression of the TSGs combined with the MT depolymerizing action of VRL inhibited metabolic activity and DNA synthesis of tumor cells according to MTT and BrdU.Immunological analysis exhibited ADCC.There was induction of PCD in IDC cells according to TUNEL and TEM.A large number of tumor cells formed apoptotic bodies which were phagocytosed by adjacent tumor cells leading to a bystander killing effect.Concluding,this therapeutic approach with immunovinorelbine (IVRL) may revolutionize IDC treatment adding significantly to the current clinical armamentarium. No significant financial relationships to disclose.

Immunochemoconjugate of anti-DNMT1/HDAC2 bispecific F(ab)2-bsAb linked with cleavable disulfide to vinorelbine induces apoptosis in chemoresistant infiltrating ductal carcinoma (IDC) characterised by HDAC2 overexpression and 5'CpG island hypermethylation of the FHIT, RAR-β2, BRCA-1, APC, p16(CDKN2A), RASSF1A, CDH1(E-cadherin), 14–3-3-σ (stratifin), HIC1 and MDG1 tumor suppressor genes

2088 One of the reasons for chemoresistance to IDC is deacetylation and DNA methylation which causes silencing of tumor suppressor genes(TSGs).DNMT1 interacts with HDAC2 to repress transcription of TSGs. IDC cells obtained by surgical excision from a patient were analysed by IHC,PCR-based LOH, ChIP, MSP,RT-PCR and NB.There was loss of FHIT expression,LOH at FHIT and 5' CpG island methylation of the FHIT .There was transcriptional silencing of the following TSGs: BRCA1,RAR-β2,APC,p16 (CDKN2A), RASSF1A,CDH1(E-cadherin),14–3-3-σ (stratifin),HIC1 and MDG1.There was overexpression of DNMT1 and HDAC2 suggesting a link between histone deacetylation,cytosine methylation,local chromatin condensation and subsequent transcriptional repression.This chemoresistant IDC was defined as CIMP+.We treated IDC cells with anti-DNMT1/HDAC2 bispecific F(ab)2-bsAb linked with cleavable disulfide to VRL . Post-treatment, there was inhibition of HDAC2 and DNMT1 blocking the 5'CpG island methylation of the tumour suppressor genes resulting to transcriptional activation by upregulation of their mRNA. There was histone hyperacetylation which opens chromatin structure in which the DNA is more loosely wrapped around the histones making it more receptive to interaction with transcription factors.Overexpression of the TSGs combined with the MT depolymerizing action of VRL inhibited metabolic activity and DNA synthesis of tumor cells according to MTT and BrdU.Immunological analysis exhibited ADCC.There was induction of PCD in IDC cells according to TUNEL and TEM.A large number of tumor cells formed apoptotic bodies which were phagocytosed by adjacent tumor cells leading to a bystander killing effect.Concluding,this therapeutic approach with immunovinorelbine (IVRL) may revolutionize IDC treatment adding significantly to the current clinical armamentarium. No significant financial relationships to disclose.

Understanding the mechanisms of FHIT inactivation in cervical cancer for biomarker development.

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. 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 transcriptase (RT)-PCR. Western blot analysis and immunohistochemistry were performed to characterize Fhit protein expression. Homozygous deletions were found in six of nine cervical cancer cell lines, but only one 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. 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.