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1.
Diabet Med ; 34(4): 582-585, 2017 04.
Article in English | MEDLINE | ID: mdl-27477181

ABSTRACT

AIMS: An early genetic diagnosis of neonatal diabetes guides clinical management and results in improved treatment in ~ 40% of patients. In the offspring of individuals with neonatal diabetes, a prenatal diagnosis allows accurate estimation of the risk of developing diabetes and, eventually, the most appropriate treatment for the baby. In this study, we performed non-invasive prenatal genetic testing for a fetus at risk of inheriting a paternal KCNJ11 p.R201C mutation causing permanent neonatal diabetes. METHODS: A droplet digital polymerase chain reaction assay was used to detect the presence of the mutation in cell-free circulating DNA (cfDNA) extracted from maternal plasma at 12 and 16 weeks' gestation. RESULTS: The mutation was not detected in the cfDNA samples, suggesting that the fetus had not inherited the KCNJ11 mutation. The fetal DNA fraction was estimated at 6.2% and 10.7%, which is above the detection limit of the assay. The result was confirmed by Sanger sequencing after the baby's birth, confirming that the baby's risk of developing neonatal diabetes was reduced to that of the general population. CONCLUSIONS: We report the first case of non-invasive prenatal testing in a family with neonatal diabetes. A prenatal diagnosis in families at high risk of monogenic diabetes informs both prenatal and postnatal management. Although the clinical impact of this novel technology still needs to be assessed, its implementation in clinical practice (including cases at risk of inheriting mutations from the mother) will likely have a positive impact upon the clinical management of families affected by monogenic diabetes.


Subject(s)
Cell-Free Nucleic Acids/analysis , Diabetes Mellitus/genetics , Potassium Channels, Inwardly Rectifying/genetics , DNA Mutational Analysis , Diabetes Mellitus/congenital , Diabetes Mellitus/diagnosis , Female , Fetus/metabolism , Genetic Testing , Humans , Polymerase Chain Reaction , Pregnancy , Pregnancy Trimester, First , Pregnancy Trimester, Second , Prenatal Diagnosis/methods , Sensitivity and Specificity
2.
Diabetologia ; 56(9): 1958-63, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23771172

ABSTRACT

AIMS/HYPOTHESIS: Current genetic tests for diagnosing monogenic diabetes rely on selection of the appropriate gene for analysis according to the patient's phenotype. Next-generation sequencing enables the simultaneous analysis of multiple genes in a single test. Our aim was to develop a targeted next-generation sequencing assay to detect mutations in all known MODY and neonatal diabetes genes. METHODS: We selected 29 genes in which mutations have been reported to cause neonatal diabetes, MODY, maternally inherited diabetes and deafness (MIDD) or familial partial lipodystrophy (FPLD). An exon-capture assay was designed to include coding regions and splice sites. A total of 114 patient samples were tested--32 with known mutations and 82 previously tested for MODY (n = 33) or neonatal diabetes (n = 49) but in whom a mutation had not been found. Sequence data were analysed for the presence of base substitutions, small insertions or deletions (indels) and exonic deletions or duplications. RESULTS: In the 32 positive controls we detected all previously identified variants (34 mutations and 36 polymorphisms), including 55 base substitutions, ten small insertions or deletions and five partial/whole gene deletions/duplications. Previously unidentified mutations were found in five patients with MODY (15%) and nine with neonatal diabetes (18%). Most of these patients (12/14) had mutations in genes that had not previously been tested. CONCLUSIONS/INTERPRETATION: Our novel targeted next-generation sequencing assay provides a highly sensitive method for simultaneous analysis of all monogenic diabetes genes. This single test can detect mutations previously identified by Sanger sequencing or multiplex ligation-dependent probe amplification dosage analysis. The increased number of genes tested led to a higher mutation detection rate.


Subject(s)
Diabetes Mellitus, Type 2/diagnosis , Diabetes Mellitus, Type 2/genetics , Genetic Testing/methods , High-Throughput Nucleotide Sequencing/methods , Female , Humans , Infant, Newborn , Male , Mutation
3.
Diabetologia ; 55(9): 2381-5, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22660720

ABSTRACT

AIMS: The gene SLC2A2 encodes GLUT2, which is found predominantly in pancreas, liver, kidney and intestine. In mice, GLUT2 is the major glucose transporter into pancreatic beta cells, and biallelic Slc2a2 inactivation causes lethal neonatal diabetes. The role of GLUT2 in human beta cells is controversial, and biallelic SLC2A2 mutations cause Fanconi-Bickel syndrome (FBS), with diabetes rarely reported. We investigated the potential role of GLUT2 in the neonatal period by testing whether SLC2A2 mutations can present with neonatal diabetes before the clinical features of FBS appear. METHODS: We studied SLC2A2 in patients with transient neonatal diabetes mellitus (TNDM; n = 25) or permanent neonatal diabetes mellitus (PNDM; n = 79) in whom we had excluded the common genetic causes of neonatal diabetes, using a combined approach of sequencing and homozygosity mapping. RESULTS: Of 104 patients, five (5%) were found to have homozygous SLC2A2 mutations, including four novel mutations (S203R, M376R, c.963+1G>A, F114LfsX16). Four out of five patients with SLC2A2 mutations presented with isolated diabetes and later developed features of FBS. Four out of five patients had TNDM (16% of our TNDM cohort of unknown aetiology). One patient with PNDM remains on insulin at 28 months. CONCLUSIONS: SLC2A2 mutations are an autosomal recessive cause of neonatal diabetes that should be considered in consanguineous families or those with TNDM, after excluding common causes, even in the absence of features of FBS. The finding that patients with homozygous SLC2A2 mutations can have neonatal diabetes supports a role for GLUT2 in the human beta cell.


Subject(s)
Diabetes Mellitus/genetics , Fanconi Syndrome/genetics , Glucose Transporter Type 2/genetics , Insulin/metabolism , Mutation , Base Sequence , Carbohydrate Metabolism, Inborn Errors/genetics , DNA Primers , Diabetes Mellitus/diagnosis , Fanconi Syndrome/diagnosis , Female , Humans , Infant, Newborn , Insulin/genetics , Insulin Secretion , Male
4.
Cell Death Differ ; 14(10): 1780-91, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17627285

ABSTRACT

The sphingoplipid ceramide is responsible for a diverse range of biochemical and cellular responses including a putative role in modulating cell cycle progression. Herein, we describe that an accumulation of ceramide, achieved through the exogenous application of C(6)-ceramide or exposure to sphingomyelinase, induces a G(2) arrest in Rhabdomyosarcoma (RMS) cell lines. Utilizing the RMS cell line RD, we show that this G(2) arrest required the rapid induction of p21(Cip1/Waf1) independent of DNA damage. This was followed at later time points (48 h) by the commitment to apoptosis. Apoptosis was prevented by Bcl-2 overexpression, but permitted the maintenance of elevated p21(Cip1/Waf1) protein expression and the stabilization of the G(2) arrest response. Inhibition of p21(Cip1/Waf1) protein synthesis with cyclohexamide (CHX) or silencing of p21(Cip1/Waf1) with siRNA, prevented ceramide-mediated G(2) arrest and the late induction of apoptosis. Further, adopting the recent discovery that murine double minute 2 (MDM2) controls p21(Cip1/Waf1) expression by presenting this CDK inhibitor to the proteasome for degradation, RD cells overexpressing MDM2 abrogated ceramide-mediated p21(Cip1/Waf1) induction, G(2) arrest and the late ensuing apoptosis. Collectively, these data further support the notion that ceramide accumulation can modulate cell cycle progression. Additionally, these observations highlight MDM2 expression and proteasomal activity as key determinants of the cellular response to ceramide accumulation.


Subject(s)
Ceramides/pharmacology , Cyclin-Dependent Kinase Inhibitor p21/metabolism , G2 Phase/drug effects , Proto-Oncogene Proteins c-mdm2/metabolism , Rhabdomyosarcoma/metabolism , Rhabdomyosarcoma/pathology , Apoptosis/drug effects , Apoptosis/physiology , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Damage , G2 Phase/physiology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Proto-Oncogene Proteins c-mdm2/genetics , RNA, Small Interfering/pharmacology
5.
Genetica ; 123(3): 295-302, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15954500

ABSTRACT

The androgen receptor (AR) gene, located on the X chromosome, is an important regulator of human spermatogenesis. In the past decade, the link between the CAG polyglutamine tract, situated on exon one of the AR gene, and reduced spermatogenesis has become a controversial one. Alterations in the length of the CAG polyglutamine tract have been associated with prostate cancer at a reduced intrinsic length and neuromuscular diseases at a CAG repeat length of > or = 40. Minimal intermediate increases have been linked with depressed spermatogenesis in infertile males. Asian and Australian groups have published an association between increased CAG repeat length and reduced spermatogenesis while many European studies have found no such association. The aim of this study was to document the association between increased CAG repeat length and reduced spermatogenesis in a group of Irish infertile males and controls known to have fathered at least one child. The study employed the ABI 377 DNA sequencer to size the CAG repeat region of exon one of the AR gene in each group. Statistical analysis revealed no actual link between the length of the CAG tract and a reduction of spermatogenesis in a cohort of infertile patients (n = 66) of Irish ethnic origin when compared to a fertile control group (n = 77) (p = 0.599).


Subject(s)
Infertility, Male/genetics , Receptors, Androgen/genetics , Trinucleotide Repeats/genetics , Electrophoresis, Polyacrylamide Gel , Humans , Ireland , Male , Polymerase Chain Reaction , Spermatogenesis/genetics
6.
Genet Test ; 7(2): 155-8, 2003.
Article in English | MEDLINE | ID: mdl-12885340

ABSTRACT

The variable clinical manifestations of cystic fibrosis (CF) suggest the influence of modifier genes. Genetic and environmental factors that determine whether an individual will develop associated complications are still being determined. It has been proposed that the gene for hemochromatosis, HFE, may be a modifier locus for CF disease phenotype. Recent research has suggested a relationship between mutations to the HFE gene and the development of meconium ileus (MI) and liver disease in CF. This study aims to expand our knowledge of the HFE mutations C282Y and H63D carrier rate in an Irish population of CF allele carriers. PCR restriction enzyme analysis was performed on blood samples from CF patients to identify the C282Y and H63D mutations. HFE status of CF allele carriers and CF patients (Delta F508) homozygotes with and without meconium ileus was determined. The carrier frequency for C282Y was 30.8% for the Delta F508 homozygote MI positive group, as compared to 12.5% for the non-Delta F508 MI positive group but did not reach statistical significance (p = 0.27). Interestingly, no Delta F508 homozygote patients were homozygous for the C282Y mutation.


Subject(s)
Cystic Fibrosis/genetics , Histocompatibility Antigens Class I/genetics , Membrane Proteins/genetics , Alleles , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA Mutational Analysis , Gene Frequency , Hemochromatosis Protein , Heterozygote , Homozygote , Humans , Ileus/genetics , Ireland , Liver Diseases/genetics , Phenotype
7.
Cell Death Differ ; 10(6): 729-39, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12761581

ABSTRACT

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.


Subject(s)
Adaptor Proteins, Signal Transducing , Caspases/metabolism , Membrane Glycoproteins/metabolism , Rhabdomyosarcoma/metabolism , Tumor Necrosis Factor-alpha/metabolism , Apoptosis Regulatory Proteins , BH3 Interacting Domain Death Agonist Protein , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase 8 , Caspases/drug effects , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Fas-Associated Death Domain Protein , Humans , Membrane Glycoproteins/genetics , Mitochondria/enzymology , Models, Biological , Mutation/drug effects , Mutation/genetics , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Rhabdomyosarcoma/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , Sphingomyelins/metabolism , Sphingosine/metabolism , Sphingosine/pharmacology , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/genetics , bcl-X Protein
8.
Cell Death Differ ; 10(2): 211-7, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12700649

ABSTRACT

Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (-575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco(2) (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC(3)/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2'-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco(2), and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.


Subject(s)
Azacitidine/analogs & derivatives , Colonic Neoplasms/genetics , DNA Methylation , Enhancer Elements, Genetic , Gene Expression Regulation, Neoplastic , Promoter Regions, Genetic , fas Receptor/metabolism , Apoptosis , Azacitidine/pharmacology , Caco-2 Cells , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , CpG Islands/genetics , DNA Methylation/drug effects , DNA Modification Methylases/antagonists & inhibitors , DNA, Neoplasm/drug effects , DNA, Neoplasm/metabolism , Decitabine , HCT116 Cells , HT29 Cells , Humans , Sensitivity and Specificity , fas Receptor/immunology
9.
Clin Genet ; 63(2): 121-5, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12630958

ABSTRACT

The incidence of cystic fibrosis (CF) at birth in Ireland is 1/1461. Neonate CF genetic testing is not routinely performed in Ireland. Currently, screening is only carried out where there is clinical evidence or a family history to suggest disease. Here we report the frequencies of common CF mutations occurring in an Irish population composed of samples collected from western, mid-western and southern regions of Ireland. Rarer CF mutations were also identified in a selected number of CF patients. In addition, a number of polymorphisms were identified, some of which are reported to be functionally and phenotypically important.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Gene Frequency/genetics , Cystic Fibrosis/epidemiology , DNA Mutational Analysis/methods , DNA Primers , Humans , Ireland/epidemiology , Polymorphism, Genetic
10.
Int J Androl ; 25(1): 59-64, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11869379

ABSTRACT

In this study, reverse transcription-polymerase chain reaction (RT-PCR) was optimized to analyse the presence of DAZ, RBMY1, USP9Y, protamine-2, SRY and actin messenger RNA (mRNA) in testicular cells of men suffering from idiopathic azoospermia. All samples (n=28), including five controls, showed normal expression of actin, SRY and USP9Y. Sperm was not recovered from eight patients after testicular biopsy. Of these, four patients showed altered mRNA levels for the fertility genes, DAZ, RBMY1 and protamine-2. One patient, who was previously shown to be azoospermia factor region (AZF)b deleted, lacked RBM mRNA and presented with reduced amplification of protamine-2 mRNA. This correlated with previous studies, which proposed that RBM expression is exclusive to AZFb and that the lack of testicular RBMY1 mRNA results in suppressed spermatogenesis. Two patients were each lacking DAZ mRNA but did show expression of RBMY1 mRNA at a reduced level, suggesting that there might be residual spermatogenesis in the absence of DAZ expression. Protamine-2 mRNA was detected in one patient and was absent in the second patient. Finally, one patient lacked DAZ, RBMY1 and protamine-2 mRNA. The 19 remaining azoospermic patients presented with normal expression patterns for each of the fertility genes studied. This study demonstrates that the expression of spermatogenesis-specific genes varies in azoospermia. The study of the expression of such genes in a larger number of patients might be useful in characterizing and identifying subpopulations of azoospermic men.


Subject(s)
Nuclear Proteins , Oligospermia/metabolism , Protamines/genetics , RNA-Binding Proteins/genetics , Testis/metabolism , Transcription Factors , Biopsy , DNA Primers , DNA-Binding Proteins/genetics , Deleted in Azoospermia 1 Protein , Fertility/genetics , Fertilization in Vitro , Gene Expression , Humans , Karyotyping , Male , Oligospermia/genetics , Oligospermia/pathology , Reference Values , Reverse Transcriptase Polymerase Chain Reaction , Sex-Determining Region Y Protein , Testis/pathology , Y Chromosome
12.
Pathol Oncol Res ; 7(2): 95-106, 2001.
Article in English | MEDLINE | ID: mdl-11458271

ABSTRACT

Death ligands (TNF, FasL, TRAIL) and their respective death receptor signaling pathways can be used to induce tumor cells to undergo apoptosis. Chemotherapeutic drugs can induce apoptosis and the upregulation of death ligands or their receptors. Downstream events following cytotoxic stress-induced DNA damage and the signaling pathways that lead to the induction of apoptosis may be either dependent or independent of death receptor signaling. The involvement of the Fas signaling pathway in chemotherapy-induced apoptosis has been the most extensively studied, with the current emergence of information on the TRAIL signaling pathway. Fas-mediated and chemotherapy-induced apoptosis can converge at the level of the receptor, FasL, DISC formation, activation of the initiator caspase-8, at the level of the mitochondria, or at the level of downstream effector caspase activation. Convergence is influenced by the specific form of DNA damage, the cellular environment, and the specific pathway(s) by which death receptor-mediated or drug-mediated apoptosis are induced. This review discusses the different levels of interaction between signaling pathways in the different forms of cell death.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Membrane Glycoproteins/physiology , Neoplasms/drug therapy , Receptors, Tumor Necrosis Factor/physiology , Tumor Necrosis Factor-alpha/physiology , fas Receptor/physiology , Antineoplastic Agents/therapeutic use , Apoptosis Regulatory Proteins , Caspases/metabolism , Ceramides/physiology , DNA Damage , Enzyme Activation/drug effects , Fas Ligand Protein , Humans , MAP Kinase Signaling System/drug effects , Mitochondria/drug effects , Mitogen-Activated Protein Kinases/physiology , Models, Biological , NF-kappa B/physiology , Neoplasms/pathology , Receptors, Tumor Necrosis Factor/drug effects , Signal Transduction/drug effects , TNF-Related Apoptosis-Inducing Ligand
13.
Int J Androl ; 24(1): 31-6, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11168648

ABSTRACT

The region of the Y chromosome most critical for male fertility is called the azoospermia factor (AZF) region and it is located within subintervals five and six on the long arm of the Y chromosome. Several genes, all residing here, contribute to spermatogenesis and deletions in these genes are thought to be pathogenetically involved in some cases of male infertility associated with azoospermia or oligozoospermia. The aim of this study was to establish the prevalence of microdeletions in the AZF region of the Y chromosome in an Irish male population undergoing fertility treatment. To do this, we applied and compared two independent polymerase chain reaction (PCR) based screening methods, namely, a PCR protocol using several sequence-tagged site (STS) primer sets and a recently published multiplex PCR Y chromosome screening protocol. A total of 78 patients, attending the IVF unit at University College Hospital, Galway, were included in this study. Of them, 56 suffered from idiopathic azoospermic/oligozoospermic infertility. The remaining 22 patients had various conditions, which may have contributed to their infertility. A total of 50 age-matched normospermic men were included as controls. Two microdeletions were found; one in the AZFa region and one in AZFb region. These deletions were observed among the truly idiopathic cases. Further analysis was performed to study the extent of the deletions and it was confirmed that each deletion encompassed the respective AZF region including the AZF candidate gene.


Subject(s)
Chromosome Deletion , Y Chromosome , Adult , Humans , Infertility, Male/genetics , Ireland , Male , Oligospermia/genetics , Polymerase Chain Reaction
14.
Clin Cancer Res ; 6(11): 4432-41, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11106264

ABSTRACT

We examined the patterns of induction of apoptosis, Fas expression, and the influence of the status of the p53 tumor suppressor gene, in response to treatment of human colon carcinoma cell lines to 5-fluorouracil (FUra) combined with leucovorin (LV) under conditions of both DNA-directed (HT29, VRC5/c1, and RKO) and RNA-directed (HCT8 and HCT116) cytotoxicity. Acute apoptosis was induced in cell lines expressing wtp53 (RKO, HCT8, and HCT116), independent of the mechanism of FUra action. In HT29 cells that expressed mp53, apoptosis was a delayed event. Cell lines undergoing DNA-directed FUra cytotoxicity demonstrated marked accumulation of cells in S-phase (HT29 and RKO), whereas those lines undergoing RNA-directed cytotoxicity (HCT8 and HCT116) demonstrated marked cell cycle phase arrest in G2-M, both reversible by dThd. dThd partially protected HCT8 and HCT116 cells from FUra-LV-induced apoptosis but had no influence on FUra-LV-induced loss in clonogenic survival. In cells expressing wtp53, the Fas death receptor was induced in response to FUra-LV treatment. FUra-LV sensitized RKO cells to the anti-Fas monoclonal antibody CH-11 that was completely reversed by dThd, demonstrating the involvement of DNA damage in FUra-LV-induced, Fas-dependent sensitization to CH-11. In contrast, FUra-LV sensitized HCT116 cells to CH-11-induced apoptosis, which was not dThd reversible. Transduction of HT29 cells with Ad-wtp53 induced elevated Fas expression and sensitized the cells to FUra-LV-induced apoptosis. Data indicate that the presence of a wtp53 gene determines FUra-LV-induced Fas expression, the kinetics of FUra-LV-induced apoptosis and not the extent of apoptosis induced, both being independent of the mechanism of FUra action. Therefore, in colon carcinomas that express wtp53, the approach to sensitize tumors to Fas-mediated apoptosis may be further enhanced from the effect of FUra-LV in elevating Fas expression in a p53-dependent manner.


Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Apoptosis , Colonic Neoplasms/drug therapy , Fluorouracil/administration & dosage , Genes, p53/physiology , Leucovorin/administration & dosage , fas Receptor/biosynthesis , Caspase Inhibitors , Cell Cycle/drug effects , Colonic Neoplasms/pathology , DNA Damage , Humans , Tumor Cells, Cultured
15.
Clin Cancer Res ; 6(10): 4119-27, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11051265

ABSTRACT

Seven pediatric rhabdomyosarcoma (RMS) cell lines were resistant to the induction of apoptosis via the Fas death receptor. In contrast, four of seven lines (RD, Rh1, Rh18, and Rh30) were highly sensitive to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). TRAIL induced apoptosis within 4 h and also reduced clonogenic survival, both reversible by caspase inhibitors. DR5 (but not DR4) was expressed at high level in all cell lines. Expression of the decoy receptors DcR1 and DcR2 did not correlate with TRAIL sensitivity. All RMS lines expressed the adapter molecule FADD, and six of seven expressed procaspase-8. Expression of the inhibitory proteins c-FLIPL and c-FLIPs was high in three TRAIL-sensitive (RD, Rh1, and Rh30) and two TRAIL-resistant (Rh28 and Rh41) lines. All RMS lines expressed Bid and procaspases-3, -6, -7, and -9. Procaspases-8 and -10 were highest in TRAIL-sensitive RMS (RD, Rh1, and Rh30), and procaspase-10 was not expressed in Rh18, Rh36, or Rh41. TRAIL induced loss of mitochondrial membrane potential in TRAIL-sensitive Rh1 but not in TRAIL-resistant Rh41 cells. There was no correlation between expression of members of the Bcl-2 family (Bcl-2, Bcl-xL, Bax, and Bak) and TRAIL sensitivity. TRAIL-sensitive Rh18 expressed procaspase-8 in the absence of procaspase-10 and c-FLIP, and procaspase-10 was not detected in TRAIL-resistant Rh41 in the presence of procaspase-8 and c-FLIP. Data suggest that caspase-8 may be sufficient to deliver the TRAIL-induced apoptotic signal in the absence of both caspase-10 and c-FLIP (Rh18) but not in the presence of c-FLIP (Rh41). In RD, Rh1, and Rh30, the presence of c-FLIP may require amplification of the apoptotic signal via caspase-10.


Subject(s)
Apoptosis , Drug Resistance, Neoplasm , Membrane Glycoproteins/metabolism , Rhabdomyosarcoma/pathology , Tumor Necrosis Factor-alpha/metabolism , fas Receptor/metabolism , Apoptosis Regulatory Proteins , Blotting, Western , Caspases/metabolism , Cell Division , Child , Child, Preschool , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Membrane Potentials , Mitochondria/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleases/metabolism , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand , Time Factors , Tumor Cells, Cultured
16.
Cancer Res ; 60(10): 2643-50, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10825136

ABSTRACT

In thymidylate synthase-deficient (TS-) colon carcinoma cells, thymineless death is mediated via Fas/Fas ligand (FasL) interactions after thymidine deprivation and inhibited by the Fas-inhibitory monoclonal antibody NOK-1. The objective of the study was to elucidate whether other modes of DNA damage induced by doxorubicin, topotecan, and etoposide (VP-16) could elicit a similar cytotoxic response in TS- cells by signaling via the Fas death receptor. After a 72-h drug exposure, a loss in clonogenic survival that was not prevented by NOK-1 was induced by each agent in the absence of acute apoptosis, yielding IC50 values of 5 (doxorubicin), 10 (topotecan), and 150 nM (VP-16). Furthermore, TS- cell clones selected for resistance to Fas-mediated apoptosis (CH-11) were cross-resistant to the induction of thymineless death after thymidine deprivation but were not cross-resistant to doxorubicin, topotecan, or VP-16. A close correlation was found between acute induction of apoptosis (24 h) and up-regulated expression of FasL at high concentrations of each of the three agents (0.3-3 microM doxorubicin, 0.3-3 microM topotecan, and 10-90 microM VP-16), which was caspase dependent but Fas independent. At all drug concentrations, cell cycle distribution analyses demonstrated marked accumulation of cells in the G2-M phase. At nanomolar drug concentrations, prolonged arrest of TS- cells in G2-M phase resulted in the up-regulation of FasL expression and the delayed appearance of apoptotic cells (6 days), which could also be inhibited by the general caspase inhibitor Z-VAD-FMK, but not by NOK-1 or Fas-Fc. In clonogenic assays, Z-VAD-FMK did not rescue cells treated with VP-16 in contrast to treatment with CH-11 or thymineless stress, suggesting an irreversible commitment to cell death in G2-M phase. Expression of FasL at all drug concentrations appeared to be unrelated to the mechanism of drug-induced apoptosis. This was in contrast to the Fas-dependent regulation of thymineless death, which could be inhibited by blocking Fas/FasL interactions.


Subject(s)
Cell Death , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA Damage , fas Receptor/physiology , Amino Acid Chloromethyl Ketones/pharmacology , Antigens, Surface , Apoptosis , Caspases/metabolism , Clone Cells , Fas Ligand Protein , Humans , Membrane Glycoproteins/metabolism , Signal Transduction , Thymine/metabolism , Tumor Cells, Cultured
17.
J Biol Chem ; 275(14): 10023-9, 2000 Apr 07.
Article in English | MEDLINE | ID: mdl-10744679

ABSTRACT

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS(-) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.


Subject(s)
Apoptosis , I-kappa B Proteins , Membrane Glycoproteins/metabolism , NF-kappa B/metabolism , Thymine/metabolism , Transcription Factor AP-1/metabolism , Cell Survival , Clone Cells , Colonic Neoplasms , DNA-Binding Proteins/metabolism , Fas Ligand Protein , Humans , Membrane Glycoproteins/genetics , NF-KappaB Inhibitor alpha , Promoter Regions, Genetic , Recombinant Proteins/metabolism , Thymidylate Synthase/deficiency , Transcriptional Activation , Transfection , Tumor Cells, Cultured
18.
Cancer Res ; 60(1): 92-100, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10646859

ABSTRACT

Recent studies of fluoropyrimidine (FP)-mediated radiosensitization (RS) have focused on the molecular mechanisms underlying regulation of the cell cycle, particularly at the G1-S transition. Although thymidylate synthase (TS) inhibition by FP is necessary, we hypothesize that FP-RS is temporally dependent on progression of cells into S-phase under conditions of altered deoxynucleotide triphosphate pools, particularly an increased dATP:dTTP ratio, which subsequently results in enhanced DNA fragmentation and cell death. To better understand the mechanism of FP-RS, we characterized the cellular and biochemical responses to ionizing radiation (IR) alone, using different synchronization techniques in two isogenic, TS-deficient mutant cell lines, JH-1 (TS-) and JH-2 (Thy4), derived previously from a human colon cancer cell line. After G0 synchronization by leucine deprivation, these clones differ under subsequent growth conditions and dThd withdrawal: JH-2 cells have an intact G1 arrest (>72 h) and delayed cell death (>96 h), whereas JH-1 cells progress rapidly into early S-phase and undergo acute cell death (<24 h). No difference in the late S-phase and G2-M cell populations were noted between these growth-stimulated, G0-synchronized TS-deficient cell lines with dThd withdrawal. Biochemically, the intracellular ratio of dATP:dTTP increased substantially in JH-1 cells as cells progressed into early S-phase compared with JH-2 cells, which remained in G1 phase. Synchronized JH-1 cells showed significantly decreased clonogenic survival and an increase in DNA fragmentation after IR when compared with JH-2 cells. RS was demonstrated by an increase in alpha and decrease in beta, using linear quadratic analyses. An alternative synchronization technique used mimosine to induce a block in late G1, close to G1-S border. Both JH-1 and JH-2 cells, synchronized in late G1 and following growth stimulation, now progressed into S-phase identically (<24 h), with similarly increased dATP:dTTP ratios under dThd withdrawal conditions. These late G1-synchronized JH-1 and JH-2 cells also showed a comparable reduction in clonogenic survival and similar patterns of increased DNA fragmentation following IR. We suggest, based on the cellular and biochemical differences in response to IR between G0- and late G1-synchronized cells, that S-phase progression through the G1 restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.


Subject(s)
Deoxyribonucleosides/metabolism , G1 Phase/radiation effects , Neoplasm Proteins/deficiency , Resting Phase, Cell Cycle/radiation effects , S Phase/radiation effects , Thymidylate Synthase/deficiency , Adenosine Triphosphate/metabolism , Apoptosis , Cell Survival , DNA Damage , Deoxyribonucleosides/pharmacology , Flow Cytometry , G1 Phase/drug effects , G1 Phase/genetics , Humans , Pyrimidines , Radiation Tolerance , Resting Phase, Cell Cycle/drug effects , Resting Phase, Cell Cycle/genetics , S Phase/drug effects , Thymine Nucleotides/metabolism
19.
Curr Opin Oncol ; 11(6): 475-81, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10550011

ABSTRACT

Recent investigation further defines the role of p53 and of signaling events upstream and downstream of p53 in apoptosis following drug-induced DNA damage. The transcription factors NF-kappaB and AP-1 can be activated, and then directly transactivate FasL in response to chemotherapeutic agents. Death receptors for FasL (Fas) and for TRAIL (DR4, DR5) are emerging as important regulators of drug-induced apoptosis in human cancers, mediated by caspase activation. Apoptosis has been accepted as the predominant mechanism of drug-induced cell death in preclinical experimental models and in clinically sensitive tumors. However, drug-induced cell death can include acute or delayed apoptosis, necrosis, or a delayed mitotic death, and require further delineation for their relative contribution to tumor responses in vivo.


Subject(s)
Apoptosis/drug effects , Neoplasms/drug therapy , Neoplasms/pathology , Animals , Apoptosis/genetics , DNA Damage , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology
20.
Clin Cancer Res ; 5(2): 425-30, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10037193

ABSTRACT

We have shown previously (J. A. Houghton et al., Proc. Natl. Acad. Sci. USA, 94: 8144-8149, 1997) that thymineless death in thymidylate synthase-deficient (TS-) colon carcinoma cells is mediated via Fas/FasL interactions after deoxythymidine (dThd) deprivation, and that Fas-dependent sensitivity of human colon carcinoma cell lines may be dependent upon the level of Fas expressed. The objective of this study was to elucidate whether a Fas-dependent component exists in 5-fluorouracil (FUra)/leucovorin (LV)-induced cytotoxicity of colon carcinoma cells, and whether this may be potentiated by IFN-gamma-induced elevation in Fas expression, using the HT29 cell line as a model. The cytotoxic activity of FUra/LV was inhibited by dThd in HT29 cells and also, in part, by NOK-1+NOK-2 MoAbs that prevent Fas/FasL interactions. FUra/LV-induced cytotoxicity was significantly potentiated by IFN-gamma, reversed by exposure to NOK-1+NOK-2 antibodies, and correlated with a 4-fold induction of Fas expression in the presence of IFN-gamma and significant elevation in expression of FasL. Using five additional human colon carcinoma cell lines, FUra/LV-induced cytotoxicity was dThd-dependent in GC3/c1, VRC5/c1, and Caco2 but not in HCT8 or HCT116 cells. Like HT29 cells, this cytotoxicity was potentiated by IFN-gamma in GC3/c1 and VRC5/c1 but not in Caco2, which fails to express Fas, nor in HCT8 and HCT116, in which no dThd-dependent FUra-induced cytotoxicity was demonstrated. Data suggest that a Fas-dependent component, potentiated by IFN-gamma, exists in FUra/LV-induced cytotoxicity but requires FUra/LV-induced DNA damage for IFN-gamma-induced potentiation to occur.


Subject(s)
Colonic Neoplasms/metabolism , Fluorouracil/pharmacology , Leucovorin/pharmacology , Neuropeptides/metabolism , Receptors, Tumor Necrosis Factor , Cell Division/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Drug Synergism , Fas Ligand Protein , HT29 Cells , Humans , Interferon-gamma/pharmacology , Membrane Glycoproteins/biosynthesis , Neuropeptides/biosynthesis , Tumor Cells, Cultured , Up-Regulation , fas Receptor
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