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 Dr. Markus
Christmann
Department of Toxicology
Obere Zahlbacher Str. 67
D- 55131 Mainz
Germany
Tel.
0049-6131-17-9066
e-mail:
mchristm@uni-mainz.de
Regulation of DNA repair after exposure to genotoxic noxa
During the life span cells are
exposed to different endogenous and exogenous stress factors. These are also DNA
damaging agents such as UV light, ionizing radiation, oxidative stress and
chemical mutagens. As self-defending mechanisms within a cell, different DNA
repair mechanisms have evolved in order to protect the DNA molecule from
mutagenic and toxic effects of such substances.
An overview paper describing
different DNA repair mechanisms was published some years ago.
Christmann M, Tomicic MT,
Roos WP, Kaina B. (2003) Mechanisms of human DNA repair: an update.
Toxicology 193(1-2), 3-34.
The main question still remains
how cells react on multiple insults and whether different DNA repair mechanisms
after a particular insult are induced. Molecular mechanisms regulating the DNA
repair can strongly differ, such as transcriptional induction of genes,
switching on and �off the genes by different methylation rates (hypo and
hypermethylation), translocation of proteins and their post-translational
modification.
We could show that treatment with
alkylating agents leads to translocation of mismatch repair proteins MSH2 and
MSH6 (MutSalpha complex) from thy cytoplasm to the nucleus. In addition, we
could show that these proteins are post-translationally modified via
phosphorylation. Also during chemotherapy repair capacity of a perticular tumor
can be strongly changed. So e.g. for melanoma cells treated with
chloroethylating cytostatic drug fotemustin we could show that the treatmnt led
to reactivation of the repair protein MGMT, which is due to the methylation of
the gene body.
Christmann
M, Tomicic MT, Kaina B. (2002) Phosphorylation of mismatch repair proteins MSH2
and MSH6 affecting MutSalpha mismatch-binding activity. Nucleic Acids Res.
30(9), 1959-66.
Christmann
M, Pick M, Lage H, Schadendorf D, Kaina B. (2001) Acquired resistance of
melanoma cells to the antineoplastic agent fotemustine is caused by reactivation
of the DNA repair gene MGMT. Int J Cancer. 92(1):123-9.
Christmann
M, Kaina B (2000) Nuclear translocation of mismatch repair proteins MSH2 and
MSH6 as a response of cells to alkylating agents. J Biol Chem. 2000 275(46),
36256-62.
The project is financed by German
Science Foundation (DFG).
Transcriptional regulation of DNA repair genes
As already mentioned, cells can
react on genotoxic stress by up- or down-regulation of particular genes. Proto-oncogenes
from the c-fos/c-jun family as well as the tumor suppressor gene p53 are
directly induced after DNA damage. The corresponding proteins can further induce
other genes working as transcription factors.
We could already show that the
genes coding for the repair proteins MGMT and APE are transcriptionally
regulated after genotoxic stress.
Grösch,
S., Fritz, G. and Kaina, B. (1998) Apurinic endonuclease (APE-Ref-1) is induced
in mammalian cells by oxidative stress and involved in clastogenic adaptation,
Cancer Res., 58, 4410-4416.
Fritz,
G., Tano, K., Mitra, S. and Kaina, B. (1991) Inducibility of the DNA repair gene
encoding O6-methylguanine-DNA methyltransferase in mammalian cells by DNA
damaging treatments. Mol. Cell. Biol. 11, 4660-466
Fibroblasten,
welche defizient für c-Fos und p53 sind, reagieren hypersensitiv hinsichtlich
der clastogenen, zytotoxischen und Apoptose-induzierenden Wirkung von UV-Licht
und Alkylantien.
Lackinger
D, Eichhorn U, Kaina B. (2001) Effect of ultraviolet light, methyl
methanesulfonate and ionizing radiation on the genotoxic response and apoptosis
of mouse fibroblasts lacking c-Fos, p53 or both. Mutagenesis. 16(3):233-41.
Lackinger
D, Kaina B. (2000) Primary mouse fibroblasts deficient for c-Fos, p53 or for
both proteins are hypersensitive to UV light and alkylating agent-induced
chromosomal breakage and apoptosis. Mutat Res. 457(1-2):113-23.
The question is, however, whether
the hypersensitivity of c-fos or p53-deficient cells is due to a lack in
induction of DNA repair genes, in order words, whether it is a cause of a defect
in the DNA repair. So the aim of the actual project is to clarify this question
and to identify the DNA repair genes induced upon exposure to UV-C light that
might be responsible for hypersensitivity or resistance of cells.
For this purpose we used customized DNA microarrays carrying all known DNA repair genes and
replication-associated genes (so-called DNA repair chip). The experiments
showed a p53-depent induction of the flap endonuklease 1 (Fen1). Overexpression
of Fen1 leads to an enhanced abrogation of the replication blockage, which could
improve the access of the DNA repair proteins to the UV-C induced lesions
leading to enhanced cellular resistance
Christmann
M, Tomicic MT, Origer J, Kaina B. (2005) Fen1 is induced p53 dependently and
involved in the recovery from UV-light-induced replication inhibition. Oncogene.
24(56):8304-13.
Further experiments revealed that c-Fos defecient cells
are defective in the repair of UV-C induced DNA lesions. They show a decreased
rate of sealing of repair-mediated DNA strand breaks and are unable to remove
cyclobutane pyrimidine dimers from DNA. This NER defect is due to impaired
re-synthesis of the NER-endonuclease XPF, which is mediated by a direct
activation of the xpf promoter by c-Fos. The data suggest a novwl role
for c-Fos in cells exposed to genotoxic stress. Being part of the transcription
factor AP-1, c-Fos stimulates NER via the upregulation of XPF, thus playing a
central role in the recovery of cells from UV light induced DNA damage.
Christmann M, Tomicic MT, Origer J, Aasland
D, Kaina B. (2006) c-Fos is required for excision repair of UV-light induced DNA
lesions by triggering the re-synthesis of XPF. Nucleic Acids Res.
2006;34(22):6530-9.
if c-Fos is lacking, the cells are unable to remove
cyclobutane pyrimidine dimers from DNA, they are unable torecover from
transcription blockage leading to reduced expression of the MAP kinase
phosphatase 1 (MKP1). This results in sustained JNK activation and AP-1
dependent induction of the fasL, thereby activating the death receptor pathway
and inducing apoptosis.
Christmann M, Tomicic MT, Aasland D, Kaina B.
(2007) A role for UV-light-induced c-Fos: Stimulation of nucleotide excision
repair and protection against sustained JNK activation and apoptosis.
Carcinogenesis. 2007 Jan;28(1):183-90.
The project is financed by German
Science Foundation (DFG).
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