96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

Abstracts
plification identifies a separate subgroup. The identification of distinct
molecular subgroups of AS forms a basis for the identification of novel
pathways that may help to better understand the biology of AS and may
eventually lead to the development of more specific treatments.
SA-P-061
Molecular analysis of COL1A1/PDGFB translocation and PDGFB
amplification in patients with Dermatofibrosarcoma protuberans
K . Walluks1 , S . Hauk2 , C . Wölfel1 , Y . Chen1 , T . Cui1 , L . Yang1 1 2 Universitätsklinikum Jena, Institute of Pathology, Jena, Zytovision, Bremen
Aims. Dermatofibrosarcoma protuberans (DFSP) is a dermal and subcutaneous
tumor of intermediate malignancy. The most remarkable
cytogenetic feature of DFSP is the chromosomal translocation t(17;22)
(q22;q13), causing a fusion of the platelet-derived growth factor beta
chain (PDGFB) gene on 22q13 and the collagen type 1 alpha 1 (COL1A1)
on 17q22. The aim of the study was to analyze the molecular characteristics
of DFSP
Methods. We performed Fluorescence in situ hybridization (FISH) and
multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to
detect chromosomal translocation and fusion gene transcripts in 16 formalin-fixed,
paraffin-embedded DFSP samples. Additionally, we analysed
the gene copy number of PDGFB in the 16 samples by real-time PCR.
Results. Eleven out of 16 samples (68.8%) showed fusion transcripts by
multiplex RT-PCR analysis. Various exons of the COL1A1 gene were fused
with PDGFB gene, among them, exon 25 was found to be more frequently
involved. It turned out that, PDGFB copy numbers in the DFSP
samples was slightly higher than in normal skin tissues (p=0.007).
Conclusions. Our results suggest that the COL1A1/PDGFB fusion transcripts
and amplification of PDGFB may be diagnostic markers for patients
with DFSP, and PDGFB could be a potential target for treatment
of patients with DFSP.
SA-P-062
PHD3 silencing leads to increased tumor growth accompanied by
enlarged tumor vessels
A . Kettelhake1 , M . Rezaei2 , A . Kuzmanov1 , D . Poitz3 , B . Wielockx1 , G . Breier1 1 2 Technical University of Dresden, Department of Pathology, Dresden, Technical
University of Dresden, Department of Pathology, Dresden, 3Technical University of Dresden, Department of Internal Medicine and Cardiology,
Dresden
Aims. The fast growth of solid tumors causes the development of hypoxic
areas that are very often marked by the upregulation of the hypoxia-inducible
factor (HIF). HIF enables the tumor cells to survive under hypoxic
conditions for example by increasing the production of angiogenic
factors. HIF is regulated by prolyl hydroxylase domains proteins (PHD1,
2, 3, 4). However, the function of PHD3 during tumor progression and
angiogenesis has not been investigated in detail so far. It is our aim to
address these open questions.
Methods. We stably silenced PHD3 in a murine osteosarcoma cell line
(LM8) using a lentiviral transduction system and analyzed the cell clones
by quantitative real-time PCR and Western blotting. To investigate the
behavior of these clones in vivo we injected them subcutaneously on the
back of C3H mice and measured the tumor size every 2 days for a period
of 14 days. Perfusion of tumor tissue, immunohistochemical staining as
well as immunofluorescent staining was performed to analyze tumors.
Results. Surprisingly, downregulation of PHD3 does neither affect the
protein level of HIF nor the expression levels of known HIF-target genes.
The other PHD isoforms (PHD1, PHD2, PHD4) as well as factor inhibiting
HIF-1 (FIH) show equal mRNA levels in sh-PHD3 clones compared
to controls. However, transforming growth factor α (TGF-α) and platelet-derived
growth factor c (PDGF-C) are markedly upregulated, whereas
the angiopoietin 2 (Ang2) mRNA level is decreased in the sh-PHD3
158 | Der Pathologe · Supplement 1 · 2012
clones. In vivo experiments show the development of significantly larger
tumors compared to control tumors. Interestingly, the density of tumor
vessels is decreased but the vessels are enlarged as evidenced by PECAM
staining. α-smooth muscle actin (αSMA) immunofluorescence staining
reveals that more vessels in the PHD3 silenced tumors are covered with
αSMA-positive cells. We found no difference in vessel perfusion or leakiness
between shPHD3-tumors versus control tumors.
Conclusions. Our data suggest that PHD3 plays an important role as tumor
suppressor because knocking it down leads to accelerated tumor
growth. Unexpectedly, this function seems to be HIF-independent. The
structure of the shPHD3-tumor vasculature is completely altered indicating
that PHD3 is involved in tumor angiogenesis as well. At the moment
we are determining which factors are responsible for the observed
phenotype. For this, we are using clones simultaneously knocking down
PHD3 and one of the differentially expressed growth factors.
SA-P-063
Ubiquitin, SH2 and UBA domains of p62 regulate interaction of
p62 with K8/18 and aggregation properties
V . Mahajan1 , C . Stumptner1 , A . Thueringer1 , T . Klingstedt2 , P . Nilsson2 ,
K . Metchler3 , K . Kashofer1 , H . Denk1 , K . Zatloukal1 , J . Haybaeck1 1 2 Medical University of Graz, Institute of Pathology, Graz, Austria, Linkoping
University, Sweden, 3Medical University of Vienna, Institute of Molecular
Pathology, Vienna, Austria
Aims. Steatohepatitis and other liver diseases are characterized by presence
of cytoplasmic protein aggregates termed Mallory-Denk bodies
(MDBs) and Intracellular Hyaline Bodies (IHBs. Sequestosome 1/p62,
ubiquitin, Keratin 8 (K8) and Keratin 18 (K18) are the major constituents
of MDBs. We investigated whether interaction of p62 with K8/18
depends on a) ubiquitination, b) phosphorylation, c) conformational alterations,
or d) structural domains of p62.
Methods. The SH2/PB1, ZIP/PB1 and UBA domains of p62 were deleted.
Specific phosphorylation sites (S24 and S152) of p62 were mutated. The
phosphorylation and deletion constructs were co-transfected along with
K8 and K18 in presence or absence of ubiquitin. We used Luminescent
conjugated oligothiophenes (LCOs) to investigate conformational changes
of p62 deletion and phosphorylation mutants.
Results. Deletion of the SH2 domain or partially of the PB1 domain leads
to loss of the filamentous ultrastructure of p62 but resembles an IHBlike
aggregation pattern. Deletion of the ZIP domain or the remaining
PB1 domain lead to irregularly shaped intracytoplasmic aggregates
whereas UBA deleted p62 displayed a diffuse distribution pattern but
only a partial loss of filaments ultrastructure and did not interact with
K8/18 anymore. CHO-K1 cells transfected with various combinations of
SQSTM1/p62, ubi and Krt8/Krt18 demonstrated that SH2 domain deleted
p62 co-localizes with K8 in the absence of ubiquitin. The phosphorylation
sites S24 and S152 do not seem to regulate the interaction of p62
with ubiquitinated keratins but mutation at S24 created a diffuse distribution
pattern of p62. LCO analysis demonstrated the presence of cross
beta-sheet conformation in SH2 domain deleted p62 and K8. Additional
oxidative stress may interfere with MDB components but not with their
interaction.
Conclusions. These findings explain the observation that SH2 and UBA
domains govern the aggregation property of p62 and influence the interaction
patterns with K8/K18. Thus it is clear that filamentous assemblies
of type I MDBs are predominantly due to p62 while type II MDBs may
need p62 and ubiquitin. Alternatively the amorphous granular nature
of type III MDBs results from insoluble K8/K18 aggregates. K8 and SH2
deleted p62 can undergo conformational changes from predominantly
Alpha-helical to cross β-sheet structures which allow their interaction
even in the absence of additional ubiquitin. Therefore the SH2 domain
might regulate the interaction between K8 and p62wt.