Cancer Research in Switzerland - Krebsliga Schweiz

Nägeli Hanspeter | Regulation of nucleotide excision
repair by protein modifiers (KLS 01827­02­2006)
The frequency of skin cancer due to sunlight exposure is
increasing dramatically. The ultraviolet (UV) radiation of
sunlight induces damage in the genome by causing the
crosslinking of adjacent DNA bases. According to the type
of chemical link, these UV lesions are known as cyclobutane
dimers or 6,4 photoproducts. They lead to genetic
abnormalities and result in cancer unless they are readily
removed by DNA repair. This removal of UV crosslinks
from the genome is carried out by a multi­component
DNA repair system generally known as nucleotide excision
repair. However, this DNA damage excision machinery
faces the important problem that the cellular DNA
substrate is tightly packaged in chromatin, raising the
question of how UV lesions may be detected in such a
poorly accessible environment. Another problem is that
cyclobutane dimers cause minimal changes of the DNA
structure, and therefore, this particular type of crosslink is
poorly recognized by XPC protein, which is the initial
damage sensor component of the DNA repair machinery.
In the frame of our research project, we used cell biology
methods to analyze the dynamic assembly and disassembly
of active repair complexes in the chromatin context.
This approach led us to discover two novel regulatory
mechanisms that coordinate the DNA repair of UV lesions
in human skin cells. As a master regulator in both control
systems we have identified DDB2, a previously enigmatic
factor known on the basis of its binding preference for
UV­damaged DNA but whose function in the DNA damage
response remained unclear. We discovered that DDB2
is able to inspect the cellular chromatin to search for
highly accessible sites and demarcate these hotspots for
DNA repair activity. For that purpose, DDB2 recruits an
enzyme complex that mediates the modification of the
XPC sensor protein with ubiquitin residues. These attached
ubiquitin modifiers mediate the retention of XPC
protein at DNA repair hotspots, thus preventing its futile
migration to more densely packed chromatin that is refractory
to DNA repair. By this mechanism, DDB2 ensures
the fast repair of DNA sites that are actively engaged in
critical cellular processes, thus providing more time for the
long­term and slow repair of less accessible chromatin
sites. Independently of ubiquitin modifiers, we found that
DDB2 is also able to exert a lever action on the DNA double
helix to allow for the docking of the XPC sensor to the
otherwise unrecognizable cyclobutane dimers and thus
initiate their repair.
In summary, our results show that DDB2 is a master organizer
that optimizes the spatiotemporal distribution of
DNA repair activity in the chromatin of human skin cells.
In future experiments, we will test how vitamins, UV filters,
antioxidants and other ingredients of cosmetics or
sunscreens influence the quantity and function of DDB2
in human cells. Knowledge of these regulatory systems
provides a rational basis for preventive interventions to increase
the efficiency and precision of DNA repair and
hence to reduce the risk of skin cancer.
Project coordinator
Prof. Dr. Hanspeter Nägeli
Institut für Veterinärpharmakologie und ­toxikologie
Universität Zürich
Winterthurerstrasse 260
CH­8057 Zürich
Phone +41 (0)44 635 8763
naegelih@vetpharm.uzh.ch
Ochsenbein Adrian F. | Immunosurveillance of chronic
myeloid leukemia in mice (OCS 01627­02­2005)
Chronic myelogenous leukaemia (CML) is a malignant
clonal myeloproliferative disease arising from a haematopoietic
stem cell expressing the BCR/ABL fusion protein.
CML is characterized by a chronic phase lasting for several
years. During this time period, immune responses co­exist
with the CML and probably control the disease. Eventually
CML progresses from the chronic phase to terminal
blast crisis. This might be due to failure in immunosurveillance.
We identified peripheral and central tolerance
mechanisms that impair the immunosurveillance of CML
and contribute to disease progression in a murine CML
model. To this end, we transduced bone marrow cells
from donor mice with a retroviral construct that expresses
the oncogene BCR/ABL and transferred these cells to recipient
mice.
We found that CML­specific CTLs were functionally impaired.
CTL did not produce effector cytokines, did not
proliferate after antigenic stimulation and displayed very
limited cytolytic function. This functional impairment of
CTL is termed exhaustion. CTL exhaustion has been attributed
to the signal transduction of different inhibitory
receptors such as PD­1. Indeed, CTL exhaustion in CML
was mediated by the PD 1/PD L1 interaction. Blocking
PD­1 signal transduction by using PD­1­deficient recipient
mice or by administration of aPD­L1 antibody reversed
CML­specific T cell tolerance, and the time to disease progression
was increased. In addition, PD­1 was upregulated
on CD8 + T cells from CML patients, suggesting that
this inhibitory pathway may also be of importance in the
regulation of CML­specific CTLs in humans.
The BCR/ABL fusion protein provides novel and potentially
immunogenic tumour­specific antigens. The leukaemia
BCR/ABL expressing stem cell differentiates to different
cell populations including professional antigen­presenting
dendritic cells (DC). BCR/ABL­expressing DCs were found
in various organs. However, BCR/ABL­expressing DCs
failed to efficiently induce leukaemia­specific T cell responses
due to low DC maturation and impaired migration
to secondary lymphoid organs. Moreover, we demonstrated
that BCR/ABL­expressing DCs preferentially
migrated to the thymus where they induced a deletion of
leukaemia­specific CD8 + T.
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