The principal psychoactive component of marihuana is Δ-9-tetrahy-drocannabinol. This compound at 10-5 molar concentration in the medium of human cell cultures appeared to inhibit DNA, RNA, and protein synthesis by 50, 40, and 30% respectively, as measured by incorporation of radioactive precursors into acid-insoluble cell fractions in human diploid fibroblasts, human neuroblastoma cells, and mouse neuroblastoma cells. While Δ-9-tetrahydrocannabinol inhibited semiconservative DNA synthesis, it had no effect on DNA repair synthesis in human cells as assayed by the photolysis of 5-bromodeoxyuridine incorporation into DNA during repair after ultraviolet radiation damage. Δ-9-tetrahydrocannabinol also had no effect on rejoining of DNA single-strand breaks induced by γ-rays. The nonspecificity of the inhibition of macromolecular synthesis by Δ-9-THC suggested a possible interference with uptake of radioactive precursors. However, experimentation has shown that this depression of macromolecular synthesis cannot be accounted for by reduced transport of radioactive precursors into the cell because the rate of transport of these precursors into the cell is essentially the same in the presence or absence of Δ-9-THC. Pool sizes of macromolecular precursors as measured radioisotopically (3Hthymidine, 3H-uridine, 14C-leucine) appear to be reduced about 50%, and this reduced pool size could fully account for the reduced macromolecular synthesis seen in the presence of Δ-9-THC. We do not know what causes this apparent reduction of pool sizes in the presence of Δ-9-THC.

A diet rich in polyphenols has been proposed to have beneficial health effects and to reduce risk of disease. Ellagic acid, a polyphenol common in red berries and pomegranates, has potential anti-tumorigenic effects that make it interesting to further study in different cancer cell systems. Neuroblastoma is a childhood cancer that arises during development of the peripheral nervous system. Neuroblastoma, being an embryonal tumor, show loss of function of genes controlling differentiation and apoptosis. Neuroblastoma is a heterogenic tumor disease, and highly malignant neuroblastomas are difficult to treat despite different treatment modalities, identifying a need for new and combinatory treatments. A common model for human neuroblastoma is the SH-SY5Y cell line resembling immature neuroblasts that can be differentiated in vitro with several agents including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the vitamin A-derivative all-trans retinoic acid. Here, the effect of ellagic acid on proliferation, cell detachment and apoptosis in non-differentiated and in vitro-differentiated SH-SY5Y cells were studied with the aim of identifying cellular target mechanisms and a possible therapeutic potential for ellagic acid. In non-differentiated cells, ellagic acid reduced cell number, inhibited cell cycle activity, and induced cell detachment and apoptosis. Apoptosis was partly mediated by the intrinsic pathway. 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid both induced morphological differentiation, while only the latter induced G0/G1-arrest. Single-cell analysis revealed that 12-O-tetradecanoylphorbol-13-acetate-treated cells continued cycling during neuritogenesis while these two read-outs were mutually exclusive in all-trans retinoic acid-treated cells. 12-O-tetradecanoylphorbol-13-acetate- and especially all-trans retinoic acid-differentiated cells showed lower sensitivity to ellagic acid-dependent cell detachment and apoptosis. / <p>Artikel 4 ("Altered sensitivity...") ingick som manuskript i avhandlingen, nu publicerad.</p>

Thesis (Ph.D.)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Predictive assays are of key importance in clinical radiotherapy, chemotherapy and toxicology. Prior to exposing malignant tissues to irradiation or drugs in the clinic, a good understanding of the damage response to the cytotoxic agent is required. Such information is necessary for effective planning and treatment. Regrettably however the methods which detect DNA damage, namely micronucleus, apoptosis and DNA repair assays do not rank cells according to their intrinsic survival response to cytotoxic agents. The application of predictive assays based on micronuclei and apoptosis in the clinic therefore remains unreliable. Using a panel of 7 neuroblastoma and 6 neuroepithelial cell lines, it is shown that damage assays also do not rank cell lines according to cell survival. However, radiosensitivity can be reconstructed from micronuclei formation and apoptosis, and a new parameter, cell death due to small deletions, chromosome aberrations and misrepair. The interrelationships between radiation-induced micronuclei, apoptosis and repair is complex and varies between cell lines. Micronuclei formation and apoptosis are exponentially interrelated. This suggests that these cell inactivation pathways are strongly correlated. Evidence exists to show that the expression of apoptosis and micronuclei is influenced by the extent of DNA double-strand break repair within the first 2 hours after irradiation. Cell lines which repair more damage in the first 2 hours express more micronuclei and less apoptosis. Micronuclei formation and apoptosis and are not significantly correlated with the 20 hours slow repair component. There is however a strong correlation between 20 hours of repair and radiosensitivity, with the more radioresistant cell lines being more repair proficient. This suggests that the 2 hours (fast) DNA repair component is more error prone, and that cells lines repairing more damage late after irradiation tend to show better survival. In conclusion, micronuclei formation, apoptosis and DNA repair are strictly cell type specific and are not suitable for predicting radiosensitivity in terms of cell survival. However, these assays are very useful for studies on the influences of dose modifying agents i.e. oxygen tension, radiation modality, pH, cytotoxic sensitisers and radiation protectors which alter cellular responses and provide insight into damage mechanisms. / AFRIKAANSE OPSOMMING: Toetse wat kliniese gevolge kan voorspel is van uiterse beking in stralingsterapie, chemoterapie en toksikologie. Voordat kwaadaardige weefsels aan bestraling of chemise middels blootgestel can word in die kliniek, moet daar 'n goeie begrip van die skade weerstand wees van die selgiftige middel. Hierdie inligting is noodsaaklik vir effektiewe beplanning en behandeling. Ongelukkig stem die metodes wat ONS skade, apoptose en ONS hersteltoetse, nie ooreen met die selle se inherente straling sensitiwiteit nie. Die aanwending van voorspelbare toetse gebaseer op mikrokerne en apoptose in die kliniek bly dus onbetroubaar. Deur gebruik te maak van 'n paneel van 13 neurologiese sellyne, is daar bewys dat ONS skade toetse nie sellyne rangskik volgens seloorlewing nie. Radiosensitiwiteit kan herbou word deur 'n neiging om mikrokerne te vorm, apoptose, en sel sterftes weens klein vermiste ONS volgordes, chromosoom aberrasies en verkeerd herstelde ONS. Die verhouding tussen straling-geïnduseerde mikrokerne, apoptose en selgenees is kompleks en varieer tussen sellyne. Die ontstaan van mikrokerne en apoptose is eksponensiel verbind. Dit dui aan dat hierdie seltraagheidsbane streng gekorreleer word. Daar is bewys dat die uitdrukking van apoptose en mikrokerne deur die mate van herstel van die ONS dubbelstring-breuke binne die eerste 2 ure na bestraling beïnvloed is. Daar is gevind dat sellyne wat meer skade herstel binne die eerste 2 ure meer mikrokerne en minder apoptose toon. Die ontstaan van mikrokerne en apoptose is nie betekenisvol gekorreleer met die 20-uur stadige herstel komponent nie. Daar is inderdaad 'n sterk korrelasie tussen die 20-uur herstel komponent en radiosensitiwiteit, en die meer radioweerstandbiedende sellyne net In hoër herstel bekwaamheid. Dit laat mens dink dat die 2 uur (vinnige) DNS herstel komponent meer geneig is om foutief te wees, en dat sellyne wat meer skade, laat na bestraling herstel, beter oorlewing toon. Ten slotte, die ontstaan van mikrokerne, apoptose en DNS herstel is strenggesproke seltipe spesifiek en is nie toepaslik om radiosensitiviteit, in terme van seloorlewing, te voorspel nie. Hierdie toetse is nuttig vir studies waar die invloed van dosismodifiseringsagente, soos suurstof-spanning, straling-tipe, pH, sitotoksieke sensiteerders en stralingsbeskermers, wat sellulêre gevoeligheid verander en insig gee tot skade meganismes.

Das Neuroblastom ist der dritthäufigste maligne Tumor im Kindesalter und ist für 15% der Todesfälle durch Krebs bei Kindern unter 14 Jahren verantwortlich. Viele molekularbiologische Vorgänge, die zu der heterogenen Prognose der Patienten beitragen, sind noch nicht verstanden. Als Hauptrisikomerkmal stellt sich die Amplifikation und erhöhte Expression von MYCN dar. In Vorarbeiten der Arbeitsgruppe von Prof. Christiansen zeigte MYCN Einfluss auf die Genregion von Thyroid Cancer 1 (TC1), das als neuer Marker für maligne Schilddrüsenkarzinome erkannt wurde. In der vorliegenden Arbeit wurden erste Untersuchungen zur prognostischen Bedeutung von TC1 im Neuroblastom, sowie die Charakterisierung eines TC1 Überexpressionsphänotyps humaner Neuroblastomzelllinien unter Einfluss von MYCN durchgeführt. Es wurde ein Überexpressionsvektor von TC1 in die Neuroblastomzelllinie SH-EP eingebracht, welche über ein aktivierbares MYCN- Konstrukt verfügt. Dieser neue Phänotyp wurde bezüglich der Proliferation, des Zellzyklus und der Apoptose im Vergleich zu einer Kontrollzelllinie ohne Überexpression untersucht. Eine In-silico Recherche in der Versteeg Neuroblastomdatenbank ergab eine deutlich bessere Überlebenswahrscheinlichkeit für Patientin mit hoher TC1 Expression. Es konnte gezeigt werden, dass MYCN Amplifikation und Expression in einem Panel von Neuroblastom Zelllinien nicht mit der TC1 Expression korrelieren. Die spezifische Aktivierung von MYCN führte hingegen zu einer Expressionssteigerung von TC1. Weiterhin zeigte sich, dass eine TC1 Überexpression die Proliferation hemmt, indem es die MYCN induzierte G1- S- Phasen- Transition inhibiert. TC1 zeigt antiproliferative Eigenschaften im Zellkulturmodell und stellt sich als neuer prognostisch günstiger Parameter im Neuroblastom dar.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

<p>Neuroblastoma is a pediatric cancer arising from undifferentiated neural crest-derived precursor cells. Treatment strategies for neuroblastoma aim to promote neuroblast differentiation, however current therapies available are only modestly effective. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that act to promote neuroblast differentiation, though the precise factors released and their mechanism of action in neuroblastoma remains unclear. Here, we identify a novel component of the differentiating stroma secretome and harness stroma biology to inform the use of a combination therapy for neuroblastoma treatment.</p><p>HBEGF expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from late-stage disease containing very little stroma or HBEGF. Addition of soluble HBEGF to neuroblastoma cell lines leads to increased neuroblast differentiation and decreased proliferation. Heparan sulfate proteoglycans (HSPGs) and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor (EGFR), leading to activation of the ERK1/2 and STAT3 pathways and upregulation of the inhibitor of DNA binding 1 transcription factor. </p><p>Expression of the type III TGF-β receptor (TβRIII), an HSPG, is epigenetically regulated in neuroblastoma cells via direct binding of the N-Myc transcription factor to Sp-1 sites on the TβRIII promoter. Analysis of patient microarray data demonstrate that other members of the differentiating stroma secretome, including HBEGF and EGFR, are positively correlated with TβRIII expression, suggesting that these proteins may be co-regulated. Treatment with inhibitors aimed at blocking N-Myc function, including inhibitors of histone deacetylases, DNA methyltransferases (DNMTs), and aurora kinase A (AurkA) can promote neuroblast differentiation and decrease proliferation. The combination of the DNMT inhibitor decitabine with the AurkA inhibitor MLN8237 enhances differentiation and reduces proliferation compared to either agent alone. Importantly, the combination of clinically achievable doses of these targeted agents dramatically reduces tumor growth in orthotopic xenograft models of neuroblastoma, identifying a novel combination therapy that may benefit children with this disease.</p><p>In conclusion, these studies dissect the tumor microenvironment to identify an important member of the differentiating stroma secretome, while also revealing a combination therapy for clinical development that has the potential to decrease adverse side effects and increase effectiveness of neuroblastoma treatment.</p> / Dissertation

Le neuroblastome (NB) représente 8% de tous les cancers pédiatriques et est caractérisé par sa grande hétérogénéité clinique. Afin d’évaluer son pronostic, plusieurs facteurs génétiques sont utilisés : amplification de MYCN, délétion 1p, gain 11q et gain 17q. Les buts de notre travail étaient d’abord de vérifier si l’hybridation in situ en fluorescence (FISH) permet une analyse complète de ces anomalies et ensuite, en utilisant une analyse globale du génome telle le polymorphisme nucléotidique simple (SNP), de vérifier la concordance avec les résultats de la FISH et le pronostic potentiel des anomalies du 14q, en particulier du gène AKT. Nous avons donc établi un panel de sondes pour la FISH qui a été appliqué sur 16 tumeurs non-fixées. Après isolation de l’ADN de 36 tumeurs, nous avons effectué une analyse génotypique par SNP utilisant les puces « Affymetrix Genome-Wide Human SNP Array 6.0 » contenant 945,826 sondes non polymorphiques et 906,000 sondes polymorphiques. Nos résultats ont démontré que la FISH permet l’évaluation complète des anomalies génétiques importantes du NB et que les anomalies déséquilibrées sont détectées très précisément par SNP. Les anomalies du 14q tendent à être associées avec des facteurs cliniques comme le grade et l’évolution, contrairement aux anomalies d’AKT. L’analyse du 14q a révélé trois gènes d’intérêt, MAX, BCL11B et GPHN, qui devraient être analysés sur un plus grand échantillon. Ainsi, l’étude par FISH semble adaptée pour détecter les anomalies génétiques classiques du NB, alors que celles retrouvées en 14q représentent de potentielles cibles thérapeutiques pour cette tumeur. / Neuroblastoma (NB) accounts for 8% of all childhood cancers and is characterized by its clinical heterogeneity. To evaluate its prognostic, many genetic markers are used: MYCN amplification, 1p deletion, 11q gain and 17q gain. Our goals were first to verify if fluorescence in situ hybridization (FISH) allows a complete analysis of these abnormalities and, second, using a global genomic analysis as single nucleotide polymorphism (SNP), to verify the concordance with FISH results and the prognostic potential of 14q abnormalities, especially these of AKT gene. We then established a FISH panel that has been applied on 16 unfixed tumors. After DNA isolation of 36 tumors, we made a genotypic analysis by SNP using « Affymetrix Genome-Wide Human SNP Array 6.0 » containing 945,826 nonpolymorphic probes and 906,000 polymorphic probes. Our results have demonstrated that FISH allows a complete evaluation of the NB’s important genetic abnormalities and that unbalanced abnormalities are detected very precisely by SNP. 14q abnormalities seem to be associated with clinical factors such as tumor grading and evolution, unlike AKT abnormalities. Analysis of 14q abnormalities revealed three genes of interest, MAX, BCL11B and GPHN, which should be analyzed on a larger sample. Thereby, FISH study seems appropriate to detect the NB’s classic genetic abnormalities, while those found in 14q represent potential therapeutic targets for this tumor.

Histone deacetylase inhibitor trichostatin A (TSA) increases cytotoxicity of antineoplastic agent ellipticine to human neuroblastoma cells. Its mechanism of action has not yet been explained. One of the possible mode of action is conformational change in chromatin, which leads to changes in DNA that is more accessible to covalent modification and intercalation. The aim of this work is to study another mode of action, which can explain this phenomenon. The question is, if TSA can increase cytotoxicity of ellipticine to human neuroblastoma cells by modulation of activities and expression of cytochromes P450 and peroxidases. These enzymes are responsible for cytotoxicity of ellipticine to human neuroblastoma cells. TSA has no effect on oxidation of ellipticine mediated by cytochromes P450 leading to metabolites responsible for formation of ellipticine-DNA adducts and detoxication metabolites. TSA increases formation of ellipticine dimer, which is a detoxication metabolite, forming during its oxidation by peroxidases. TSA has no effect on activities of CYP1A1, CYP1A2, CYP3A, which significantly participate in oxidation of ellipticine. TSA modulates expression of enzymes oxidizing ellipticin in human neuroblastoma cells. TSA in the presence of ellipticine increases expression of CYP1A1 a CYP3A4 in...

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and inhibition of topoisomerase II. Ellipticine was also found to form covalent DNA adducts mediated by its enzymatic activation with cytochromes P450 (CYP) and peroxidases. The next study demonstrated increasing formation of these ellipticine-DNA adducts by histone deacetylase inhibitor valproate (VPA) in neuroblastoma cells. This phenomenon correlates with increasing cytotoxicity of ellipticine induced by this histone deacetylase inhibitor. This observation can be explained by several mechanisms. One of them can be loosening the structure of chromatine, which leads to accessing DNA for modification. Another one is the effect of VPA on activities and expression of enzymes metabolizing ellipticine. This study was aimed to test the second hypothesis. Since VPA has been shown to be metabolized by similar enzymes as ellipticine is, we have studied the effect of VPA (i) on oxidation of ellipticine by cytochromes P450 and peroxidases, (ii) on activities of the CYP enzymes, which significantly participate in oxidation of ellipticine (CYP1A, CYP3A) and (iii) on expression of enzymes oxidizing ellipticine (CYP1A1, CYP3A4, lactoperoxidase). Oxidation of ellipticine in vitro by model...

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