Materials and methods :
Cells
AML cells, normal bone marrow, and umbilical cord blood cells were obtained after
approval by the institutional review boards and appropriate informed consent from
patients at the Hematology and Obstetric Department at the CHU-Nice (Centre
Hospitalier Universitaire, Nice, France). Patient blasts and cord blood mononucleated
cells were isolated within 24 hours after collection by Ficoll-Paque-Plus density
gradient (Amersham Biosciences, Buckinghamshire, UK). Fresh cells were
resuspended in RPMI 1640 medium containing 2 mM glutamine, 50U/mL penicillin,
50mg/mL streptomycin, 1mM pyruvate, 100µM -mercaptoethanol, and 10% fetal calf
serum before use.
Purification of normal CD34 positive cells.
Cells were incubated for 15min at 4°C with StemSep® Human CD34 Positive
Selection Cocktail (StemCell Technologies, Vancouver, BC), washed and incubated
with StemSep® Magnetic Colloid before magnetic separation on positive selection
columns (MACS Miltenyi Biotec, Bergisch Gladbach, Germany). Enrichment was
assessed by flow cytometry.
Assay for myeloid clonogenic progenitors
Cells were plated in triplicate in 35-mm tissue culture dishes ( VWR, Fontenay sous
Bois, France) in methylcellulose enriched media 1.3% methycellulose, 25% fetal
bovine serum, 2% bovine serum albumine, and 70% Iscove’s Modified Dulbecco’s
Media, already supplied with recombinant human SCF (rhu SCF 50 ng/ml), rhu IL-3
(20 ng/ml), rhu IL-6 (20 ng/ml), rhu-G-CSF(20 ng/ml), rhu EPO (3 U/ml) (R&D
systems Europe, Lille, France). Cells were plated at 500 to 3.105 cells/ml. For normal
CB samples, erythroid colonies (burst-forming and colony-forming units-erythroid:
BFU-E, CFU–E) were counted separately. AS602868 (3-10 µM) was directly added
to methylcellulose. Plates were incubated for 14 days at 37°C, 100% humidity, and
5% CO2. Clonogenic assays were also performed on bone marrow cells from
AS602868 treated or untreated mice using Complete Medium with murine Cytokines
MethoCult® GF M3434 (StemCell Technologies, Vancouver, BC). Colonies were
scored using an inverted microscope. All recombinant cytokines were from Peprotech
( Neuilly/Seine, France). Leukemic colonies were identified because of their smaller
size and the presence of blast cells.
Bulk-culture LTC-IC assays.
Long term culture–initiating cell assays (LTC-IC) in bulk culture were carried out
using a modification of a previously described procedure (1).
Briefly, M210B4 murine feeder cells genetically engineered to produce human IL-3,
G-CSF (kind gift of Dr N. Rochet, CNRS FRE 2720, Nice) were irradiated with 30 cGy
from a
137Cs
source and immediately plated in collagen-coated 12-well plates with
MyeloCult H5100 medium supplemented with fresh hydrocortisone (10 µM
hydrocortisone 21-hemisuccinate; Sigma). As AS602868 appeared to promote
detachment of feeder cells, normal or leukemic cells were removed and washed after
a 1 week culture, before replating on fresh M210B4 feeders, without further addition
of AS602868. Half of the cell suspension was replaced by fresh medium every week
in order to exhaust AML blasts and differentiated progenitors. After 5 weeks in
culture, the average number of CFC derived from LTC-ICs were quantified in a
clonogenic assay (2). Bars indicate means of quintuplet CFC counts ± standard
deviation. Leukemic colonies were identified because of their smaller size and the
presence of blast cells.
Mice and NOD/SCID Repopulation Assay
Animals : C57BL/6 and NMRI-nu/nu nude mice (Janvier, Le Genest St Isle, France)
and NOD/LtSz-scid/scid mice were from Charles River (L’Arbresle, France).
NOD/SCID xenografts : one day before transplantation, 5 week old NOD/SCID mice
were conditioned with 375 cGy total body irradiation (30 cGy/min) from a 60Co
source.
Normal or leukemic cells were thawed, and recovered in StemSpan® SFEM medium
for 18h. For cord blood xenograft, enrichment of Lin- hematopoietic cells was
performed using StemSep® negative selection kit. One day later, human cells were
injected in mouse tail vein (5. 106 AML cells, 5.104 cord blood cells). Every two weeks
chimerism was assessed by femoral bone marrow sampling. When human cells
could be significantly detectable, mice were treated with AS602868 resuspended in
0.5% carboxymethylcellulose 0.25% tween 80 for 4-6 weeks by oral gavage everyday
at
30
mg/kg.
Control
mice
were
administrated
with
vehicule
(0.5%
carboxymethylcellulose 0.25% tween 80). Leukemic cells were identified because of
their blast morphology and of a CD45/SSC gating characteristic of leukemic cells (3).
Flow cytometry analysis : after treatment mice were sacrificed, BM was harvested
and analysed. Cells were filtered through a 70 µm cell strainer (Falcon; BD
Biosciences), treated with ammonium chloride to lyse red blood cells, washed and
labeled with desired mAb. For chimerism evaluation samples were resuspended in
Hanks’ balanced salt solution with 5% human serum and anti-mouse IgG Fc receptor
monoclonal antibody (2.4G2; BD Pharmingen, Le-Pont-de-Claix, France) for blocking
of nonspecific binding to mouse Fc receptors. Samples were then incubated on ice
with human-specific pan-leukocyte marker CD45 TRI-COLOR® ( Invitrogen, Cergy
Pontoise, France) and anti-CD34-PE (BD Pharmingen) to detect human cells. Half of
the cells were incubated with mouse IgG1 TRI-COLOR® and IgG1-PE as isotypes
control for nonspecific immunofluorescence. Flow cytometric analysis was performed
on a Becton Dickinson FACScan flow cytometer. A gate was set to exclude cells
labeled with isotypes control. Statistical analysis : the Mann-Whitney nonparametric
test was used. Values were considered significantly different for P values less than
0.05.
Evaluation of NF-B target gene expression by semi quantitative and
quantitative RT-PCR :
After incubation with AS602868 for 24h, total RNA was isolated from 5·10 6 cells
using an RNeasy Mini Kit (Qiagen Inc., Courtaboeuf, France) 1µg of total RNA was
reverse-transcribed with
Superscript II (Invitrogen, Cergy Pontoise, France). For
semi-quantitative PCR, the amplification was performed for 25 cycles with an initial
hot start at 94 C° for 3 min, followed by 40 s of denaturation at 94 C°, 40 s of
annealing at 55 C°, 1 min of extension at 73 C°, and a final extension period of 7 min.
Primers sequences are available upon request. Fifteen-microlitre aliquots were run
on a 1 % agarose gel. Quantitative real time PCR, was performed on an ABI PRISM
7000 Sequence Detection System using SYBR Green I Dye (Applied Biosystems,
FosterCity, California, USA) as described (4). cDNAs (5 µl) or water as control, were
amplified in duplicate by real-time PCR in a final volume of 25 µl using the SYBR
Green Master Mix reagent (Eurogentec, Angers, France) and 300 nM of forward and
reverse primers. Primers were designed using PRIMER Express Software (Applied
Biosystems) IB- mRNA expression was used as reporter of NF-B activation (5).
Fluorescence differences were collected by the 7000 ABI thermal cycler during each
60°C cycle. Semi-logarithmic plots were constructed of delta fluorescence versus
cycle number. For analysis, a threshold was set for change in fluorescence at a point
in the linear PCR-amplification phase (Ct) (4).
Supplementary reference list :
1.
Sutherland H, Landsdorp P, Henkelman R, Eaves A, CJ E. Functional characterization
of individual human hematopoietic stem cells cultured at limiting dilution on supportive
marrow stromal layers. Proc Natl Acad Sci USA 1990;87:3584-3588.
2.
Ailles L, Gerhard B, Hogge D. Detection and characterization of primitive malignant
and normal progenitors in patients with acute myelogenous leukemia using long-term
coculture with supportive feeder layers and cytokines. Blood 1997;90:2555-2564.
3.
Lacombe F, Durrieu F, Briais A, Dumain P, Belloc F, Bascans E, et al. Flow
cytometry CD45 gating for immunophenotyping of acute myeloid leukemia. Leukemia
1997;11:1878-1886.
4.
Frelin C, Imbert V, Griessinger M, Peyron A, Rochet N, Philip P, et al. Targeting NFB activation via pharmacological inhibition of IKK2, induced apoptosis of human acute
myeloid leukemia cells. Blood 2005;105:804-811.
5.
Bottero V, Imbert V, frelin C, Formento J, Peyron J-F. Monitoring NF-kappaB
transactivation potential via real-time PCR quantification of IkappaB-alpha gene expression.
Mol. Diagn 2003;7:187-194.