12th Congress of the European Hematology ... - Haematologica

12 th Congress of the European Hematology Association
0156
ONCOGENETIC FUNCTION OF KTS + ISOFORM OF WT1 IN ACUTE AND CHRONIC
LEUKEMIAS
D. Cilloni, I. Defilippi, S. Carturan, F. Messa, V. Rosso, F. Arruga,R.
Catalano, C. Bittoto, C. Boveri, E. Messa, P. Nicoli, E. Bracco, G. Saglio
University of Turin, TURIN, Italy
Background. The Wilms’ tumour gene (WT1) is overexpressed in a
variety of hematological malignancies including acute and chronic
leukemias, myeloproliferative disorders and myelodysplastic syndromes
and nowadays it is considered a sort of universal marker of leukaemia.
WT1 was originally identified as responsible for the kidney tumour of
Wilms and described as a tumour suppressor gene but, in the setting of
leukaemia, it seems to function as an oncogene. WT1 has different isoforms.
In particular, KTS + and KTS – are 2 isoforms derived from alternative
spicing of exon 9. In normal cells the two isoforms are approximately
equally represented. Aims. the aim of the study was to analyze
the different function and distribution of the two isoforms. Methods.
After informed consent, 132 BM samples were collected from 86 AML
and 46 CML patients at diagnosis and 20 samples from healthy subjects.
62 patients were also evaluated during follow-up. WTS + and KTS –
isoforms were quantified by capillary electrophoresis The relative
amount of two isoforms was calculated by measuring the picks area of
electropherogram . NIH3T3 and 293T cell lines were transfected with
WT1 KTS + or WT1 KTS – plasmids. WT1 protein was studied by Western
blot and immunofluorescence in BM cells and transfected cell lines.
Downstream genes transcriptionally activated by WT1 such as Spred-2
and E-Cadherin were evaluated by Real Time PCR. Results. We demonstrated
that AML and CML patients have an unbalanced KTS + /KTS – ratio
with a significant increase of KTS + isoform as compared to KTS – . The
ratio observed ranges from 1.6 to 6.1 in AML from 1.6 to 9.5 in CML.
In 10% of the patients we observed a complete disappearance of the
KTS- isoform . Western blot and immunofluorescence carried out in BM
cells and transfected cell lines allow to establish that the KTS+ isoform
is mainly localized in the cytoplasm and KTS – isoform is mainly nuclear
localized. In BM cells carrying the isoform KTS + or in cells transfected
with KTS + isoform we observed the lack of transcription of downstream
genes such as Spred1 or E-cadherin. In addition, in patients who
achieved a complete remission after chemotherapy, WT1 KTS + /KTS –
ratio returned within the normal range and Spred1 and E-cadherin transcript
and protein were significantly upregulated. Finally, cells transfected
with KTS + isoform presented morphology changes, altered adhesion
properties and increased proliferation as compared to KTS- transfected
cells. Conclusions. This study demonstrates that in leukemic cells there
is a disruption of the normal transcription activity of WT1 and this is
mainly due to the unbalanced ratio between the two isoforms KTS + and
KTS – with different localization and function. This alteration results in
a defective transcriptional activity of WT1 which can probably play an
oncogenic role in leukemic cells.
56 | haematologica/the hematology journal | 2007; 92(s1)
0157
ANKHD1 PROTECTS LEUKEMIA CELLS FROM APOPTOSIS AND BINDS TO SIVA, A
PROAPOPTOTIC PROTEIN
FT Traina, P.R.M. Lima, F.F. Costa, S.T.O. Saad
State University of Campinas, CAMPINAS, Brazil
Background. Ankyrin-repeat-containing proteins regulate multiple cellular
functions including transcription, cell-cycle, cell survival and participate
in protein'protein interactions via their repeat motifs. Ankyrin
Repeat and KH Domain Containing 1, ANKHD1, has been recently
described, in humans, as a cytoplasmic protein overexpressed in prostate
cancer cell line and in leukemia cells compared to normal hematopoietic
cells. Its homologous protein, MASK, was described in Drosophila
melanogaster as an essential protein for differentiation, proliferation and
cell survival. However, the role of ANKHD1 in leukemia cells has not
been fully elucidated. Aims. The aim of this study was to identify new
proteins associated with ANKHD1 and the role of ANKHD1 in the
apoptotic process of leukemia cells. Methods. In order to identify possible
targets of the ANKHD1 protein, we performed a yeast two-hybrid
screen using ANKHD1 protein (amino acids 1130-1243) in pGBKT7 vector,
as the bait, and a Matchmaker pACT2-cDNA library from normal
human bone marrow (Clontech), as the prey. The protein interaction
detected was confirmed using the yeast two-hybrid assay, through cotransfections
of AH109 yeast with the ANKHD1-pGBKT7 bait and the
new candidate for protein interaction identified in pGADT7 vector. Posttranscriptional
ANKHD1 gene silencing was done using small interfering
RNA, SMARTpool siRNA duplexes (Dharmacon), at a concentration
of 400 nM. Transient transfections of Jurkat cells were performed by
electroporation in a Bio-Rad Gene Pulser II (300V, 975 microfarads).
Cells were cultured for 48 h after transfections and then submitted to
Western blotting and apoptosis analysis. Apoptotic cell death was evaluated
using Annexin V-FITC/PI staining and FACS analysis. Results. The
yeast two-hybrid screening identified the new protein interaction
between ANKHD1 and SIVA. Co-transfections of AH109 with pGBKT7-
ANKHD1 and different SIVA-pGADT7 constructs (SIVA1, SIVA2, SIVA
C-terminal, SIVA N-terminal, SIVA Dead Domain) confirmed the association
between ANKHD1/SIVA1 and ANKHD1/SIVA2, and the need
for both the N-terminal and C-terminal regions of SIVA for the interaction
with ANKHD1. Western blotting confirmed that ANKHD1 expression
was reduced by 80% in the Jurkat cells transfected with ANKHD1
siRNA compared with controls cells (electroporated cells). Treatment
of Jurkat cells with the ANKHD1 siRNA resulted in increased apoptosis
(27% of apoptotic cells) compared with control cells (14% of apoptotic
cells). Conclusions. The association between ANKHD1 and SIVA
isoforms suggests that ANKHD1 participates in the apoptotic signaling
in leukemia cells, since we know that SIVA1 and SIVA2 are overexpressed
in acute lymphoblast leukemia cell lines and induce apoptosis
in Jurkat cells. The increased apoptotic rate after posttranscriptional
ANKHD1 gene silencing indicates an anti-apoptotic function of
ANKHD1 in Jurkat cells. In conclusion, ANKHD1 protects leukemia
cells from apoptosis and binds to SIVA, possibly inhibiting the proapoptotic
function of SIVA. These results indicate that ANKHD1 is associated
with the abnormal phenotype of leukemia cells; the identification of
new disease-specific targets for acute leukemia immunotherapy expands
treatment options and increases our chances of successfully treating this
heterogeneous disease and lowering the unacceptably high mortality
rate.