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A Dritschilo

Publications and source records attributed to A Dritschilo.

At least 19 recordsLinked to original sources
PubMed2001

Ionizing radiation-induced apoptosis in ataxia-telangiectasia fibroblasts. Roles of caspase-9 and cellular inhibitor of apoptosis protein-1.

Ionizing radiation (IR) has been shown to induce apoptosis to a greater extent in a fibroblast cell line AT5BIVA derived from an individual with ataxia-telangiectasia (AT) than in control fibroblasts. However, the signaling pathway that underlies IR-induced apoptosis in AT cells has remained unknown. The mechanism of apoptosis in response to gamma-irradiation has now been examined in three AT fibroblast lines (AT3BIVA, AT4BIVA, and AT5BIVA) derived from different individuals with AT. The apoptotic indexes of these cell lines at 72 h after irradiation were 12, 31, and 35%, respectively, compared with a value of 2.3% for control fibroblasts. Immunoblot analysis and fluorometric assays revealed that the extents of IR-induced activation of caspase-3 and caspase-9 were markedly greater in AT4BIVA and AT5BIVA cells than in AT3BIVA and control cells. Furthermore, the basal abundance of the apoptotic inhibitor, a cellular inhibitor of apoptosis proteins (c-IAP-1), was markedly reduced in AT4BIVA and AT5BIVA cells compared with that in AT3BIVA and control cells. The overexpression of either caspase-9 mutant forms or recombinant c-IAP-1 in AT5BIVA cells inhibited the IR-induced activation of caspases-3 and 9 and reduced the apoptotic index of the irradiated cells. These results indicate that the extent of IR-induced apoptosis in different AT cell lines is inversely related to the abundance of c-IAP-1 and directly related to the extent of activation of caspases-3 and 9.

Amino Acid Substitution↗
PubMed2001

Role of ATM in oxidative stress-mediated c-Jun phosphorylation in response to ionizing radiation and CdCl2.

Ionizing radiation-induced phosphorylation of the transcription factor c-Jun is impaired in cells derived from individuals with ataxia telangiectasia (AT), in which the ATM gene is mutated. We demonstrate here that ATM modulates c-Jun phosphorylation following exposure to ionizing radiation as well as treatment with CdCl(2), a potent pro-oxidant. Exposure of AT and control fibroblasts to CdCl(2) induced a biphasic increase in c-Jun phosphorylation on serine residues 63 and 73, with the extent of the second phase being markedly greater in AT cells than in control cells. Heme oxygenase-1, a marker of oxidative stress, was also significantly induced in AT fibroblasts. Expression of recombinant ATM in AT fibroblasts, however, reduced the extent of the effects of CdCl(2) on both c-Jun phosphorylation and heme oxygenase-1 induction. Our data suggest that ATM contributes to oxidative stress-mediated signaling that leads to c-Jun phosphorylation by acting as a sensor of ionizing radiation-induced oxidative stress and by modulating intracellular redox homeostasis.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2001

NF-kappa B signaling pathway as a target for human tumor radiosensitization.

NF-kappa B is a critical nuclear transcriptional factor that is activated in response to cellular stresses and regulates the expression of genes involved in cell proliferation and cell death. When regulated NF-kappa B activation is disrupted, cells undergo apoptosis. That is, constitutively elevated or dysregulated NF-kappa B activation leads to cell death in response to stress. These mechanisms have been shown experimentally by expressing dominant negative inhibitors of NF-kappa B (I kappa B-alpha) in cancer cells exposed to chemotherapeutic agents or to ionizing radiation. NF-kappa B also plays an important role in a novel, radiation-inducible signaling pathway that involves the ataxia-telangiectasia mutated (ATM) protein kinase. Cells from patients with ataxia-telangiectasia (AT) are exquisitely sensitive to ionizing radiation and exhibit impaired NF-kappa B activation in response to this stress. Restoration of NF-kappa B regulation in AT fibroblasts by introducing a dominant negative form of I kappa B-alpha has resulted in correction of radiation sensitivity and a reduction of ionizing radiation-induced apoptosis. Expression of introduced ATM in AT cells results in correction of NF-kappa B regulation and an increase in postradiation survival without reduction in radiation-induced apoptosis. Taken together, these observations support a central role for NF-kappa B regulation in cellular intrinsic radiation sensitivity and apoptosis after exposure to ionizing radiation. Therefore, we hypothesize that the signaling pathway involving ATM/NF-kappa B/I kappa B offers attractive potential molecular targets for radiation sensitization in strategies to enhance the therapeutic ratio in cancer treatment.

Apoptosis↗
PubMed2000

Limitations of reduced-field irradiated volume and technique in conventional radiation therapy of prostate cancer: implications for conformal 3-D treatment.

In order to define technical limitations of conventional external beam irradiation for clinically localized prostate cancer, we evaluated the impact of several reduced-field treatment factors, such as reduced-field (RF) irradiated volume, RF technique, photon energy of treatment, and dose on survival endpoints and local control in a retrospective series. Several survival endpoints, such as disease-specific survival, freedom from relapse survival, biochemical no-evidence of disease (bNED) survival, and local control were associated with several treatment variables using univariate and multivariate analyses in 329 patients. Reduced-field technique appeared to predict survival outcome, with patients treated by bilateral 120 degrees arcs faring less well than those treated by full 360 degrees rotational fields. The irradiated volume of the reduced-field was also significantly associated with survival outcome, with patients treated with smaller volumes faring less well. Local failure rates also appeared increased, although not statistically, in patients treated with smaller RF sizes. In an attempt to explain these detected deficiencies, dose-volume histograms for prostate coverage were created for a small sample of patients. The deficiencies related to small reduced-field volume appeared to be largely attributable to poor dosimetric coverage of the prostate. These results underscore the limitations of conventional external beam treatment for prostate carcinoma when conventional techniques are employed, particularly if small reduced fields are used, and further supports the development of improved treatment techniques, such as conformal irradiation, as alternatives.

Adenocarcinoma↗
PubMed2000

Atomic force microscopy examination of conformations of polynucleotides in response to platinum isomers: significance of GC content at broken ends.

Atomic force microscopy is a technique that enables visualization of macromolecular conformations of polynucleotides at nanometer resolution. We investigated the results of interactions of cisplatin, a DNA binding anticancer drug, and its inactive counterpart, transplatin isomer, on the molecular conformation of polynucleotides: poly d(G-C). poly d(G-C) (polyGC) and poly d(A-T). poly d(A-T) (polyAT). We observed that polyAT exhibited an increased number of enlarged ends of molecules, which we attribute to unwound and/or collapsed regions of polyAT. PolyGC molecules did not show such ends unless cisplatin was added to the PolyGC polymers. Transplatin had the apparent effect of causing overlapping or stacking of the polymer molecules. Addition of exonuclease-III to these polymers removed the visible enlarged ends. The effects of cisplatin as compared to transplatin on the polyGC duplex polymers provide support for the presence of intrastrand covalent linkages, consistent with known N7 guanine interaction of the cis isomer on molecular conformation. Furthermore, our results indicate that the mechanism of interactions of DNA with cisplatin may be dependent on the GC content of the molecules. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 68-72, 2000.

Antineoplastic Agents↗
PubMed1999

Sensing of ionizing radiation-induced DNA damage by ATM through interaction with histone deacetylase.

The ATM gene is mutated in individuals with ataxia telangiectasia, a human genetic disease characterized by extreme sensitivity to radiation. The ATM protein acts as a sensor of radiation-induced cellular damage and contributes to cell cycle regulation, signal transduction, and DNA repair; however, the mechanisms underlying these functions of ATM remain largely unknown. Binding and immunoprecipitation assays have now shown that ATM interacts with the histone deacetylase HDAC1 both in vitro and in vivo, and that the extent of this association is increased after exposure of MRC5CV1 human fibroblasts to ionizing radiation. Histone deacetylase activity was also detected in immunoprecipitates prepared from these cells with antibodies to ATM, and this activity was blocked by the histone deacetylase inhibitor trichostatin A. These results suggest a previously unanticipated role for ATM in the modification of chromatin components in response to ionizing radiation.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1999

Inhibition of homologue of Slimb (HOS) function sensitizes human melanoma cells for apoptosis.

Homologue of Slimb (HOS)/beta-transducin repeats containing proteins up-regulate nuclear factor kappaB activity by targeting its inhibitor (IkappaB) for ubiquitination and subsequent degradation. We investigated whether inhibition of HOS function may modulate apoptosis in human melanoma cells. Forced expression of the dominant negative HOSdeltaF construct inhibited IkappaB degradation and led to sensitization of melanoma cells to apoptosis induced by tumor necrosis factor alpha with cycloheximide, as well as by cisplatin and ionizing and UV irradiation. These data indicate that HOS plays an important role in controlling the IkappaB-dependent apoptotic pathways in human melanoma.

Apoptosis↗
PubMed1999

Regulation of the human poly(ADP-ribose) polymerase promoter by the ETS transcription factor.

Ewing's sarcoma (EWS) cells accumulate elevated steady-state levels of poly (ADP-ribose) polymerase (PARP) mRNA and protein. To understand the molecular mechanisms underlying PARP upregulation, we cloned and analysed the 5'-flanking region of the PARP gene from EWS cells. Nucleotide sequence analysis demonstrated no variations in the PARP promoter region in EWS cells. The PARP promoter encompasses multiple binding motifs for the ETS transcription factor. We have also observed that there is a coordinated up-regulation of the expression of both PARP and ETS1, relative to cells of other human tumor types expressing lower levels of PARP. Transient co-expression of ETS1 in EWS cells resulted in a strong enhancement of PARP-promoter activity. The participation of ETS in the regulation of PARP gene expression was further demonstrated in EWS cells stably transfected with Ets1 antisense cDNA constructs. Antisense-mediated down-regulation of endogenous ETS1 resulted in the inhibition of PARP expression in EWS cells, and sensitized these cells to ionizing radiation. These data provide support for ETS regulation of PARP expression levels, and implicate ETS transcription factors in the radiation response of EWS cells.

Base Sequence↗
PubMed1999

Enhanced radiation late effects and cellular radiation sensitivity in an ATM heterozygous breast cancer patient.

We observed severe late effects in a patient treated with radiation therapy for breast cancer. Radiation survival studies of patient fibroblasts show an enhanced cellular radiation sensitivity (Do = 0.95 Gy). Genetic analysis reveals that the patient is heterozygous for a mutated ATM gene. Protein truncation test (PTT) and sequence analysis identified a truncation within the leucine zipper domain, corresponding to a fragment previously reported to exhibit dominant negative function. These findings demonstrate that ATM heterozygosity may be associated with enhanced clinical radiation sensitivity and suggest a clinical relevance to this truncation that results in a dominant negative-acting protein.

Adenocarcinoma, Mucinous↗
PubMed1999

Detection of heterogeneity of apoptotic fragments of poly (ADP-ribose) polymerase in MDA-MB-468 breast cancer cells: two-dimensional gel analysis.

Caspace-mediated proteolysis of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) (EC 2.4, 2.30) is a biochemical marker of cell death in response to various apoptotic stimuli. Anti-PARP antibodies identifying the 89 kDa polypeptide from the C-terminus as well as the 113 kDa native enzyme are often used to demonstrate evidence of apoptosis-associated, interleukin converting enzyme (ICE)-mediated limited cleavage. Recent evidence points to redundancy of caspases, heterogeneity of their cleavage sites, and a possibility of generating distinct context-specific, and cell-specific PARP fragments. In the present study, we employed antibodies directed to multiple sites in PARP and probed two-dimensionally resolved proteins of the estrogen receptor negative MDA-MB-468 breast tumor cells, induced to undergo apoptosis by ionizing radiation (IR). Our results revealed that the 24 kDa apoptotic fragment of PARP, from the N-terminus, consists of at least three isoforms, located at a p/more basic than the full length enzyme. We also report a hitherto unrecognized feature of an anti-PARP antiserum, VIC-5, detecting both the 89 kDa and the 24 kDa caspase-generated fragments of PARP. Thus, application of two-dimensional electrophoresis combined with antisera directed to multiple sites would be valuable in distinguishing PARP cleavage site- and inhibitor specificities of proteases during apoptosis.

Apoptosis↗
PubMed1999

Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo.

Raf-1, a cytosolic protein serine/threonine kinase, plays important roles in cell growth, proliferation, transformation, and cell survival. The aim of the present study was to evaluate the radiotherapeutic efficacy of a fully phosphorothioated and well-characterized antisense raf oligodeoxyribonucleotide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mRNA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposomal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 microg/ml and 13.5 microg/ml for LE-5132 and 5132, respectively. The decrease in plasma concentration of LE-5132 and 5132 followed a biexponential pattern, with initial distribution half-lives (t1/2alpha) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t1/2beta) with LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. The area under the plasma concentration-time curve (AUC) was 5.8 times higher with LE-5132 than with 5132. Significantly higher intact ODN levels could be measured in most organs within 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-fold, spleen, 31-fold, heart 3-fold, lungs 1.5-fold). In kidneys, the level was lower with LE-5132 (0.77-fold). LE-5132 composition, unlike 5132, did not affect clotting time in vitro. Significant decline in the level of Raf-1 protein was observed in vitro in relatively radioresistant human laryngeal squamous cell carcinoma cells (SQ-20B) treated with LE-5132 compared with SQ-20B cells treated with equimolar concentration of 5132 or liposome-encapsulated mismatched 5132 (0.5 microM LE-5132, 71.3%+/-22.5%; 1.0 microM LE-5132, 79.6%+/-16.7%). In addition, LE-5132 appeared to be a more potent antitumor compound than 5132 (p < 0.001). These data established the suitability of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous administration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf-1 expression in tumor tissue compared with blank liposome-treated or untreated control groups. LE-5132 or ionizing radiation (IR) treatment alone caused significant but transient inhibition of SQ-20B tumor growth but not tumor regression. Remarkably, a combination of LE-5132 and IR treatments led to significant and sustained tumor regression for at least 27 days after the last treatment (< 0.001). Histopathologic examination of tumor samples revealed a significant proportion of cells containing fragmented chromatin in the LE-5132 + IR treatment group as compared with single agent and untreated control groups. These in vivo data support the notion that Raf-1 has proliferative and survival functions and advance the scientific and technologic bases for the use of antisense raf ODN in the management of radioresistant malignancies.

3' Untranslated Regions↗
PubMed1999

Differential expression of stathmin during neoplastic conversion of human prostate epithelial cells is reversed by hypomethylating agent, 5-azacytidine.

In a variety of human tumor tissues, including those of prostate and breast, CpG hypermethylation represents one of the mechanisms downregulating the expression of specific proteins, including tumor suppressor proteins. Using 267B1-XR cells generated by ionizing radiation-induced transformation of epithelial cells, derived from neonatal human prostate and immortalized by SV40 (267B1), we now report markedly low levels of expression of the cytoplasmic phosphoprotein stathmin, in addition to several proteins of the actin microfilaments and intermediate filaments that characterize the altered phenotype. Stathmin is emerging as a relay protein integrating signals from diverse pathways during differentiation and neoplastic progression. In this in vitro prostate carcinogenesis model system, where loss of specific-protein expression is a major feature of the transformed 267B1-XR cells, we employed 5-azacytidine treatment followed by 2D-PAGE to reveal if experimental genomic hypomethylation reinstated the levels of any of the differentially expressed proteins. Our data suggest that stathmin represents one such example.

Antimetabolites, Antineoplastic↗
PubMed1999

Intermediate filament proteins during carcinogenesis and apoptosis (Review).

The intermediate filament network spreading from the cell periphery to the nucleus forms dynamic linkages between nuclear matrix, actin microfilaments, and the extracellular matrix. The six different types (types I-VI) of IF proteins consisting of nearly 50 different proteins form at least nine different kinds of filaments depending on the tissue types: keratins, lamins, vimentin, desmin, neurofilaments, peripherin, alpha-internexin, glial fibrillary acidic protein and nestin. Their tissue specific expression in normal cells and differential expression/assembly in neoplasia has been of immense value in tumor diagnosis. At the same time, recent in vitro studies point out that keratins, lamins and vimentin are subject to caspase-mediated proteolysis in an apoptosis-related manner. We reviewed the experimentally demonstrated P4-P1 motif specificities of caspases in the selection of substrates in the IF protein family. In addition, we provided clues to possible cleavage of additional IF proteins during programmed cell death, based on acceptable cut site motifs indicated by searches using the PIR protein sequence database. The present review concludes with presentation of evidence on the emerging roles of IFs in association with intermediate filament associated proteins in the dynamic remodeling of the cell during development of neoplastic phenotype and execution of apoptosis.

Amino Acid Sequence↗
PubMed1998

Impaired ionizing radiation-induced activation of a nuclear signal essential for phosphorylation of c-Jun by dually phosphorylated c-Jun amino-terminal kinases in ataxia telangiectasia fibroblasts.

The c-Jun amino-terminal kinases (JNKs) participate in intracellular signaling in response to cytokines and cellular stresses. JNKs are activated by phosphorylation on two critical residues, the threonine 183 and tyrosine 185, within the TPY motif. The activated JNKs, in turn, phosphorylate the nuclear protein c-Jun, a major component of the transcription factor AP1. In vitro studies have revealed a defect in ionizing radiation-induced activation of the JNK signaling pathway in lymphoblastoid cells from individuals with ataxia telangiectasia (AT). However, the biochemical basis for this signaling defect is not clear. Here, we show that ionizing radiation induces the phosphorylation of endogenous c-Jun in normal fibroblasts but not in AT fibroblasts. The p46 isoforms of dually phosphorylated JNKs were detected in the nuclei of both normal and AT fibroblasts following exposure to ionizing radiation or sham radiation. However, c-Jun kinase activity was detected in normal cells but not in AT cells. Furthermore, an exogenous purified active JNK protein was able to phosphorylate endogenous c-Jun in nuclear extracts only of normal cells and only after the cells were irradiated. Electrophoretic mobility shift assays also showed that the ionizing radiation-induced increase in the DNA binding activity of AP1 observed in normal cells was absent or markedly reduced in AT cell lines. These data suggest that the defect in ionizing radiation-induced signaling through c-Jun in AT cells is the result of impaired function of an unknown nuclear protein or proteins that negatively regulate both JNK and c-Jun.

Ataxia Telangiectasia↗
PubMed1998

A novel ionizing radiation-induced signaling pathway that activates the transcription factor NF-kappaB.

The signaling pathway through which ionizing radiation induces NF-kappaB activation is not fully understood. IkappaB-alpha, an inhibitory protein of NF-kappaB mediates the activation of NF-kappaB in response to various stimuli, including cytokines, mitogens, oxidants and other stresses. We have now identified an ionizing radiation-induced signaling pathway that is independent of TNF-alpha. IkappaB-alpha degradation is rapid in response to TNF-alpha induction, but it is absent in response to ionizing radiation exposure in cells from individuals with ataxia-telangiectasia (AT). Overexpression of wild-type ATM, the product of the gene defective in AT patients, restores radiation-induced degradation of IkappaB-alpha. Furthermore, phosphorylation of IkappaB-alpha by immunoprecipitated ATM kinase is increased in control fibroblasts and transfected AT cells following ionizing radiation exposure. These data provide support for a novel ionizing radiation-induced signaling pathway for activation of NF-kappaB and a molecular basis for the sensitivity of AT patients to oxidative stresses.

Acetylcysteine↗