Biomedical subjects

Kay Huebner

Publications and source records attributed to Kay Huebner.

At least 37 records · Page 2Linked to original sources

PubMed2004

Differentially expressed genes execute zinc-induced apoptosis in precancerous esophageal epithelium of zinc-deficient rats.

Zinc deficiency (ZD) in rats increases esophageal cell proliferation and the incidence of N-nitrosomethylbenzylamine-induced esophageal tumors. Conversely, zinc replenishment (ZR) rapidly induces apoptosis in esophageal epithelia and reverses cancer development. We investigated gene expression changes in ZR versus ZD esophageal epithelia to identify differentially expressed genes associated with the antitumor effect of ZR. Weanling rats were fed a ZD diet for 6 weeks to establish esophageal cell proliferation or a zinc-sufficient (ZS) diet. Then, 10 ZD rats were treated with zinc gluconate intragastrically and switched to ZS diet; the remaining 10 ZD and ZS animals were treated with saline. All animals were killed 26-28 h later. Using cDNA microarrays, real-time polymerase chain reaction amplification and RNA hybridization techniques, we identified novel differentially expressed genes, including a RNA-binding protein with two RNA recognition motifs and a zinc knuckle (ZD7), and a DNA/RNA helicase with a DEAD box (ZD10) with two splice variants, ZD10a and ZD10b. In situ hybridization detected increased mRNA expression of ZD7, ZD10a and ZD10b in ZR esophageal epithelia, which displayed markedly increased occurrence of apoptotic cells, relative to ZD epithelia. Overexpression of ZD7 in human esophageal cancer cells resulted in induction of apoptosis and activation of caspase-3 and -7, activities that were inhibited by caspase-specific inhibitors. In addition, ZD7 mRNA levels and zinc-induced apoptosis in rat squamous carcinoma cells were reduced by specific small interfering ribonucleic acids. Thus, ZR rapidly induces ZD7 and ZD10 expression, which in turn stimulates apoptosis. These results provide the beginnings of a molecular pathway for zinc-induced apoptosis under conditions that reverse esophageal tumor initiation.

Animals↗

PubMed2004

CpG methylation in the Fhit regulatory region: relation to Fhit expression in murine tumors.

To determine if: (1) 5' CpG island methylation is related to Fhit inactivation; (2) there are tumor or carcinogen-specific methylation patterns, we examined 35 CpG sites in the promoter, exon and intron 1 of the mouse Fhit gene. In primary tumors of lung, urinary bladder and tongue, induced by different carcinogens, 15-35% of sites were methylated, with specific methylation patterns associated with each cancer type, suggesting cancer- or tissue-specific methylation patterns. The methylation patterns were associated with reduced Fhit expression, as determined by immunohistochemical analyses. Methylation of rat Fhit 5' CpGs in mammary adenocarcinomas, detected by methylation specific PCR amplification, also correlated with reduced gene expression. Thus, there was an overall association between promoter/exon 1 methylation and decreased Fhit expression. In contrast, in cancer-derived cell lines 70-95% of the CpG sites were methylated. This is the first detailed study of the relationship between Fhit 5' CpG island methylation and Fhit expression in murine tumors, our main models for preclinical cancer studies, and provides evidence that loss of Fhit expression and methylation are correlated in these mouse models and these models will be useful to examine the complex relationships among gene expression, methylation patterns and organ specificity.

Acid Anhydride Hydrolases↗

PubMed2004

Loss of WWOX expression in gastric carcinoma.

PURPOSE: WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene that maps to the common fragile site FRA16D on chromosome 16q23.3-24.1. To investigate the role of the WWOX gene in the development of gastric carcinoma, we examined a large series of primary adenocarcinomas and nine gastric cancer cell lines for the expression of Wwox. EXPERIMENTAL DESIGN: Loss of heterozygosity, reverse-transcription-PCR, and immunohistochemistry were used to assess the role of WWOX in stomach cancer. A total of 81 primary gastric adenocarcinoma were analyzed. RESULTS: Loss of heterozygosity was observed in 31% of the cases and loss of Wwox protein expression was found in 65% of gastric adenocarcinoma primary specimens and 33% of gastric cancer cell lines. In addition, we found a high correlation between Wwox and Fhit protein expression. CONCLUSIONS: Our results indicate that alterations of the WWOX gene may be involved quite frequently in gastric tumorigenesis. Our data could be used in future studies to develop diagnostic and targeted therapy of stomach cancer.

Adenocarcinoma↗

PubMed2004

The fragile genes FHIT and WWOX are inactivated coordinately in invasive breast carcinoma.

BACKGROUND: FHIT and WWOX are a tumor suppressor and a candidate suppressor that encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3-24.1, respectively. Reduced or absent Fhit expression has been reported in two-thirds of invasive breast tumors in association with adverse prognostic factors. Loss of 16q has been reported frequently in low-grade, invasive breast tumors. METHODS: Expression of Fhit and Wwox was evaluated by immunohistochemical staining in 97 archived breast carcinoma specimens. Expression levels were analyzed for correlations with each other, as well as with various patient and tumor characteristics. RESULTS: Reduced Fhit and Wwox expression in tumors was observed in 54.6% and 63.2% of specimens, respectively. Fhit and Wwox expression were correlated strongly (P = 0.001). Reduced Fhit staining was seen more frequently in premenopausal patients (P = 0.010), estrogen receptor (ER)-negative or scantly ER-positive tumors (P = 0.058 borderline), high-grade tumors (P = 0.005), and tumors with metastases (P = 0.041). Reduced Wwox staining was more common in tumors with less favorable ER status (P = 0.033). Wwox expression in normal tissue was reduced in 32.9% of specimens, especially in patients with higher stage disease (P = 0.033). Severely reduced Wwox staining (extent < 10%) in normal tissue was found only in postmenopausal women, but reduced Wwox staining (11-75%) was more common in premenopausal women (P = 0.012). Tumor status, lymph node status, and intensity of Fhit expression in tumors were related independently to survival (P = 0.003, P < 0.001, and P = 0.046, respectively). CONCLUSIONS: The strong correlation observed between Fhit and Wwox expression was consistent with the common elevated susceptibility of fragile sites to DNA damage. Reduced Fhit expression is associated with adverse prognostic factors. The current results suggest that Wwox also has an important and complex association with steroid hormone expression and breast carcinogenesis.

Acid Anhydride Hydrolases↗

PubMed2004

Functional association between Wwox tumor suppressor protein and p73, a p53 homolog.

The WWOX gene is a recently cloned tumor suppressor gene that spans the FRA16D fragile region. Wwox protein contains two WW domains that are generally known to mediate protein-protein interaction. Here we show that Wwox physically interacts via its first WW domain with the p53 homolog, p73. The tyrosine kinase, Src, phosphorylates Wwox at tyrosine 33 in the first WW domain and enhances its binding to p73. Our results further demonstrate that Wwox expression triggers redistribution of nuclear p73 to the cytoplasm and, hence, suppresses its transcriptional activity. In addition, we show that cytoplasmic p73 contributes to the proapoptotic activity of Wwox. Our findings reveal a functional cross-talk between p73 and Wwox tumor suppressor protein.

Cell Line↗

PubMed2004

Fhit is a physiological target of the protein kinase Src.

The FHIT gene is a tumor suppressor that is frequently inactivated by genomic alterations at chromosomal region 3p14.2. In the last few years, a considerable amount of data describing inactivation of FHIT in a variety of human malignancies and demonstrating the tumor suppressor potential of Fhit have been reported. Despite the demonstration that FHIT functions as a tumor suppressor, the pathway through which Fhit induces apoptosis and inhibits growth of cancer cells is not known. Our data demonstrate that Fhit is a target of tyrosine phosphorylation by the Src protein kinase. We show that Src phosphorylates Y114 of Fhit in vitro and in vivo, providing insight into a biochemical pathway involved in Fhit signaling.

3T3 Cells↗

PubMed2004

Cancer prevention and therapy in a preclinical mouse model: impact of FHIT viruses.

A link between common chromosome fragile sites and frequent chromosomal deletions in cancer was observed two decades ago and led to the hypothesis that genes at fragile sites may play a role in tumor development. In 1996, the human fragile histidine triad gene, FHIT, was identified by positional cloning of the chromosome region spanning the carcinogen-sensitive, common fragile site, FRA3B at 3p14.2. Loss or inactivation of the FHIT gene in a large fraction of human tumors results in absence or reduction of Fhit protein. In vitro analyses and in vivo tumorigenicity studies show that restoration of Fhit protein induces tumor suppression in 50% of tumor cell lines tested. Viral vector-mediated FHIT gene transfer to Fhit-deficient mice not only prevents but reverses the carcinogen-induced tumor development in vivo, in accordance with the oncosuppressive properties of Fhit protein. The strong proapoptotic activity following Fhit infection of cancer cells strengthens the case for further exploration of FHIT gene therapy in cancer prevention and treatment.

Acid Anhydride Hydrolases↗

PubMed2003

Fragile site orthologs FHIT/FRA3B and Fhit/Fra14A2: evolutionarily conserved but highly recombinogenic.

Common fragile sites are regions that show elevated susceptibility to DNA damage, leading to alterations that can contribute to cancer development. FRA3B, located at chromosome region 3p14.2, is the most frequently expressed human common fragile site, and allelic losses at FRA3B have been observed in many types of cancer. The FHIT gene, encompassing the FRA3B region, is a tumor-suppressor gene. To identify the features of FHIT/FRA3B that might contribute to fragility, sequences of the human FHIT and the flanking PTPRG gene were compared with those of murine Fhit and Ptprg. Human and mouse orthologous genes, FHIT and Fhit, are more highly conserved through evolution than PTPRG/Ptprg and yet contain more sequence elements that are exquisitely sensitive to genomic rearrangements, such as high-flexibility regions and long interspersed nuclear element 1s, suggesting that common fragile sites serve a function. The conserved AT-rich high-flexibility regions are the most characteristic of common fragile sites.

Acid Anhydride Hydrolases↗

PubMed2003

Combined cyclin D1 overexpression and zinc deficiency disrupts cell cycle and accelerates mouse forestomach carcinogenesis.

Overexpression of cyclin D1 and disruption of cell cycle control in G(1) occur frequently in human esophageal cancer. Transgenic (TG) mice with cyclin D1 overexpression targeted to the oral-esophageal tissue by the EBV ED-L2 promoter showed increased severity in esophageal dysplasia without cancer development, after multiple doses of N-nitrosomethylbenzylamine (NMBA). Dietary zinc deficiency (ZD) in mice enhances cellular proliferation in esophagus/forestomach and susceptibility to NMBA-induced carcinogenesis. We investigated whether cyclin D1 overexpression in TG mice, together with ZD, might lead to unchecked cell proliferation and accelerated NMBA-induced tumorigenesis. Five-week-old TG and wild-type (WT) mice were fed a ZD- or -sufficient (ZS) diet, forming four groups: ZD:TG; ZS:TG; ZD:WT; and ZS:WT. After 4 weeks, animals were given a single intragastric NMBA dose and were sacrificed 25 and 77 days later. Without NMBA, cell proliferation was greatest in ZD:TG esophagus/forestomach, followed by ZD:WT, and then ZS:TG>/=ZS:WT. The high rate of cell proliferation was accompanied by overexpression of cell cycle progression and tumorigenesis biomarkers, including proliferating cell nuclear antigen, cyclin D1, cyclin-dependent kinase 4, p53, cytokeratin 14, epidermal growth factor receptor, and by a reduced rate of apoptosis. ZD substantially increased forestomach tumor incidence in TG mice: 85% of ZD:TG versus 14% of ZS:TG mice had forestomach tumors (P < 0.001), with progression to malignancy occurring only in ZD:TG tumors. Additionally, 14% of ZD:TG mice developed esophageal tumors and esophageal intestinal metaplasia at 77 days. Thus, cyclin D1 overexpression, in cooperation with ZD, decontrols cell proliferation, ensuring cell expansion, a prerequisite for cancer development.

Animals↗

PubMed2003

Regression of upper gastric cancer in mice by FHIT gene delivery.

Fhit expression is reduced in most cancers, and Fhit replacement by FHIT expression viruses in lung, esophageal, pancreatic, and cervical cancers induces apoptosis in the cancer cells. Mice carrying one or two inactivated Fhit alleles are hypersensitive to development of N-nitrosomethylbenzylamine (NMBA)-induced forestomach tumors. In the present study, we investigated the kinetics and mechanism of tumor reversal and intervention by oral delivery of FHIT expression viruses. Tumor analysis showed that: a) by 37 days post-NMBA, control mice showed approximately 7 tumors and by 84 days approximately 10 tumors/forestomach; b) mice receiving FHIT virus at 2 or 42 days post-NMBA showed significantly reduced tumor burdens; c) Fhit was still expressed at 82 days postinfection; d) control viral infection had no effect on tumor development; and e) reduced Bcl2, increased Bax expression, and increased TUNEL-positive apoptotic nuclei characterized the restored epithelia of FHIT transduced forestomachs. Thus, FHIT viral gene delivery prevents or retards development of carcinogen-induced forestomach tumors and reverses development of established tumors by 60-70% through an apoptotic pathway. This dramatic reduction in tumor burden emphasizes the efficacy of targeting the FHIT apoptotic pathway for tumor eradication.

Acid Anhydride Hydrolases↗

PubMed2003

WW domain containing oxidoreductase gene expression is altered in non-small cell lung cancer.

WWOX (WW domain containing oxidoreductase), a putative tumor suppressor gene that maps to the common fragile site FRA16D on chromosome 16q23.3-24.1, is altered in breast, esophageal, and ovarian cancer. Because the FRA3B/FHIT locus at 3p14.2 is a preferential target for genetic changes caused by tobacco smoke, we intended to evaluate the status of the FRA16D/WWOX gene in non-small cell lung cancer; we have analyzed 27 paired normal and tumor lung tissues and 8 lung cancer cell lines for WWOX alterations by reverse transcriptase-PCR, loss of heterozygosity, and mutation analysis. Transcripts missing WWOX exons were detected in 7 primary tumors (7 of 27; 25.9%) and 5 of 8 cell lines. In addition, loss of heterozygosity at the WWOX locus was observed in 10 primary tumors (10 of 27; 37.0%). We conclude that WWOX alterations occur in a significant fraction of lung cancers and may contribute to the pathogenesis of non-small cell lung cancer.

Carcinoma, Non-Small-Cell Lung↗

PubMed2003

Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding.

The FHIT gene is inactivated early in the development of many human tumors, and Fhit-deficient mice have increased cancer incidence. Viral reexpression of Fhit kills Fhit-deficient cells by induction of apoptosis. Fhit, a member of branch 2 of the histidine-triad superfamily of nucleoside monophosphate hydrolases and transferases, is a diadenosine polyphosphate hydrolase, the active-site histidine of which is not required for tumor suppression. To provide a rigorous test of the hypothesis that Fhit function depends on forming a complex with substrates, we designed a series of alleles of Fhit intended to reduce substrate-binding andor hydrolytic rates, characterized these mutants biochemically, and then performed quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in k(cat) and increases as large as 30-fold in K(M). Nonetheless, when mutant FHIT genes were expressed in two human cancer cell lines containing FHIT deletions, reductions in apoptotic activity correlated exclusively with K(M). Mutants with 2- and 7-fold increases in K(M) significantly reduced apoptotic indices, whereas the mutant with a 30-fold increase in K(M) retained little cellular function. These data indicate that the proapoptotic function of Fhit is limited by substrate binding and is unrelated to substrate hydrolysis.

Acid Anhydride Hydrolases↗

PubMed2003

p53 deficiency accelerates induction and progression of esophageal and forestomach tumors in zinc-deficient mice.

The p53 tumor suppressor protein plays a pivotal role in preventing uncontrolled cellular proliferation. By contrast, zinc deprivation enhances esophageal cell proliferation and the induction of esophageal tumors in rodents by N-nitrosomethylbenzylamine (NMBA). We investigated whether p53 deficiency rendered zinc-deficient (ZD) mice more susceptible to NMBA-induced esophageal/forestomach carcinogenesis. At 6-7 weeks of age, p53 null (-/-), heterozygous (+/-), and wild-type (+/+) mice were placed on ZD or zinc-sufficient (ZS) diets to form six experimental groups: ZD:p53-/-; ZD:p53+/-; ZD:p53+/+; ZS:p53-/-; ZS:p53+/-; and ZS:p53+/+. After 3 weeks, 15-23 mice in each group were treated once with NMBA (2 mg/kg body weight). Control animals were untreated. Zinc deficiency alone induced unrestrained cellular proliferation in the esophagus and forestomach of p53-/- mice. Forestomach tumors were first detected in a ZD:p53-/- mouse at 13 days. By 30 days, 100% (21 of 21) of ZD:p53-/- mice developed forestomach tumors and 38% showed esophageal tumors versus 42 and 0% in ZS:p53-/- mice (P < 0.004, esophagus; P < 0.001, forestomach). ZD:p53-/- mice showed an accelerated progression to malignancy, with 10% of esophageal tumors and 38% of forestomach tumors presenting as carcinomas. Nearly 20% of ZD:p53-/- mice developed esophageal Barrett's metaplasia, a lesion not previously seen in NMBA-induced neoplasia. ZD:p53+/- mice had significantly higher tumor incidence than ZS:p53+/- mice. The order of tumor incidence in forestomach was as follows: naught incidence in ZS:p53+/+ mice; ZD:p53-/- > ZD:p53+/- > ZS:p53-/- > ZD:p53+/+ >/= ZS:p53+/- > ZS:p53+/+. The rapid rate of tumor induction/progression in ZD:p53-/- mice was accompanied by an increase in the rate of cell proliferation and a decrease in apoptosis. cDNA array expression analysis of known genes identified a 5-fold up-regulation of cytokeratin 14 mRNA expression in ZD:p53-/- versus ZS:p53-/- forestomach, a result showing gene-modulating effects of zinc deficiency. Cytokeratin 14 is a biomarker in human esophageal carcinogenesis. Our findings provide in vivo evidence for the collaboration of a deficiency of both p53 and zinc in esophageal carcinogenesis and reveal molecular targets of this collaboration.

Animals↗

PubMed2003

Candidate tumor suppressor genes at FRA7G are coamplified with MET and do not suppress malignancy in a gastric cancer.

Common fragile sites predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer. Chromosomal fragile sites not only are susceptible to DNA instability in cancer cells, but may also be associated with genes that contribute to the neoplastic process. FRA7G is a common fragile site containing the candidate tumor suppressor genes CAV1, CAV2, and TESTIN (TES). The human gastric cancer cell line GTL-16 has an amplification of this genomic region and was used to seek evidence for the suppressor candidacy of one of these genes. Our results demonstrate that CAV1, CAV2, and TESTIN are coamplified with the MET oncogene and overexpressed in GTL-16. Somatic mutation was not detected in the coding regions of these genes, although they were each overexpressed. The results show that CAV1, CAV2, and TESTIN are not tumor suppressor genes in this gastric cancer.

Chromosome Fragility↗

PubMed2003

The histidine triad protein Hint is not required for murine development or Cdk7 function.

The histidine triad (HIT) protein Hint has been found to associate with mammalian Cdk7, as well as to interact both physically and genetically with the budding yeast Cdk7 homologue Kin28. To study the function of Hint and to explore its possible role in modulating Cdk7 activity in vivo, we have characterized the expression pattern of murine Hint and generated Hint-deficient (Hint(-/-)) mice. Hint was widely expressed during mouse development, with pronounced expression in several neuronal ganglia, epithelia, hearts, and testes from embryonic day 15 onward. Despite this widespread expression, disruption of Hint did not impair murine development. Moreover, Hint-deficient mice had a normal life span and were apparently healthy. Histological examination of tissues with high Hint expression in wild-type animals did not show signs of abnormal pathology in Hint(-/-) mice. Functional redundancy within the HIT family was addressed by crossing Hint(-/-) mice with mice lacking the related HIT protein, Fhit, and by assaying the expression levels of the HIT protein gene family members Hint2 and Hint3 in Hint(+/+) and Hint(-/-) tissues. Finally, Cdk7 kinase activity and cell cycle kinetics were found to be comparable in wild-type and Hint(-/-) mouse embryonic fibroblasts, suggesting that Hint may not be a key regulator of Cdk7 activity.

Acid Anhydride Hydrolases↗

PubMed2003

Expression of FRA16D/WWOX and FRA3B/FHIT genes in hematopoietic malignancies.

The WW domain containing oxidoreductase (WWOX) gene was recently identified as a candidate tumor suppressor gene at a common fragile site, FRA16D. Because the fragile histidine triad (FHIT) gene, a tumor suppressor gene encompassing the most active, common fragile site FRA3B, is frequently deleted in various cancers, we evaluated the expression of WWOX and FHIT in 74 cases of primary hematopoietic neoplasias and 20 leukemia cell lines. Aberration or absence of WWOX transcripts was detected in 51% of the primary cases and 55% of cell lines, and three WWOX nucleotide variants were detected among the leukemia cell lines. FHIT expression was absent or altered in 36% of the primary cases and 15% of cell lines. The occurrence of aberrant FHIT reverse transcription-PCR products correlated significantly with the occurrence of WWOX alterations. Wild-type transcripts of both genes were expressed in normal hematopoiesis along with a small fraction of short transcripts. A DNA blot study showed that WWOX and FHIT genes were deleted in 2 of 18 cases with primary acute leukemias; both genes were not expressed in the 2 cases. Furthermore, treatment of cells with a demethylating or histone acetylating agent in culture resulted in increased expression of WWOX and FHIT mRNA in leukemia cells. Conclusions are that WWOX expression is frequently altered or absent in hematopoietic disorders, often in association with FHIT alterations, and that alterations of these fragile genes may result not only from genomic deletions but also from epigenetic modifications associated with expression of fragility.

Acetylation↗

PubMed2002

The fragile histidine triad/common chromosome fragile site 3B locus and repair-deficient cancers.

In various studies of sporadic breast cancers, 40-70% were strongly positive for fragile histidine triad (Fhit) protein expression, whereas only 18% of BRCA2 mutant breast cancers demonstrated strong Fhit expression, suggesting that the BRCA2 repair function may be necessary to retain intact fragile common chromosome fragile site 3B(FRA3B)/FHITloci. In the current study, 22 breast tumors with deleterious BRCA1 mutations were analyzed for Fhit expression by immunohistochemistry in a case-control matched pair analysis. Loss of Fhit expression was significantly more frequent in the BRCA1 cancers compared with sporadic breast tumors (9% Fhit positive versus 68% Fhit positive), suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To investigate the relationship between repair gene deficiencies and induction of chromosome fragile sites in vitro, we have analyzed the frequency of aphidicolin induction of chromosome gaps and breaks in PMS2-, BRCA1-, MSH2-, MLH1-, FHIT-, and TP53-deficient cell lines. Each of the repair-deficient cell lines showed elevated expression of chromosome gaps and breaks, consistent with the proposal that proteins involved in mismatch and double-strand break repair are important in maintaining the integrity of common fragile regions. Correspondingly, genes at common fragile sites may sustain elevated levels of DNA damage in cells with deficient DNA repair proteins such as those mutated in several familial cancer syndromes.

Acid Anhydride Hydrolases↗

PubMed2002

FHIT: from gene discovery to cancer treatment and prevention.

Chromosomal abnormalities, including homozygous deletions and loss of heterozygosity, are among the most common features of human tumours. The short arm of human chromosome 3, particularly the region 3p14.2, is a major site of such rearrangements. The 3p14.2 region spans the most active common fragile site of the human genome, encompassing a familial-kidney-cancer-associated breakpoint and a papilloma virus integration site. 6 years ago, the FHIT gene was identified in this region. Subsequent studies have shown that FHIT is commonly the target of chromosomal aberrations involving the long arm of human chromosome 3 and is thereby inactivated in most of the common human malignant diseases, including cancers of the lung, oesophagus, stomach, breast, and kidney. During the past 5 years, evidence has accumulated in support of a tumour-suppressor function for FHIT. In this review, we describe the recent findings in the molecular biology of FHIT with particular focus on the opportunities for treatment and prevention of cancer that have emerged.

Acid Anhydride Hydrolases↗