Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells

Hauptbestandteil der Arbeit war die strahlenbiologische Charakterisierung muriner LLC1-Zellen in vitro. Dabei handelt es sich um in Kultur überführte Einzelzellen eines bei in vivo Experimenten weit verbreiteten Tumormodells der Maus. Die soliden, entdifferenzierten Tumorzellverbände in vivo ents...

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1. Verfasser: Eberle, Fabian
Beteiligte: Engenhart-Cabillic (Prof.Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Deutsch
Veröffentlicht: Philipps-Universität Marburg 2013
Medizin
Ausgabe:http://dx.doi.org/10.17192/z2013.0138
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topic Lunge
Lewis-Lung
Adjuvante Strahlentherapie
Medizin, Gesundheit
Fabian Eberle
Lewis-Lung
Strahlenbiologie
Fabian Eberle
Krebs <Medizin>
spellingShingle Lunge
Lewis-Lung
Adjuvante Strahlentherapie
Medizin, Gesundheit
Fabian Eberle
Lewis-Lung
Strahlenbiologie
Fabian Eberle
Krebs <Medizin>
Eberle, Fabian
Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells
Major component of the study was the radiobiological characterization of murine LLC1 cells in vitro. These cells are culture transferred single cells emanated from a widely used in vivo mouse tumor model. The solid, undifferentiated tumor clusters in vivo corresponded to a non-small cell lung cancer, the culture transferred individual cells were classified as malignant due to nuclear pleomorphism, hyperchromasia, atypical mitoses and moved nuclear- cytoplasmic ratio. If obtained under optimal conditions in culture the LLC1-cells showed a fast, stable growth pattern - with a doubling time of 16 hours- even over long culture periods, but reacted very sensitively to changes in the cell culture environment, such as composition of the growth medium, seeding density or type and coating of the growth surface. In this respect, they expressed divergent phenotypes, which differed mainly in terms of their morphology, adhesion capability and efficiency of plating. In genotypic terms the majority of the cells showed a tetraploid chromosome set with 80 single chromosomes. Ensuring optimal culture conditions reduced the confounding effect of culture associated cellular impairment and guaranteed the validity and reproducibility of further radiobiological analysis. Mathematically determined (based on Single-Hit-Multi-Target-Model & Linear- Quadratic-Model) characteristics of generated DEKs, suggested high radiation sensitivity, moderate to poor pronounced repair capacity. Considered radiobiological the LLC1-cells corresponded to an early responsive cell model most likely. Despite a generally rather weak distinctive capacity of repair the mechanisms of rapid DNA DSB repair suggested to be intact and conform to repair-kinetics and –dimension of malignant cells already described in the literature. For LLC1 cells the gene expression analysis showed no sufficient regulation of p53 dependent genes after photon irradiation. The repair genes ERCC1 and RRM2b, the apoptosis related gene BAX and the cell cycle regulatory gene CDKN1A were not induced by irradiation with photons. Genes with p53-independent regulatory mechanisms such as cell cycle regulating gene GADD45 and apoptosis related genes BCL2 and BIRC5 were irradiation induced and resulted in G2 arrest and induction of apoptosis after photon irradiation in LLC1 cells. The shift of the cellular balance towards proapoptotic mechanisms for photon irradiation is not due to overexpression of the pro-apoptotic BAX gene, which could not be induced by exposing to ionizing radiation in LLC1-cells, but mainly by underexpression of anti-apoptotic genes such as BCL2/BIRC5, with consequently missing inhibition of apoptosis. We observed significant increase in G2-population initially after photon irradiation, both with 2 Gy and 6 Gy. After exposure to high doses (6 Gy), a longrunning, potentially terminal G2 arrest was initiated, whereas after irradiation with low doses (2 Gy) a decreasing G2-population could be observed 18 h to 24 h post rad. No G1-arrest occurred in LLC1 cells even after exposure to 6 Gy photons. Quantitative screening by Western blot showed a p21 deficiency causing the lack of G1 arrest in LLC1-cells, a phosphorylation of p53 on serine 15 was detectable, and showed a radiation induced increase on protein level. The subsequently performed p53 gene sequencing shows a heterozygous mutation to UAA STOP codon in sequence 1 (94-115 forward) on base position 120. This mutation prevents a sufficient protein synthesis in LLC1-cells which is why a functionality of the protein is not given. This defective protein structure of p53 explains the lack of G1 arrest, the p21- deficiency as well as the lack of inducibility of p53-dependent genes such as CDKN1A, RRM2b, ERCC1 and BAX at the mRNA level, while p53 is electrophoretically detectable.
url http://archiv.ub.uni-marburg.de/diss/z2013/0138/pdf/dfbe.pdf
author2 Engenhart-Cabillic (Prof.Dr.)
author2_role ths
last_indexed 2013-03-12T23:59:59Z
institution Medizin
oai_set_str_mv ddc:610
open_access
doc-type:doctoralThesis
xMetaDissPlus
dewey-raw 610
dewey-search 610
genre Medical sciences, Medicine
genre_facet Medical sciences, Medicine
topic_facet Medizin, Gesundheit
author Eberle, Fabian
doi_str_mv http://dx.doi.org/10.17192/z2013.0138
edition http://dx.doi.org/10.17192/z2013.0138
description Hauptbestandteil der Arbeit war die strahlenbiologische Charakterisierung muriner LLC1-Zellen in vitro. Dabei handelt es sich um in Kultur überführte Einzelzellen eines bei in vivo Experimenten weit verbreiteten Tumormodells der Maus. Die soliden, entdifferenzierten Tumorzellverbände in vivo entsprachen einem nichtkleinzelligen Bronchialkarzinom, auch die in Kultur überführten Einzelzellen konnten aufgrund von Kernpleomorphie, Hyperchromasie, atypischen Mitosen und verschobener Kern-Plasma-Relation als maligne eingestuft werden. In Kultur zeigten die LLC1-Zellen unter optimalen Bedingungen ein schnelles, stabiles Wachstumsverhalten- mit einer Verdopplungszeit von 16 h- auch über lange Kultivierungszeiträume hinweg, reagierten aber Wachstumsmedium, Aussaatdichte sowie Art und Beschichtung der Wachstumsfläche betreffend, ausgesprochen sensibel auf Veränderungen des Kultur-Milieus. Dabei prägten sie divergente Phänotypen aus, die sich vor allem hinsichtlich ihrer Morphologie, Adhäsionsfähigkeit und Plattierungseffizienz voneinander unterschieden. Genotypisch wies der Großteil der Zellen tetraploide Chromosomensätze mit 80 Einzelchromosomen auf. Die exakte Orientierung an den ermittelten optimalen Kulturbedingungen war, durch Reduktion von Störfaktoren einer kultivierungsbedingten zellulären Beeinträchtigung, Grundlage der Aussagekraft und Reproduzierbarkeit weiterführender strahlenbiologischer Analyseverfahren. Anhand mathematisch (Single-Hit-Multi-Target-Modell & Linearquadratisches Modell) ermittelter Kenngrößen aus generierten DEKs ließen sich die LLC1-Zellen als strahlensensible Zellen mit mäßig bis schlecht ausgeprägter Reparaturkapazität einstufen, die strahlenbiologisch eher einem frühreagierenden Zellsystem entsprechen. Trotz einer generell eher schwach ausgeprägten Reparaturkapazität waren die Mechanismen der schnellen DNA-DSB Reparatur intakt und entsprachen sowohl zeitkinetisch als auch dimensional denen anderer bereits in der Literatur beschriebener maligner Zellen. In der Genexpressionsanalyse zeigte sich für LLC1-Zellen keine suffiziente Genregulation p53 abhängiger Gene nach Photonenbestrahlung. Die reparaturrelevanten Gene RRM2b und ERCC1, das apoptoserelevante BAX und das zellzyklusregulierende CDKN1A waren durch Photonenbestrahlung nicht induzierbar. Gene mit p53 unabhängigen Regulationsmechanismen wie das zellzyklusregulierende Gen GADD45 und die apoptoserelevanten Gene BCL2 und BIRC5 waren induzierbar und führten in LLC1-Zellen zu G2-Arretierung und Apoptoseinduktion nach Photonenbestrahlung. Dabei erfolgte die Verschiebung des zellulären Gleichgewichts hin zu proapototischen Mechanismen nach Photonenbestrahlung nicht durch Überexpression des, in LLC1-Zellen nicht strahleninduzierbaren, proapoptotischen Gens BAX, sondern primär durch Unterexpression antiapoptotischer Gene wie BCL2/BIRC5 mit konsequenterweise fehlender Hemmung der Apoptoseinduktion. Nach Exposition gegenüber hohen Dosen (6 Gy) leiten LLC1-Zellen einen GADD45 vermittelten, lang andauernden, potentiell zeitlich unbegrenzten G2-Arrest ein. Hingegen war nach Bestrahlung mit geringeren Dosen von 2 Gy ein Ende des G2-Arrests zwischen 18 h und 24 h post rad festzustellen. Eine G1-Arretierung erfolgt in LLC1-Zellen auch nach Exposition gegenüber 6 Gy Photonen nicht. Im quantitativen screening mittels Westernblot zeigte sich in LLC-Zellen eine p21 Defizienz als Ursache der fehlenden G1-Arretierung, eine Phosphorylierung von p53 an Serin 15 war hingegen nachweisbar und zeigte einen strahleninduzierten Anstieg auf Proteinebene. Die daraufhin durchgeführte p53 Mutationsanalyse mittels Gensequenzierung zeigte in Sequenz 1 (94-115 forward) an Basenposition 120 eine heterozygote Mutation zum STOP-Codon UAA. Durch diese Mutation ist eine suffiziente Proteinbiosynthese des p53-Proteins in LLC1-Zellen nicht möglich und eine Funktionalität des Proteins nicht gegeben. Diese fehlerhafte Proteinstruktur des p53 erklärt sowohl den fehlenden G1- Arrest, die p21-Defizienz als auch die fehlende Induzierbarkeit p53- abhängiger Gene wie CDKN1A, RRM2b, ERCC1 und BAX auf mRNA-Ebene, bei gleichzeitig gelelektrophoretisch nachweisbarem p53.
building Medizin
title_alt Lewis-Lung-Model radiobiological characterization
language German
license_str http://archiv.ub.uni-marburg.de/adm/urhg.html
title Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells
title_short Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells
title_full Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells
title_fullStr Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells
title_full_unstemmed Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells
title_sort Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells
contents Major component of the study was the radiobiological characterization of murine LLC1 cells in vitro. These cells are culture transferred single cells emanated from a widely used in vivo mouse tumor model. The solid, undifferentiated tumor clusters in vivo corresponded to a non-small cell lung cancer, the culture transferred individual cells were classified as malignant due to nuclear pleomorphism, hyperchromasia, atypical mitoses and moved nuclear- cytoplasmic ratio. If obtained under optimal conditions in culture the LLC1-cells showed a fast, stable growth pattern - with a doubling time of 16 hours- even over long culture periods, but reacted very sensitively to changes in the cell culture environment, such as composition of the growth medium, seeding density or type and coating of the growth surface. In this respect, they expressed divergent phenotypes, which differed mainly in terms of their morphology, adhesion capability and efficiency of plating. In genotypic terms the majority of the cells showed a tetraploid chromosome set with 80 single chromosomes. Ensuring optimal culture conditions reduced the confounding effect of culture associated cellular impairment and guaranteed the validity and reproducibility of further radiobiological analysis. Mathematically determined (based on Single-Hit-Multi-Target-Model & Linear- Quadratic-Model) characteristics of generated DEKs, suggested high radiation sensitivity, moderate to poor pronounced repair capacity. Considered radiobiological the LLC1-cells corresponded to an early responsive cell model most likely. Despite a generally rather weak distinctive capacity of repair the mechanisms of rapid DNA DSB repair suggested to be intact and conform to repair-kinetics and –dimension of malignant cells already described in the literature. For LLC1 cells the gene expression analysis showed no sufficient regulation of p53 dependent genes after photon irradiation. The repair genes ERCC1 and RRM2b, the apoptosis related gene BAX and the cell cycle regulatory gene CDKN1A were not induced by irradiation with photons. Genes with p53-independent regulatory mechanisms such as cell cycle regulating gene GADD45 and apoptosis related genes BCL2 and BIRC5 were irradiation induced and resulted in G2 arrest and induction of apoptosis after photon irradiation in LLC1 cells. The shift of the cellular balance towards proapoptotic mechanisms for photon irradiation is not due to overexpression of the pro-apoptotic BAX gene, which could not be induced by exposing to ionizing radiation in LLC1-cells, but mainly by underexpression of anti-apoptotic genes such as BCL2/BIRC5, with consequently missing inhibition of apoptosis. We observed significant increase in G2-population initially after photon irradiation, both with 2 Gy and 6 Gy. After exposure to high doses (6 Gy), a longrunning, potentially terminal G2 arrest was initiated, whereas after irradiation with low doses (2 Gy) a decreasing G2-population could be observed 18 h to 24 h post rad. No G1-arrest occurred in LLC1 cells even after exposure to 6 Gy photons. Quantitative screening by Western blot showed a p21 deficiency causing the lack of G1 arrest in LLC1-cells, a phosphorylation of p53 on serine 15 was detectable, and showed a radiation induced increase on protein level. The subsequently performed p53 gene sequencing shows a heterozygous mutation to UAA STOP codon in sequence 1 (94-115 forward) on base position 120. This mutation prevents a sufficient protein synthesis in LLC1-cells which is why a functionality of the protein is not given. This defective protein structure of p53 explains the lack of G1 arrest, the p21- deficiency as well as the lack of inducibility of p53-dependent genes such as CDKN1A, RRM2b, ERCC1 and BAX at the mRNA level, while p53 is electrophoretically detectable.
publishDate 2013
era_facet 2013
publisher Philipps-Universität Marburg
first_indexed 2013-03-12T00:00:00Z
ref_str_mv references
format Dissertation
thumbnail http://archiv.ub.uni-marburg.de/diss/z2013/0138/cover.png
spelling diss/z2013/0138 opus:4739 urn:nbn:de:hebis:04-z2013-01383 2013-02-20 2013-03-12 http://dx.doi.org/10.17192/z2013.0138 Hauptbestandteil der Arbeit war die strahlenbiologische Charakterisierung muriner LLC1-Zellen in vitro. Dabei handelt es sich um in Kultur überführte Einzelzellen eines bei in vivo Experimenten weit verbreiteten Tumormodells der Maus. Die soliden, entdifferenzierten Tumorzellverbände in vivo entsprachen einem nichtkleinzelligen Bronchialkarzinom, auch die in Kultur überführten Einzelzellen konnten aufgrund von Kernpleomorphie, Hyperchromasie, atypischen Mitosen und verschobener Kern-Plasma-Relation als maligne eingestuft werden. In Kultur zeigten die LLC1-Zellen unter optimalen Bedingungen ein schnelles, stabiles Wachstumsverhalten- mit einer Verdopplungszeit von 16 h- auch über lange Kultivierungszeiträume hinweg, reagierten aber Wachstumsmedium, Aussaatdichte sowie Art und Beschichtung der Wachstumsfläche betreffend, ausgesprochen sensibel auf Veränderungen des Kultur-Milieus. Dabei prägten sie divergente Phänotypen aus, die sich vor allem hinsichtlich ihrer Morphologie, Adhäsionsfähigkeit und Plattierungseffizienz voneinander unterschieden. Genotypisch wies der Großteil der Zellen tetraploide Chromosomensätze mit 80 Einzelchromosomen auf. Die exakte Orientierung an den ermittelten optimalen Kulturbedingungen war, durch Reduktion von Störfaktoren einer kultivierungsbedingten zellulären Beeinträchtigung, Grundlage der Aussagekraft und Reproduzierbarkeit weiterführender strahlenbiologischer Analyseverfahren. Anhand mathematisch (Single-Hit-Multi-Target-Modell & Linearquadratisches Modell) ermittelter Kenngrößen aus generierten DEKs ließen sich die LLC1-Zellen als strahlensensible Zellen mit mäßig bis schlecht ausgeprägter Reparaturkapazität einstufen, die strahlenbiologisch eher einem frühreagierenden Zellsystem entsprechen. Trotz einer generell eher schwach ausgeprägten Reparaturkapazität waren die Mechanismen der schnellen DNA-DSB Reparatur intakt und entsprachen sowohl zeitkinetisch als auch dimensional denen anderer bereits in der Literatur beschriebener maligner Zellen. In der Genexpressionsanalyse zeigte sich für LLC1-Zellen keine suffiziente Genregulation p53 abhängiger Gene nach Photonenbestrahlung. Die reparaturrelevanten Gene RRM2b und ERCC1, das apoptoserelevante BAX und das zellzyklusregulierende CDKN1A waren durch Photonenbestrahlung nicht induzierbar. Gene mit p53 unabhängigen Regulationsmechanismen wie das zellzyklusregulierende Gen GADD45 und die apoptoserelevanten Gene BCL2 und BIRC5 waren induzierbar und führten in LLC1-Zellen zu G2-Arretierung und Apoptoseinduktion nach Photonenbestrahlung. Dabei erfolgte die Verschiebung des zellulären Gleichgewichts hin zu proapototischen Mechanismen nach Photonenbestrahlung nicht durch Überexpression des, in LLC1-Zellen nicht strahleninduzierbaren, proapoptotischen Gens BAX, sondern primär durch Unterexpression antiapoptotischer Gene wie BCL2/BIRC5 mit konsequenterweise fehlender Hemmung der Apoptoseinduktion. Nach Exposition gegenüber hohen Dosen (6 Gy) leiten LLC1-Zellen einen GADD45 vermittelten, lang andauernden, potentiell zeitlich unbegrenzten G2-Arrest ein. Hingegen war nach Bestrahlung mit geringeren Dosen von 2 Gy ein Ende des G2-Arrests zwischen 18 h und 24 h post rad festzustellen. Eine G1-Arretierung erfolgt in LLC1-Zellen auch nach Exposition gegenüber 6 Gy Photonen nicht. Im quantitativen screening mittels Westernblot zeigte sich in LLC-Zellen eine p21 Defizienz als Ursache der fehlenden G1-Arretierung, eine Phosphorylierung von p53 an Serin 15 war hingegen nachweisbar und zeigte einen strahleninduzierten Anstieg auf Proteinebene. Die daraufhin durchgeführte p53 Mutationsanalyse mittels Gensequenzierung zeigte in Sequenz 1 (94-115 forward) an Basenposition 120 eine heterozygote Mutation zum STOP-Codon UAA. Durch diese Mutation ist eine suffiziente Proteinbiosynthese des p53-Proteins in LLC1-Zellen nicht möglich und eine Funktionalität des Proteins nicht gegeben. Diese fehlerhafte Proteinstruktur des p53 erklärt sowohl den fehlenden G1- Arrest, die p21-Defizienz als auch die fehlende Induzierbarkeit p53- abhängiger Gene wie CDKN1A, RRM2b, ERCC1 und BAX auf mRNA-Ebene, bei gleichzeitig gelelektrophoretisch nachweisbarem p53. Lewis-Lung-Model radiobiological characterization Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells Major component of the study was the radiobiological characterization of murine LLC1 cells in vitro. These cells are culture transferred single cells emanated from a widely used in vivo mouse tumor model. The solid, undifferentiated tumor clusters in vivo corresponded to a non-small cell lung cancer, the culture transferred individual cells were classified as malignant due to nuclear pleomorphism, hyperchromasia, atypical mitoses and moved nuclear- cytoplasmic ratio. If obtained under optimal conditions in culture the LLC1-cells showed a fast, stable growth pattern - with a doubling time of 16 hours- even over long culture periods, but reacted very sensitively to changes in the cell culture environment, such as composition of the growth medium, seeding density or type and coating of the growth surface. In this respect, they expressed divergent phenotypes, which differed mainly in terms of their morphology, adhesion capability and efficiency of plating. In genotypic terms the majority of the cells showed a tetraploid chromosome set with 80 single chromosomes. Ensuring optimal culture conditions reduced the confounding effect of culture associated cellular impairment and guaranteed the validity and reproducibility of further radiobiological analysis. Mathematically determined (based on Single-Hit-Multi-Target-Model & Linear- Quadratic-Model) characteristics of generated DEKs, suggested high radiation sensitivity, moderate to poor pronounced repair capacity. Considered radiobiological the LLC1-cells corresponded to an early responsive cell model most likely. Despite a generally rather weak distinctive capacity of repair the mechanisms of rapid DNA DSB repair suggested to be intact and conform to repair-kinetics and –dimension of malignant cells already described in the literature. For LLC1 cells the gene expression analysis showed no sufficient regulation of p53 dependent genes after photon irradiation. The repair genes ERCC1 and RRM2b, the apoptosis related gene BAX and the cell cycle regulatory gene CDKN1A were not induced by irradiation with photons. Genes with p53-independent regulatory mechanisms such as cell cycle regulating gene GADD45 and apoptosis related genes BCL2 and BIRC5 were irradiation induced and resulted in G2 arrest and induction of apoptosis after photon irradiation in LLC1 cells. The shift of the cellular balance towards proapoptotic mechanisms for photon irradiation is not due to overexpression of the pro-apoptotic BAX gene, which could not be induced by exposing to ionizing radiation in LLC1-cells, but mainly by underexpression of anti-apoptotic genes such as BCL2/BIRC5, with consequently missing inhibition of apoptosis. We observed significant increase in G2-population initially after photon irradiation, both with 2 Gy and 6 Gy. After exposure to high doses (6 Gy), a longrunning, potentially terminal G2 arrest was initiated, whereas after irradiation with low doses (2 Gy) a decreasing G2-population could be observed 18 h to 24 h post rad. No G1-arrest occurred in LLC1 cells even after exposure to 6 Gy photons. Quantitative screening by Western blot showed a p21 deficiency causing the lack of G1 arrest in LLC1-cells, a phosphorylation of p53 on serine 15 was detectable, and showed a radiation induced increase on protein level. The subsequently performed p53 gene sequencing shows a heterozygous mutation to UAA STOP codon in sequence 1 (94-115 forward) on base position 120. This mutation prevents a sufficient protein synthesis in LLC1-cells which is why a functionality of the protein is not given. This defective protein structure of p53 explains the lack of G1 arrest, the p21- deficiency as well as the lack of inducibility of p53-dependent genes such as CDKN1A, RRM2b, ERCC1 and BAX at the mRNA level, while p53 is electrophoretically detectable. 2013 2013-03-12 RAHN, J. J., ADAIR, G. M. & NAIRN, R. S. 2010. Multiple roles of ERCC1-XPF in mammalian interstrand crosslink repair. Environ Mol Mutagen, 51, 567-81. 2010 Multiple roles of ERCC1-XPF in mammalian interstrand crosslink repair TAKENAGA, K. 1986. Modification of the metastatic potential of tumor cells by drugs. Cancer Metastasis Rev, 5, 67-75. 1986 Modification of the metastatic potential of tumor cells by drugs POUGET, J. P. & MATHER, S. J. 2001. General aspects of the cellular response to low-and high-LET radiation. Eur J Nucl Med, 28, 541-61. 2001 General aspects of the cellular response to low-and high-LET radiation POMP, J., WIKE, J. L., OUWERKERK, I. J., HOOGSTRATEN, C., DAVELAAR, J., SCHRIER, P. I., LEER, J. 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