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Abstract
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.
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Supervisor:
Dates
Created: 2013Issued: 2013-03-12Updated: 2013-03-12
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
Lewis-LungLewis-LungFabian EberleFabian Eberle
DFG-subjects
Adjuvante StrahlentherapieLungeStrahlenbiologieKrebs
DDC-Numbers
610
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Eberle, Fabian: Strahlenbiologische Charakterisierung des murinen Lewis-Lung-Modells. : Philipps-Universität Marburg 2013-03-12. DOI: https://doi.org/10.17192/z2013.0138.
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This item has been published with the following license: In Copyright