Basic HTML Version
International Journal of Molecular Medical Science
10
T-precursor ALL samples compiled from the archived “Microarray Innovations in Leukemia” (MILE) study (GSE13159). Expression
values for these probesets hybridized onto the Affymetrix Human Genome U133 Plus 2.0 Arrays were calculated using non-central
trimmed mean of differences between perfect match and mismatch intensities with quantile normalization (DQN3, signal normalized
with quantiles of the beta distribution with parameters p=1.2 and q=3). To determine the differential expression of each probetset,
T-tests were performed between Normal and T-precursor samples (2-sample, Unequal variance correction) exhibiting significant
reductions in
XRCC5
/
Ku80
for both probesets in T-precursor ALL cases
1.4 Low Level
Ku
Expression is a Unique Hallmark
of Childhood T-Precursor Leukemia and It is
Associated with Diminished Expression Levels of
Ikaros
Target Genes
We next examined and compared the gene expression
profiles of normal bone marrow cells in 74 healthy
bone marrow specimens vs. leukemic bone marrow
cells in primary leukemia specimens 174 patients with
T-lineage ALL for differential expression of validated
IK target genes. Thirty of 40 (75%) transcripts
representing 20 of 26 validated (77%) IK target genes
as well as all 3 IKZF1 transcripts were expressed at
markedly lower levels in leukemic bone marrow
specimens from T-lineage ALL patients (Figure 5 A
and B). These results are in accord with our original
report that children with T-lineage ALL with NCI
high-risk criteria
express
dysfunctional
dominant-negative IK isoforms (Sun et al., 1999).
Notably, leukemic bone marrow specimens from
T-lineage ALL patients also exhibited profoundly
reduced expression levels for both of the 2 transcripts
encoding the XRCC5/Ku80 component of the
heterodimeric DNA repair protein Ku (Figure 5C).
The documented association of diminished IK target
gene expression in T-lineage ALL with markedly
reduced Ku80 transcript levels uniquely indicates that
Ku80 deficiency may play a previously unknown
causal role in IK malfunction that has been implicated
in leukemogenesis of pediatric high risk T-lineage
ALL. A role of Ku80 deficiency in T-lineage ALL is
also supported by our recent study, which
demonstrated that Ku80 haploinsuffiency in mice
causes development of an LPD involving
CD2
+
CD4
+
CD8
+
CD1
+
IL7R
+
thymic T-cell precursors
with functional IK deficiency (Ozer et al., 2013).
2 Materials and Methods
2.1 Expression of Recombinant Human Ku in Sf21
Insect Ovary Cells
The 2.0-kb
ku70
and 2.2-kb
ku80
cDNA fragments
including their protein coding segments (Gell and
Jackson, 1999) were individually cloned into the
NcoI/KpnI site of the 4.9-kb pFastBacHT (PFBH)
donor vector (Life Technologies) containing a
6×-histidine (6×His) tag to construct recombinant
PFBH-ku70 and PFBH-ku80 plasmids. PFBH-
ku70
and PFBH-
ku80
were used to generate recombinant
baculoviruses by site-specific transposition in
Escherichia
(E.)
coli
DH10Bac competent cells (Life
Technologies), which harbor a baculovirus shuttle
vector (bacmid), bMON14272 with a mini-attTn7
target site for site-specific transposition using
previously reported procedures (Uckun et al., 2010,
2011; Mahajan et al., 2001). The bacterial colonies
containing recombinant bacmids were identified by
disruption of the
lacZa
gene. High molecular weight
miniprep DNA was prepared from selected
E.coli
clones containing recombinant bacmid and transfected
into Sf21 cells using the Cellfectin reagent (Life
Technologies) as previously described (Ozer et al.,
2013; Uckun et al., 2010, 2011; Mahajan et al., 2001).
Sf21 cells were infected with both recombinant
baculoviruses to produce the Ku70/Ku80 heterodimer
(Ozer et al., 2013). Cultures were incubated at 28
℃
and stirred at 80~100 rpm using a magnetic stirrer
(Bellco Glass, Inc.,Vineland, NJ) for 48 h. Infected
cells were harvested by gentle centrifugation in a
Beckman GS-6 centrifuge at 500 × g for 7 min at
room temperature. Cells from 1-liter cultures were
flash-frozen at -80
℃
and stored at -80
℃
until
purification of recombinant Ku70 and Ku80 proteins.
Frozen Sf21 insect cells were lysed in 1× Triton
X-100 extraction buffer (1% Triton X-100, 10 mM
Tris, 130 mM NaCl, 10 mM NaF, 10 mM sodium
phosphate, pH 7.5) (1 mL lysis buffer per 20 × 106
cells). One pellet of Complete
TM
protease inhibitors
(Roche Molecular Biochemicals)was added for each
25 mL of the lysate and the mixture was rotated for 2
h at 4
℃
. The cell pellets were centrifuged at 45 000
rpm × 1 h a Beckman Optima LE-80K ultracentrifuge
Molecular Medical Science, Int’l Journal of