download report - Istituto Pasteur

P a r t i c i p a n t s :
Alberto Ciolfi, PhD student; Carmen Maresca, undergraduate
student.
C o l l a b o r a t i o n s :
UCSF Cancer Center, University of California, San Francisco, USA
(Prof. Joseph F. Costello); Roswell Park Cancer Institute,
Buffalo, NY, USA (Prof. Dominic J. Smiraglia).
Report of activity
The main objective of our project is the identification
of targets of aberrant DNA methylation in
acute myeloid leukemia (AML) cell lines and primary
blasts from AML patients by Restriction Landmark
Genome Scanning (RLGS). RLGS has been widely
used to identify imprinted genes, targets of DNA
amplification, deletion, and gene amplification. When
the RLGS is executed using methylation sensitive
enzymes, it represents a genome wide quantitative
approach that is uniquely suited for simultaneously
assessing the methylation status of thousands of
CpG islands within and between samples. The identification
of a large number of aberrantly methylated
loci in a variety of tumors via RLGS has been
instrumental in showing that aberrant CpG island
methylation patterns are tumor-type specific and non
random. RLGS separates radiolabeled NotI fragments
in two dimensions and allows distinction of
single-copy CpG islands from multicopy CpG-rich
sequences 2 . The methylation sensitivity of the
endonuclease activity of NotI provides the basis for
differential methylation analysis. The reduction in
spot intensity or the appearance of “novel spots” in
RLGS profiles indicates events of aberrant methylation
or demethylation respectively.
To date, we have produced a “master DNA methylation
profile” to use as control profile in our experiments and
comparisons. We obtained this normal “master DNA
methylation profile” from the integration of methyla-
73
Molecular genetics of eukaryotes - AREA 3
Identification of novel genetic and epigenetic targets in Leukemia
by genome wide approaches
Principal investigator: Giuseppe Zardo
Researcher in Clinical Chemistry
Dipartimento di Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 80319026; Fax: (+39) 06 80319054
zardo@bce.uniroma1.it
tion data from RLGS profiles of purified human bone
marrow CD34+HSC/HPCs (2 samples) and PB cells
(2 samples) isolated from consenting healthy donors.
This profile is composed of 1262 analyzable genomic
loci. These selected loci are unmethylated and present
in all analyzed RLGS profiles.
We were able to assign the chromosomal location
and corresponding DNA sequence to 757 out of
1262 genomic loci. The identification of the chromosomal
location and DNA sequence for the remaining
loci will be performed upon the recognition of these
loci as targets of aberrant DNA methylation events.
Next, with the aim to evaluate targets of aberrant
DNA methylation in human AML cell lines we have
acquired the DNA methylation profiles of several
human leukemia cell lines that differ for their FAB
classification, block of differentiation, presence of
oncogenic fusion proteins with epigenetic activity
and sensitivity to the differentiating effect of retinoic
acid. In detail, we have acquired RLGS DNA methylation
profiles of the following AML cell lines:
-HL-60, FAB M2, control and treated with 1 µM
retinoic acid for 24/48/72/96 hours
-Kasumi-1, FAB M2, AML1/ETO+
-SKNO-1, FAB M2, AML1/ETO+
-NB-4, FAB M3, PML/RAR+, control and treated
with 1 µM retinoic acid for 72 hours
-ML-2, FAB M4
-ME-1, FAB M4eo
-MV4-11, FAB M5
-AML-193, FAB M5
-HEL, FAB M6
Still, in order to complete our RLGS experiments we
need to obtain the RLGS DNA methylation profiles
from MOLM-16, FAB M0 and UT-7, FAB M7. To
date, a stringent comparison and cross-comparison has
been carried out between the DNA methylation profiles
of HL-60 FAB M2, NB-4 FAB M3 PML/RAR+
control and treated with retinoic acid for 72 hours and
the normal “master DNA methylation profile”.