Supplementary Materials and Methods
AML patient samples
Bone marrow samples were collected at diagnosis, before drug treatment and
following informed consent from patients treated intensively in the UK MRC/NCRI
AML10, 11, 12, 14 and 15 clinical trials in accordance with the 1964 Declaration of
Helsinki. Blasts and mononuclear cells were separated on a Ficoll-Hypaque density
gradient (Sigma, Poole Dorset U.K) as previously described.1 RNA was extracted
after storage in tissue banks.
Normal human hematopoietic progenitor cells
Normal human hematopoietic progenitor cells were purified from human cord blood
following informed consent and with approval from the South East Wales Research
Ethics Committee in accordance with the 1964 Declaration of Helsinki. Mononuclear
cells were separated on a Ficoll-Hypaque density gradient and CD34+ blast cells were
enriched using MiniMACS (Miltenyi Biotec, Bisley, U.K) indirect CD34+
immunomagnetic sorting as previously described.1
Murine 32D cell line
Murine 32D cells were obtained from the ATCC (LGC Standards, Teddington, U.K)
in 2000 and cultured in RPMI supplemented with 10% FCS and 10% WEHI-3Bconditioned medium (containing IL-3).
Affymetrix microarray analyses
Affymetrix (Affymetrix UK Ltd., High Wycombe, U.K) gene expression (including
wet chemistry and data analysis) was performed as previously described.1 Affymetrix
microarray expression data for 5 pair-matched day 3 RUNX1-ETO and GFP control
transduced CD34+ cells and bone marrow mononuclear cell samples from 14 t(8;21)
and 15 other FAB-M2 AML patients at diagnosis, as well as 9 normal donors was
exported into Genespring GXv11.0 (Agilent Technologies, Palo Alto, CA). Target
intensity values from each array underwent baseline transformation to the median of
all samples and were subjected to MAS5.0 normalisation, detection call filtering and a
1.5-fold expression change filter prior to statistical and post-hoc analyses. MYCT1
and GFI1B expression analyses were based on signal intensities and detection values
for Affymetrix probe-sets 220471_s_at and 208501_at, respectively.
Retroviral transduction
Ectopic expression of RUNX1-ETO and green fluorescent protein (GFP) control was
achieved using the PINCO retroviral vector2 as described previously.1, 3, 4 Novel
MYCT1 retroviral expression vectors were generated for this study and are shown in
Supplementary Figure S1. Full-length MYCT1 coding sequence was amplified by
PCR from IMAGE clone 40126673 with or without an additional C-terminal HA tag
using proof-reading Taq polymerase. Gel-purified MYCT1 cDNA sequences were
cloned into unique BamHI and EcoRI sites in the PINCO vector for retroviral LTRdriven expression and sequence verified before use. Human CD34+ progenitor or
murine 32D cells were infected with RUNX1-ETO, MYCT1 or GFP control
expression vectors by retroviral transduction over 2 consecutive days of culture, as
previously described.3 Cells were harvested for use in downstream assays on day 3 of
culture after verification of transduction efficiency by flow cytometric detection of the
GFP surrogate marker in comparison with mock-transduced control cells.
Quantitative RT-PCR (qRT-PCR)
Transduced CD34+ cell total RNA was purified and DNase-treated using the
QIAGEN RNeasy mini kit (QIAGEN Ltd., Crawley, U.K) before g was reverse
transcribed using random hexamers. Reactions for qRT-PCR were prepared using
LightCycler FastStart DNA SYBR Green I master mix (Roche, Welwyn, U.K.),
500nM MYCT1 or ABL housekeeping gene control5 primers (See Supplemental Table
1), 3mM MgCl2 and 50ng each cDNA. Normalised expression values were
determined by crossing point analysis of MYCT1 signal compared with ABL
housekeeping gene control in duplicate samples. Fold change was calculated using
the 2-Ct method. Relative transcript levels (RTL) were calculated as 1000 x 2-Ct.
Western blotting
Retroviral-driven MYCT1-HA protein expression in 100g day 3 GFP, MYCT1 and
MYCT1-HA transduced CD34+ whole cell lysates was validated by Western blotting
using an anti-HA antibody (Cambridge Biosciences, Cambridge, U.K) at 10g/ml
overnight at 4°C and compared with expression of GAPDH, detected using 10ng/ml
primary antibody (Santa-Cruz) for 3 hours at 20°C, used as a loading control.
Chemiluminescence detection was performed using AmershamTM ECL Advance
reagents (GE Healthcare, Chalfont St. Giles, U.K). GFI1B protein expression in 25g
day 3 GFP and MYCT1 transduced CD34+ whole cell lysates was determined using
400ng/ml anti-GFI1B (Santa Cruz, Heidelberg, Germany) overnight at 4°C.
Following detection with HRP-conjugated anti-rabbit (GE Healthcare) secondary
antibody, digital images were processed and analysed using Adobe Photoshop CS3
(Adobe Systems Europe Ltd., Uxbridge, U.K) to calculate absolute signal intensity
(mean intensity x pixel number). Relative signal intensity (recorded above panels)
was generated by normalising to GAPDH housekeeping signal.
Growth in bulk culture
Following retroviral transduction, MYCT1 and GFP control transgenic CD34+ cells
were cultured in medium supplemented with 5ng/ml IL-3, SCF, G-CSF and GM-CSF
(Peprotech EC Ltd., London, U.K) for 30 days at 37oC. At the time-points indicated,
liquid cultures were analysed by four-colour immunophenotypic analysis. Cells were
incubated for 30 minutes at 4°C in PBS/1% BSA with the following primary stage
antibodies; CD13-APC (Leinco Technologies, St. Louis, Missouri, U.S), CD36-biotin
(Axxora, Cambridge, U.K) and CD34-PE and streptavidin-PerCP-Cy5.5 (BD
Biosciences, Oxford, U.K) secondary antibody at manufacturer-recommended
concentrations. Background fluorescence was established by isotype control
antibodies and data were acquired using a FACSCalibur flow cytometer (BD
Biosciences) collecting a minimum of 10,000 events. Analysis was performed using
WinMDI (J Trotter, BD Biosciences).
32D trophic withdrawal assays
Following retroviral transduction, murine 32D cells were washed twice in PBS,
counted and plated at a density of 1x105 cells/ml in the presence or absence of WEHI3B-conditioned medium containing IL-3. At defined time points over 48 hours, cell
counts and viability were assessed by trypan blue dye exclusion. Transduction
efficiency was assayed by flow cytometry. Viability at each time point was
normalised to the time zero value for each specific transgene and expressed as a
percentage.
Cell survival assays
MYCT1 and GFP control transduced day 4 hematopoietic progenitor cells were
washed twice and plated at 1×104 cells/well in 96 well U-bottomed plates in serumfree IMDM containing antibiotics. Ten-fold serial dilutions of individual cytokines
were added to appropriate wells and cells were cultured for 48 hours at 37°C. Cells
were harvested following addition of APC-conjugated beads and myeloid precursorspecific antibodies (BD Biosciences). Numbers of viable monocytes (CD13hi CD36hi)
and granulocytes (CD13hi CD36lo) were assessed by timed acquisition flow cytometry
using 1g/ml 7-aminoactinomycin D and normalised to the recovery of APCconjugated beads from the same wells.
Colony-forming assays
Colony assays using day 3 GFP control and MYCT1-expressing CD34+ cells were
performed by limiting dilution in 96 well U-bottomed plates (0.3 cells/well) in
medium supplemented with 5ng/ml IL-3, SCF, G-CSF and GM-CSF at 37oC.
Individual colonies (>50 cells) and clusters (>5 cells) were harvested 7 days following
plating and were scored and analysed for lineage differentiation. Colonies were
identified as monocytes (CD15lo, CD14+, CD13hi), granulocytes (CD15hi, CD14+,
CD13lo) or mixed granulocyte/monocyte, consisting of >20% and more than 10 cells
phenotypically-characterised as each element. The frequencies of monocyte,
granulocyte and mixed granulocyte/monocyte colonies were calculated as percentages
of the total colony numbers. Colony size was measured as the number of events
collected during a 10 second timed acquisition on a FACSCalibur flow cytometer,
normalised to the recovery of APC-conjugated beads loaded into colony-containing
wells before harvest.
References
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