Blood-2011-Charfi-1899-910 Ppo

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2011 117: 1899-1910

Prepublished online December 6, 2010;
doi:10.1182/blood-2010-10-311001
Gene profiling of Graffi murine leukemia virus-induced lymphoid

leukemias: identification of leukemia markers and Fmn2 as a potential
oncogene
Cyndia Charfi, Vronique Voisin, Louis-Charles Levros, Jr, Elsy Edouard and Eric Rassart
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LYMPHOID NEOPLASIA
Gene profiling of Graffi murine leukemia virus-induced lymphoid leukemias:

identification of leukemia markers and Fmn2 as a potential oncogene
Cyndia Charfi,1 Veronique Voisin,1 Louis-Charles Levros, Jr,1 Elsy Edouard,1 and Eric Rassart1
1Laboratoire
de Biologie Moleculaire, Departement des Sciences Biologiques, Centre BioMed, Universite du Quebec a` Montreal, Montreal, QC
The Graffi murine leukemia virus induces

a large spectrum of leukemias in mice
and thus provides a good model to compare the transcriptome of all types of
leukemias. We analyzed the gene expression profiles of both T and B leukemias
induced by the virus with DNA microarrays. Given that we considered that a
4-fold change in expression level was
significant, 388 probe sets were associated to B, to T, or common to both leuke-
mias. Several of them were not yet associated with lymphoid leukemia. We confirmed
specific deregulation of Fmn2, Arntl2,
Bfsp2, Gfra2, Gpm6a, and Gpm6b in B
leukemia, of Nln, Fbln1, and Bmp7 in T
leukemias, and of Etv5 in both leukemias.
More importantly, we show that the mouse
Fmn2 induced an anchorage-independent
growth, a drastic modification in cell
shape with a concomitant disruption of
the actin cytoskeleton. Interestingly, we
found that human FMN2 is overexpressed

in approximately 95% of pre-B acute lymphoblastic leukemia with the highest
expression levels in patients with a TEL/
AML1 rearrangement. These results,
surely related to the role of FMN2 in
meiotic spindle maintenance, suggest its
important role in leukemogenesis. Finally, we propose a new panel of genes
potentially involved in T and/or B leukemias. (Blood. 2011;117(6):1899-1910)
Introduction
Methods
To understand the mechanism of induction of leukemia in humans,

the mouse has been considered to be an ideal model given the
extent of genetic similarity between these 2 species. We have
already shown that the Graffi murine leukemia virus (MuLV), when
inoculated into newborn mice, induces a broad spectrum of
leukemias of lymphoid (T or B) and nonlymphoid (myeloid,
erythroid, or megakaryoblastic) origins.1 We also demonstrated
that the Graffi MuLV directly targets the c-Myc, Fli1, Pim1, and
Spi1/P1 oncogenes2 but also the Gris1 locus that encodes an
oncogenic truncated form of cyclin D2.3,4 We took advantage of the
large spectrum of leukemias induced by the Graffi murine leukemia
virus to analyze and compare the transcriptome of these leukemias
with a DNA microarray approach. One clear advantage of microarray analysis is that genes that are not targeted through retroviral
integration but act as oncogene on deregulation can be equally
identified. Using this approach, we recently identified several genes
that are directly involved in erythroid and megakaryoblastic
leukemias in both mouse and human.5 In this report, we have
specifically compared the transcriptomes of T and B lymphoid
leukemias induced by Graffi MuLV with their corresponding
controls. We identified new relevant signatures that highlight many
potential markers or oncogenes for T and B leukemias (especially
for pre-B-leukemia), some of which were common to both types of
lymphoid leukemia. Among the selected genes, we validated the
modulation of the expression of 10 genes of 12, the functions of
which remained poorly elucidated in lymphoid leukemias. Furthermore, for the first time, we provide data supporting a role for Fmn2,
a member of the formin family in leukemogenesis in pre-B-lineage
acute lymphoblastic leukemia (B-ALL) and particularly, in pediatric pre-B-ALL harboring the t(12;21) TEL/AML1 translocation.
Flow cytometry was performed as previously described.1 Cell populations

(tumor or control) were purified from the hematopoietic organs by positive
selection using magnetic beads coated with the chosen antibody. Leukemic
cells were sorted as follow: T cells from the thymus, B cells from the
enlarged lymph nodes, and erythroid and megakaryoblastic cells from the
infiltrated spleen. Nonleukemic control cells were sorted from a pool of
12 noninfected NFS mice: T cells from the thymus, B cells from the spleen,
and erythroblasts and megakaryoblasts from the bone marrow.
Submitted September 30, 2010; accepted November 16, 2010. Prepublished

online as Blood First Edition paper, December 6, 2010; DOI 10.1182/blood-2010-10311001.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
2011 by The American Society of Hematology
BLOOD, 10 FEBRUARY 2011 VOLUME 117, NUMBER 6
Human sample collection

For this study, samples from 12 pediatric pre-B-ALL were obtained from Dr
Daniel Sinnet (Sainte-Justine Hospital, Montreal, QC), whereas samples
from 13 adult patients with different types of B leukemia were obtained
from the Quebec Leukemia Cell Bank. As a control (CH), we pooled
peripheral blood mononuclear cells isolated from 10 healthy adults.
Information relative to each patient is provided in supplemental Table 1
(available on the Blood Web site; see the Supplemental Materials link at the
top of the online article). The research protocol was approved by Ethic
Committees of all concerned institutions.
Mice sample collection
Newborn ( 24 hours) NFS mice were injected intraperitoneally with viral
particles of Graffi MuLV variant GV-1.4 (1 106 PFU) or GV-1.2
(3 106 PFU).1 Lymph nodes, thymus, bone marrows, and spleens were
harvested from moribund mice for flow cytometry analysis and RNA
extraction. All the experimental procedures were approved by the Animal
Care Committee of the University of Quebec (Montreal, QC).
Flow cytometry and cell isolation
1899

1900
CHARFI et al
RNA isolation, microarray hybridization, and

dataset normalization
Total RNA was directly isolated from spleen, thymus, lymph nodes, and
bone marrow samples or after cell sorting with the Trizol reagent
(Invitrogen) and purified using RNeasy Mini Kit (QIAGEN). Total RNA
(2 g) from each sample was prepared for hybridization to Affymetrix
Gene Chip Mouse Genome 430, Version 2.0 arrays (Genome Quebec
Innovation Center). The Affymetrix MicroArray Suite, Version 5.0 was
used to scan and quantify the arrays. Normalization of gene expression data
was performed using the Bioconductor implementation of Robust Multi
Array (B. Bolstad, University of California, Berkeley, CA) available from
the Flexarray software Version 1.2, R 2.7.2.6
Microarray data analyses
Using the gene expression profiles obtained from selected leukemias, the
Robust Multi Array values of the 45 000 probe sets were used to identify
differentially expressed genes. Genes with signal intensity significantly
higher (up-regulated) or lower (down-regulated) in leukemias versus
control cells (ie, with a 4-fold change in expression levels) were selected.
To group microarrays and/or genes based on the high degree of their
expression patterns, hierarchical clustering (complete linkage clustering,
correlation uncentered) was constructed using GeneCluster software Version 1.6.7 Probe sets selected were further analyzed and the results were
treated with the Tree View program. The NetAffx website (Affymetrix) was
also used to retrieve gene ontology annotations, probe sequences, and used
as a link to Unigene (National Center for Biotechnology Information) for
further functional studies. The microarray dataset was deposited at Gene
Expression Omnibus under the accession number GSE12581.
Semiquantitative RT-PCR
Reverse transcription (RT) reactions were performed with oligo(dT) as
primer using the Omniscript enzyme (QIAGEN) and 100 ng of total RNA.
Using an RT reaction corresponding to 10 ng of tumor RNA samples for
each selected gene and to 2 ng for actin, the polymerase chain reactions
(PCRs) were performed with the Taq polymerase kit (QIAGEN) and the
following conditions: 94C for 3 minutes, 94C for 45 seconds, 56C for
45 seconds, 72C for 30 seconds, with a final extension at 72C for
10 minutes. Annealing temperature and number of cycles were optimized
for each of the selected 12 genes with specific primers (supplemental Table
2) for semiquantitative analysis. PCR products were analyzed on agarose
gel, and band density was quantified with Quantity One Image Software
Version 4.4, using the actin gene as an internal control.
Cellular localization of Fmn2
NIH/3T3 fibroblasts, obtained from ATCC, were grown in Dulbecco
modified Eagle medium supplemented with 10% calf serum, 50 U penicillin, and 50 g of streptomycin (Invitrogen), 16 hours before transfection.
Cells were, respectively, transfected with p-EGFP-N1 (control vector) and
GFP-tagged Fmn2 using the polyfect reagent (QIAGEN). Fmn2 localization was analyzed by confocal microscopy 48 hours after transfection. For
colocalization, transfected cells were plated on glass coverslips and grown
at 50% confluence. Colocalization with the cell surface membrane was
determined after cell staining for 5 minutes with 2.5 g/mL of CellMask
Plasma Membrane Stains (Invitrogen) and washing with phosphatebuffered saline (PBS). Actin filament staining was performed after cell
fixation for 20 minutes with 4% paraformaldehyde, followed by PBS
washes and permeabilization for 5 minutes with 0.1% Triton X-100 in PBS.
Cells were incubated 1 hour in PBS with 1% bovine serum albumin, washed
twice with PBS and the coverslips, and then incubated with 0.3M of
phalloidin coupled to the AlexaFluor-555 (Invitrogen) for 20 minutes. After
2 washes with PBS, coverslips were mounted onto slides using Prolong
Gold antifade reagent (Invitrogen) and observed within 24 hours by
confocal microscopy. For -tubulin staining, the primary antibody used was
a mouse monoclonal anti--tubulin (1:2000; Sigma-Aldrich).
Colony formation in soft agar

To determine the anchorage-independent growth, colony formation was
tested in soft agar as previously described.8 Briefly, NIH/3T3 cells were
transiently transfected with 2.5 g of empty vector (pCMV), a Ras EJ 6.6,
or pCMV-Fmn2 expression vector. After 48 hours, 1 104 cells were
mixed with melted 0.3% agarose in Dulbecco modified Eagle medium and
seeded in 6-well plates on top of a 0.6% agarose base layer containing the
same medium. The top layer was covered with 1.5 mL of Dulbecco
modified Eagle medium. Cells were fed twice a week for 4 weeks and
observed with an optical microscope (40, Ernst Leitz, 6MBH Wetzlar).
Colonies whose size was at least twice larger than that of control colonies
were counted.
Results
To better elucidate the cancer signatures of B and T leukemias
induced by the murine Graffi virus and to identify new oncogene
candidates, a microarray analysis was performed on different types
of B and T leukemias induced by this virus compared with
nonleukemic B-cell populations (CB1, Cd45RCd19) and T-cell
populations (CT1, Cd4Cd8). Three B leukemias (B1 and B2,
Cd45RCd19; B3, Cd45RlowCd19Sca1) and 3 T leukemias
(T1, Cd4Cd8; T2, Cd4Cd8; and T3, Cd4Cd8) were chosen
for the microarray experiments (National Center for Biotechnology
Information GEO: GSE12581). We were especially interested to
identify genes commonly deregulated in these tumors despite their
heterogeneity and different stage of differentiation. Hierarchical
clustering analyses of genes with a 4-fold change in expression
levels compared with control samples were used to obtain a general
trend (supplemental Figure 1). This analysis allowed us to group
leukemia samples (columns) and/or genes (rows) based on the
similarity of their expression level. As a result, T leukemias and B
leukemias were clustered separately, making 2 distinguishable
groups (supplemental Figure 1). According to these data, Graffiinduced T and B leukemias showed both distinct and common gene
expression profiles. Indeed, clustering led to the formation of 6
subgroups representing probe sets either specifically deregulated in
B leukemias (188 overexpressed and 86 down-regulated), specifically deregulated in T leukemias (9 overexpressed and 48 downregulated), or commonly deregulated in both types (8 overexpressed and 32 down-regulated). The complete list of genes
presenting these lymphoid signatures is available at:
www.biomed.uqam.ca/rassart/microarray2.html.
Gene expression profile specific for pre-B leukemias
We first analyzed the expression profile of genes that could

determine the stage of differentiation of the B leukemias. For all
B leukemias, Rag2, Vpreb1, Igll1, Enpep, Ebf1, Il7R, Bst1, and
Foxp1 were overexpressed compared with control cells (Table 1).
Results in this table correspond to the average expression calculated from all samples analyzed in the microarray analysis (B and
T controls, B lymphoid, T lymphoid, myeloid, erythroid, and
megakaryoblastic tumors). Strong expression of these genes indicated that the 3 Graffi MuLV-induced B tumors were most
probably at a pre-B differentiation stage.5,9-15 In all B leukemias,
we also detected high levels of Cd79a and Cd79b mRNAs and low
levels of Cd20/Ms4a2 mRNA (Table 1). A total of 274 probe sets,
corresponding to at least 218 genes, were found highly deregulated
in all 3 B leukemias compared with control B lymphocytes. Among
these genes, 72 (86 probe sets) were down-regulated and 146
(188 probe sets) were up-regulated (supplemental Figure 1).
Cd19
Ly6a (Sca-1)
1450570_a_at
1417185_at
Tcra
Dntt (TdT)
Cd7
1441552_at
1449757_x_at
1419711_at
2.52
3.05
2.13
0.57
2.62
1.68
0.43
0.30
0.71
0.16
0.16
2.01
3.54
8.56
1.03
0.99
3.39
3.75
0.10
0.95
0.68
0.43
0.22
8.99
7.74
8.29
1.35
1.62
2.23
1.28
1.35
0.01
0.18
0.11
0.14
0.11
0.32
0.22
0.17
0.39
0.04
0.39
0.27
0.48
2.71
0.19
2.09
0.30
0.01
0.58
0.80
0.77
0.46
3.88
0.14
0.15
0.55
0.19
0.71
0.39
2.78
6.94
7.71
4.48
9.56
7.05
7.42
4.23
B2-CT*
2.49
3.53
0.29
2.31
0.12
0.11
0.55
0.73
0.72
0.46
4.40
0.37
0.94
0.37
0.01
0.29
0.04
0.80
1.04
0.04
0.11
2.39
0.64
6.37
0.29
7.82
4.48
9.58
7.08
7.48
1.40
B1-CT*
1.83
0.56
0.13
0.17
0.04
1.54
6.40
T3-CT*
2.16
2.04
2.54
0.50
1.95
0.27
0.86
2.40
0.29
1.87
0.29
1.53
0.47
3.49
0.69
1.27
1.84
0.98
4.34
3.16
5.98
4.92
3.45
3.66
0.68
4.16
0.29
0.32
0.01
1.71
0.04
1.97
3.35
3.05
0.13
0.23
0.05
0.10
1.63
0.40
0.01
0.24
2.48
0.09
0.08
3.40
0.14
0.24
0.27
0.02
3.59
1.73
5.33
T2-CT*
1.61
T1-CT*
1.05
6.32
0.05
0.11
0.27
2.52
2.42
2.25
1.84
0.03
2.21
0.07
0.41
0.11
0.97
0.82
0.49
0.66
0.20
0.58
0.77
0.14
0.83
0.11
2.22
6.21
7.05
3.95
9.18
7.00
7.42
4.83
B3-CT*
1.22
6.63
0.60
2.89
5.19
0.34
0.68
4.48
3.27
4.81
5.68
3.76
7.49
6.43
7.04
4.38
4.99
0.84
2.48
1.46
1.58
0.15
0.59
3.09
0.79
4.94
3.22
0.88
5.35
1.71
1.72
3.62
3.00
3.53
5.39
3.07
4.00
3.28
2.70
0.72
1.54
0.87
2.77
1.70
1.57
0.28
3.64
3.19
0.45
1.40
1.65
0.44
3.62
1.08
1.81
2.17
2.32
0.52
T1
0.21
1.00
1.80
1.89
0.71
4.81
CT
1.79
4.49
3.65
2.73
4.48
1.82
1.86
2.53
3.78
2.94
5.39
3.29
5.96
5.45
5.20
0.53
0.98
3.32
3.16
3.44
1.90
0.12
3.94
3.04
0.84
0.01
2.70
1.09
2.04
1.75
2.44
1.22
T2
2.32
1.94
1.63
1.90
5.49
3.06
3.06
4.58
2.33
4.13
5.25
3.54
1.49
1.30
1.25
3.03
3.37
1.39
2.11
0.42
1.29
0.19
3.11
2.45
1.39
1.36
0.77
1.13
1.97
1.93
2.25
1.59
T3
0.99
1.70
0.75
2.38
1.90
0.55
0.58
0.13
3.66
1.15
0.60
1.07
1.13
0.88
0.44
0.04
0.52
3.76
4.68
0.51
1.29
0.39
4.84
5.37
0.56
1.40
2.26
1.56
0.86
2.70
1.63
0.94
0.62
2.36
0.13
0.86
2.91
1.19
1.02
1.43
1.17
0.75
0.98
0.65
5.05
1.46
1.28
0.35
4.04
5.25
1.84
4.97
3.93
5.02
0.56
7.59
6.34
6.72
0.32
B1
2.80
1.99
0.74
0.77
1.09
CB
Samples
2.34
1.71
0.92
2.27
2.12
0.93
0.75
0.61
0.95
0.96
2.69
1.37
1.12
1.47
1.23
0.80
0.98
0.12
4.82
0.67
0.74
0.19
4.13
4.98
2.22
5.54
4.91
3.93
7.57
6.31
6.66
3.14
B2
2.03
4.62
0.80
2.27
2.17
1.97
1.84
2.13
1.82
1.12
2.81
1.14
1.54
0.99
1.41
0.86
1.01
4.42
4.88
0.07
0.52
0.25
4.02
5.26
1.66
4.81
4.25
3.39
7.18
6.26
6.65
3.74
B3
0.81
2.05
0.89
0.96
1.53
1.00
0.64
2.06
3.08
1.39
0.65
1.13
1.43
0.97
1.16
0.75
0.87
0.30
2.24
1.92
0.77
0.38
0.17
2.23
0.00
1.33
1.87
1.14
2.01
0.68
1.86
1.19
1.11
1.31
0.64
0.40
2.14
0.59
0.52
2.61
3.19
1.34
0.43
0.79
1.41
0.68
1.15
0.18
0.24
0.07
1.36
1.95
0.46
0.00
0.93
1.49
1.67
0.21
1.15
0.88
1.51
1.12
1.11
1.01
CE
3.77
1.88
0.63
0.24
2.08
0.86
0.88
2.46
3.21
1.65
2.11
1.22
1.41
1.26
1.01
0.62
0.69
0.34
1.20
2.47
2.89
0.33
1.22
1.20
1.31
1.46
1.59
0.86
0.52
0.59
1.38
1.46
E1
2.26
1.62
0.90
2.27
2.03
0.73
0.85
2.75
3.23
1.55
2.47
1.21
1.57
1.39
1.24
0.80
0.72
1.82
2.56
3.27
4.24
0.36
3.34
2.85
1.93
1.55
3.54
1.31
1.78
1.92
2.11
1.48
E2
0.75
1.75
0.63
2.74
1.99
0.78
0.99
2.45
3.25
1.12
2.57
1.43
1.58
1.13
1.35
0.57
0.95
2.75
2.48
2.76
5.76
0.13
3.86
3.24
1.54
1.57
3.22
0.96
1.83
1.64
2.22
1.21
E3
0.78
1.67
0.53
1.86
1.99
0.28
0.72
2.47
2.19
0.98
1.29
0.64
1.05
1.02
0.98
0.86
0.88
0.58
2.14
1.42
0.45
0.10
0.52
2.57
0.15
1.24
2.99
0.91
2.01
1.81
1.33
1.16
Mk1
1.72
1.83
0.87
3.37
0.63
1.03
0.70
2.30
0.73
1.77
2.55
1.38
1.50
1.33
1.37
0.83
0.93
1.15
0.98
0.10
0.98
0.83
2.03
2.02
0.53
1.24
3.24
0.79
2.10
1.94
2.22
1.40
Mk2
2.22
1.67
0.71
2.12
1.58
0.53
0.55
1.87
0.06
1.53
1.50
1.10
1.21
1.31
1.18
0.57
0.63
2.05
2.09
0.26
1.00
2.40
2.27
2.49
0.59
1.31
1.09
0.46
2.01
1.92
2.08
1.10
Mk3
Results are presented in log base 2. CT: CD4CD8; T1: CD4CD8; T2: CD4CD8; T3: CD4CD8; CB: CD45RCD19; B1-B2: CD45RCD19Sca-1; B3: CD45RlowCD19Sca-1. M: myeloid leukemia (CD11bGr1); CE: control
erythroid cells (Ter119CD71); E1-E3: erythroleukemias (Ter119CD71); Mk1-Mk3: megakaryoblastic leukemias Mk1 (cKitCD41); and Mk2-Mk3 (cKitCD41).
*Ratio of T-CT and B-CB: mean of the deviation of T1, T2, and T3/T control value and mean of the deviation of B1, B2, and B3/B control value.
Amplitude of deviation from the mean calculated from the RMA values.
Cd28
Cd96
1415797_at
1419226_at
Cd160
Cd160
1420066_s_at
1420396_at
Cd2
Cd5
1418770_at
1418353_at
Cd3d
Cd6
1422828_at
Cd8a
Cd3e
1451673_at
1422105_at
1451910_a_at
Cd8a
Cd8a
1425335_at
1444078_at
Cd4
Cd4
1419696_at
1427779_a_at
T-cell phenotype
Ptprc (CD45R)
Ptprc (CD45R)
1422124_a_at
1440165_at
Cd79b
Ms4a2
1417640_at
Cd79a
1418830_at
1421475_at
Bst1
Foxp1
1449454_at
1438802_at
Ebf1
Il7r
1439820_at
1448575_at
Igll1
Enpep
1420176_x_at
1448649_at
Rag2
Vpreb1
1418065_at
Gene
1449869_at
B-cell phenotype
Probe set
Table 1. B and T leukemia immunophenotype

GENE SIGNATURES OF LYMPHOID LEUKEMIAS
1901

1902
CHARFI et al
Several of them had already been associated with B leukemias and

are listed in Table 2.
To identify new potential oncogenes associated with the development of pre-B leukemias, we further focused our analysis on
191 probe sets (supplemental Table 3), mostly because they were
not yet associated with lymphoid leukemias. Some of them
(70 probe sets, including Crisp3 and Lphn2) were already involved in cancer but never associated with B leukemia. Others
(72 probe sets) were never associated with cancer, and 36 probe
sets had no assigned function. Finally, the 13 remaining probe sets
were involved in leukemias other than B leukemias (supplemental
Table 3).
Gene expression profile specific for T leukemias
For all analyzed T leukemias, the expression levels of Cd3, Cd4,

and Cd8 markers, compared with the other tumor samples (B lymphoid, myeloid, erythroid, and megakaryoblastic tumors), were in
accordance with a T lineage immunophenotype (Table 1). Other
markers known to be present at the T-cell surface were also
detected (Table 1). Among these, Cd6 and Cd28 were the most
specific to Graffi-induced T leukemias. We also observed that
Cd160 was expressed in all 3 T leukemias but not in doublepositive control T cells. Moreover, the 2 Cd8 tumors (T1 and T2)
also expressed Cd7 and Dntt (TdT) (Table 1).
We found that 57 probe sets corresponding to 48 genes showed
the same pattern of deregulation in all 3 T leukemias compared
with control T lymphocytes (supplemental Figure 1; supplemental
Table 3) but not in the B leukemias. Most of them (40 genes) were
down-regulated, whereas only 8 were up-regulated.
Nine probe sets were already associated with T leukemias
(Table 2), thus validating our approach. Among the remaining
48 probe sets, 15 were associated with other cancers, 5 were related
to leukemias other than T leukemias, 20 were neither associated
with leukemias nor with other types of cancer, and 8 had no
assigned function (supplemental Table 3).
Expression profiles common to both B and T leukemias
The Robust Multi Array analysis also revealed that 57 probe sets
(corresponding to 48 known genes) were modulated in both T and
B leukemias compared with controls (supplemental Figure 1).
These genes may be associated with common characteristics of
lymphoid leukemias or common oncogenic features and/or directly
related to Graffi leukemogenesis.
Several genes already known to be associated with B and
T leukemias were identified (Table 2). Moreover, among the
57 selected probe sets, 36 had never been simultaneously associated to both types of lymphoid leukemias although some were
already associated with one type (T leukemia: 10; B leukemia:
4; supplemental Table 3). Among the remaining probe sets, 8 were
identified in other cancers, 9 had never been associated with any
types of cancer, and 4 remain uncharacterized. Matr3 was the only
gene already associated with leukemia (acute myeloid leukemia).
RT-PCR validation
To validate our microarray data, the expression levels of 12 genes

specific to either B, T, or to both leukemias (Fmm2, Arntl2, Bfsp2,
Gfra2, Gmp6a, Gmp6b, Bmp7, Fbln1, Nln, Mettl1, Etv5, and
Celsr1 [Table 3; supplemental Table 3]) were measured by
semiquantitative RT-PCR in several Graffi MuLV-induced tumors
(pure cell populations, Figure 1; or unsorted cell suspensions,
supplemental Figure 2). Significant overexpression of Fmm2,

Arntl2, Bfsp2, and Gfra2 was observed in the majority of B leukemias, although being absent in T leukemias confirming their
specificity to B leukemias (Figure 1A; Table 3; supplemental Table
3). Gmp6a and Gmp6b, expected to be B leukemia-specific,
showed significant overexpression in these tumors compared with
the control samples, albeit in only 2 and 3 of the 5 tested
B leukemias, respectively. As expected, these 2 genes were not
expressed in T leukemias or control samples. High expression
levels of Bmp7 were significantly observed in all T leukemias,
whereas Fbln1 and Nln were overexpressed in 3 of 5 T leukemias
(Figure 1B). Mettl1, expected to be T leukemia-specific gene, was
expressed in both T and B leukemias (including controls; Figure
1B). For Etv5, significant higher levels of expression were observed in 4 of 5 B leukemias and 3 of 5 T leukemias (Figure 1C).
Finally, Celsr1 was significantly overexpressed in most T leukemias compared with normal control (CT2) but, in contradiction
with the microarray data, was poorly expressed in all B leukemias
(Figure 1C). Similar validation results were obtained when using
Cd19 splenic cells as normal control instead of Cd45R cells (not
shown).
Fmn2 induces anchorage-independent growth
We further focused our interest on Fmn2, a gene specifically

overexpressed in B leukemias. This gene is a member of the formin
family (FH proteins), and its product is highly conserved among
the multidomain proteins involved in a growing range of actinbased processes.16 Most importantly, they may promote cancer
cells to become invasive and metastatic through their actin
remodeling function.17
We first studied the impact of Fmn2 overexpression on the
anchorage-independent growth of NIH/3T3 cells, a classic assay to
demonstrate the oncogenic potential of proteins. As shown in
Figure 2, the number of larger colonies formed in soft agar was
significantly higher in Fmn2-expressing cells compared with
control cells and similar in cells expressing the human Ras
oncogene (Ras EJ 6.6; Figure 2). These results suggest that the
Fmn2 protein confers anchorage independence to NIH/3T3 cells.
Fmn2 colocalizes with the plasma membrane and disrupts the
actin network
We next determined the subcellular localization of Fmn2. When

GFP-tagged Fmn2 was transiently expressed in NIH/3T3, the
protein was localized at the plasma membrane and within the
cytoplasm (Figure 3A). This was further confirmed by colocalization of Fmn2 with a cell membrane marker (CellMask Plasma
Membrane Stains; Figure 3B).
We also observed that NIH/3T3 cells expressing Fmn2 showed
a reduced size and an abnormal morphology compared with control
cells (Figure 3). Moreover, Fmn2-expressing NIH/3T3 cells appeared rounded with long extensions (Figure 3A-D). Actin cytoskeleton is one of the cellular components that maintain cell shape and
oncogenic transformation causes profound modifications linked
to cell architecture.18-20 Because the overexpression of Fmn2 in
NIH/3T3 cells altered their morphology, we first tested the effect of
its overexpression on actin filaments and on the microtubule
network because of its reported action on actin cables.21 As
illustrated in Figure 3C-D, cells overexpressing Fmn2 showed a
drastic disruption of the actin and microtubule network and a
reduced number of stress fibers compared with control cells.

1903
Table 2. Probe sets already associated with B and T leukemias

Probe set IDs
T1-CT*
T2-CT*
T3-CT*
B1-CT
B2-CT
B3-CT
Gene title
Gene
symbol
Down-regulated B leukemia probe sets

0.21
0.06
0.43
6.57
6.89
6.36
Fc receptor, IgE, low affinity II, polypeptide
Fcer2a
1451713_a_at
0.05
0.09
0.18
6.26
5.85
5.85
Fc receptor, IgE, low affinity II, polypeptide
Fcer2a
1438030_at
0.28
0.01
0.06
5.23
4.94
4.27
RAS, guanyl releasing protein 3
Rasgrp3
1447998_at
0.36
0.14
0.31
4.87
4.53
4.84
Immunoglobulin heavy chain 4 (serum IgG1)
Igh-4
1422122_at
1427292_at
0.31
0.37
0.00
4.47
2.90
3.62
Immunoglobulin chain, variable 1
Igl-V1
1439221_s_at
0.18
0.15
0.00
4.44
4.21
4.85
Tumor necrosis factor receptor superfamily, member 5
Tnfrsf5
1443783_x_at
0.01
0.01
0.16
4.17
4.56
4.67
Histocompatibility 2, class II antigen A,
H2-Aa
1452521_a_at
0.01
0.54
0.20
3.98
3.90
4.23
Urokinase plasminogen activator receptor
Plaur
1449473_s_at
Tnfrsf5
0.53
0.40
0.27
3.85
3.90
4.69
Tumor necrosis factor receptor superfamily, member 5
1427306_at
0.27
0.08
0.23
3.50
3.45
3.65
Ryanodine receptor 1, skeletal muscle
Ryr1
1438031_at
0.41
0.10
0.06
3.49
3.74
3.67
RAS, guanyl releasing protein 3
Rasgrp3
1442263_at
0.15
0.27
0.25
3.48
3.56
3.09
Regulator of G-protein signaling 13
Rgs13
1419609_at
0.32
0.15
0.34
3.45
3.90
3.75
Chemokine (C-C motif) receptor 1
Ccr1
1416055_at
0.04
0.08
0.15
3.43
3.38
3.29
Amylase 2, pancreatic
Amy2
1442544_at
0.01
0.06
0.47
3.31
3.53
3.27
Immunoglobulin heavy chain 4 (serum IgG1)
Igh-4
1451965_at
0.56
0.42
0.44
3.22
3.69
3.67
Immunoglobulin chain complex
Igk
1425063_at
0.07
0.29
0.02
3.15
2.98
2.85
Fc receptor-like 1
Fcrl1
0.50
0.13
0.51
3.15
3.18
3.30
Tnf receptor-associated factor 5
Traf5
1425902_a_at
0.16
0.41
0.48
2.84
2.54
2.90
Nuclear factor of light polypeptide gene enhancer
Nfkb2
1425289_a_at
0.10
0.15
0.58
2.82
3.56
4.96
Complement receptor 2
Cr2
1420710_at
0.05
0.37
0.40
2.70
2.12
2.23
Reticuloendotheliosis oncogene
Rel
1448861_at
in B cells 2, p49/p100
1427576_at
0.52
0.50
0.23
2.60
3.04
3.10
Immunoglobulin chain, constant region
Igk-C
1450912_at
0.16
0.23
0.11
2.49
2.19
3.14
Membrane-spanning 4-domains, subfamily A, member 1
Ms4a1
1423226_at
0.03
0.32
0.48
2.32
2.03
2.79
Membrane-spanning 4-domains, subfamily A, member 1
Ms4a1
1455019_x_at
0.30
0.48
0.10
2.43
2.68
2.52
Cytoskeleton-associated protein 4
Ckap4
1421211_a_at
0.38
0.14
0.41
2.37
3.83
3.85
Class II transactivator
C2ta
1420353_at
0.36
0.57
0.53
2.36
3.66
3.51
Lymphotoxin A
Lta
1422828_at
0.29
0.29
0.43
2.31
2.09
2.21
CD3 antigen, polypeptide
Cd3d
1415899_at
0.54
0.24
0.57
2.15
2.50
3.38
Jun-B oncogene
Junb
1419714_at
0.42
0.06
0.29
2.11
2.27
2.06
Programmed cell death 1 ligand 1
Pdcd1lg1
1447839_x_at
0.56
0.55
0.08
2.08
2.23
2.22
Adrenomedullin
Adm
1425802_a_at
0.26
0.23
0.06
2.07
2.25
4.45
Fc receptor-like A
Fcrla
Smc4l1
Up-regulated B leukemia probe sets

1427276_at
0.20
0.39
0.50
2.03
2.63
2.12
SMC4 structural maintenance of chromosomes 4-like 1 (yeast)
1452499_a_at
0.48
0.09
0.16
2.03
2.17
2.34
Kinesin family member 2A
Kif2a
1436036_at
0.40
0.10
0.08
2.08
2.53
2.79
Wolf-Hirschhorn syndrome candidate 1 (human)
Whsc1
Uck2
1439740_s_at
0.20
0.01
0.14
2.09
2.62
2.16
Uridine-cytidine kinase 2
1448531_at
0.39
0.45
0.36
2.19
2.39
2.27
Lamin B2
Lmnb2
1417299_at
0.24
0.31
0.23
2.38
2.94
2.50
NIMA (never in mitosis gene a)-related expressed kinase 2
Nek2
Vegfa
1420909_at
0.20
0.21
0.01
2.40
3.59
3.57
Vascular endothelial growth factor A
1428853_at
0.37
0.09
0.25
2.50
3.08
3.26
Patched homolog 1
Ptch1
0.18
0.27
0.08
2.54
2.94
2.43
Kinesin family member 22
Kif22
Skp2
1437716_x_at
0.11
0.11
0.05
2.57
3.04
2.53
S-phase kinase-associated protein 2 (p45)
1415849_s_at
0.30
0.53
0.46
2.61
3.02
2.97
Stathmin 1
Stmn1
1449293_a_at
0.01
0.43
0.22
2.72
2.87
2.65
Skp2
Incenp
1418969_at
1423092_at
0.27
0.57
0.49
2.74
3.13
3.10
Inner centromere protein
1444031_at
0.50
0.17
0.02
2.76
3.05
2.34
Calcium/calmodulin-dependent protein kinase II,
Camk2d
1417656_at
0.29
0.06
0.51
2.77
2.90
2.52
Myeloblastosis oncogene-like 2
Mybl2
1417694_at
0.20
0.39
0.19
2.81
2.83
4.08
Growth factor receptor bound protein 2-associated protein 1
Gab1
1439436_x_at
0.01
0.44
0.18
2.83
3.38
3.08
Inner centromere protein
Incenp
1424128_x_at
0.30
0.55
0.48
2.84
3.32
3.16
Aurora kinase B
Aurkb
0.30
0.45
0.18
2.92
2.95
2.14
Early B-cell factor 1
Ebf1
Erg
1446669_at
1440244_at
0.00
0.19
0.05
2.99
4.55
5.28
Avian erythroblastosis virus E-26 (v-ets) oncogene related
1424278_a_at
0.54
0.46
0.47
3.09
3.58
3.10
Baculoviral IAP repeat-containing 5
Birc5
1460247_a_at
0.09
0.12
0.29
3.21
3.47
3.31
Skp2
1437580_s_at
Nek2
0.44
0.56
0.10
3.23
3.47
3.29
NIMA (never in mitosis gene a)-related expressed kinase 2
1458447_at
0.49
0.43
0.52
3.50
4.00
3.67
Centromere protein F
Cenpf
1415810_at
0.19
0.39
0.35
3.50
3.56
3.55
Ubiquitin-like, containing PHD and RING finger domains, 1
Uhrf1
1419943_s_at
0.50
0.52
0.03
3.50
4.23
3.84
Cyclin B1
Ccnb1
Results are presented in log base 2.

T1, T2, and T3 indicate T leukemias; B1, B2, and B3, B leukemias; CT, T control; and CB, B control.
*Ratio T-CT: mean of the deviation of T1, T2, and T3/T control value.
Ratio B-CB: mean of the deviation of B1, B2, and B3/B control value.

1904
CHARFI et al
Table 2. Probe sets already associated with B and T leukemias (continued)

Probe set IDs
T1-CT*
T2-CT*
T3-CT*
1416961_at
0.11
0.12
0.34
B1-CT
3.56
B2-CT
4.14
B3-CT
3.69
Gene
symbol
Gene title
Budding uninhibited by benzimidazoles 1 homolog,
Bub1b
(Saccharomyces cerevisiae)
1424991_s_at
0.03
0.04
0.42
3.64
3.98
4.10
Thymidylate synthase
Tyms
1417938_at
0.03
0.02
0.37
3.75
4.19
3.88
RAD51-associated protein 1
Rad51ap1
1448899_s_at
0.38
0.31
0.24
3.76
4.13
3.84
RAD51-associated protein 1
Rad51ap1
0.32
0.19
0.26
3.78
4.30
4.38
High mobility group box 3
Hmgb3
1416155_at
1415811_at
0.37
0.03
0.11
3.91
3.95
3.94
Ubiquitin-like, containing PHD and RING finger domains, 1
Uhrf1
1418264_at
0.04
0.39
0.35
4.16
4.52
4.55
SoxLZ/Sox6 leucine zipper binding protein in testis
Solt
1417910_at
0.57
0.47
0.32
4.25
4.71
4.28
Cyclin A2
Ccna2
1434748_at
0.27
0.36
0.21
4.30
4.78
4.21
Cytoskeleton-associated protein 2
Ckap2
Ccnb1-rs1///Ccnb1
1448205_at
0.15
0.23
0.15
4.42
4.85
4.49
Cyclin B1, related sequence 1 /// cyclin B1
1416076_at
0.53
0.45
0.19
4.47
5.18
4.81
Cyclin B1
Ccnb1
1452315_at
0.05
0.27
0.57
4.48
4.82
4.58
Kif11
1439820_at
0.08
0.09
0.13
4.48
4.48
3.95
Early B-cell factor 1
Ebf1
1424046_at
0.04
0.02
0.03
4.56
5.27
4.79
Budding uninhibited by benzimidazoles 1 homolog
Bub1
1452314_at
0.14
0.18
0.10
4.58
5.06
4.63
1454694_a_at
0.13
0.08
0.03
4.66
5.14
4.87
Topoisomerase (DNA) II
Top2a
1418507_s_at
0.26
0.01
0.06
4.88
5.92
4.41
RIKEN cDNA D130043N08 gene
D130043N08Rik
1447363_s_at
0.23
0.28
0.10
4.97
5.50
5.14
Budding uninhibited by benzimidazoles 1 homolog,
Bub1b
Kif11
1435306_a_at
0.20
0.32
0.22
5.05
5.50
5.04
1424967_x_at
0.06
0.08
0.27
5.23
3.63
2.48
Troponin T2, cardiac
Tnnt2
1418726_a_at
0.29
0.11
0.20
5.49
4.30
3.17
Troponin T2, cardiac
Tnnt2
1427161_at
0.31
0.43
0.28
5.67
6.30
5.92
Centromere autoantigen F
Cenpf
1426817_at
0.25
0.39
0.41
5.64
6.09
5.88
Antigen identified by monoclonal antibody Ki 67
Mki67
Kif11
1420176_x_at
0.27
0.14
0.04
7.08
7.05
7.00
Immunoglobulin -like polypeptide 1
Igll1
1448649_at
0.02
0.24
0.17
9.58
9.56
9.18
Glutamyl aminopeptidase
Enpep
Down-regulated T leukemia probe sets

1427419_x_at
2.15
8.36
8.79
0.26
0.06
0.26
Ccr9
1421919_a_at
2.68
6.65
6.65
0.03
0.06
0.30
Ccr9
Rorc
1425792_a_at
2.79
3.33
5.75
0.18
0.26
0.30
RAR-related orphan receptor gamma
1423954_at
3.21
2.52
3.89
0.26
0.13
0.24
Complement component 3
C3
1447541_s_at
2.12
2.46
2.49
0.53
0.33
0.30
Integrin, E, epithelial-associated
Itgae
1450295_s_at
2.23
2.13
2.88
0.11
0.36
0.13
Poliovirus receptor /// DNA segment, Chr 7, ERATO Doi
Pvr /// D7Ertd458e
458, expressed
1427411_s_at
2.60
2.07
2.24
0.33
0.31
0.06
Deleted in lymphocytic leukemia, 2
Dleu2
1420413_at
2.30
2.07
2.06
0.42
0.29
0.50
Aolute carrier family 7 (cationic amino acid transporter,
Slc7a11
y system), member 11
Up-regulated T leukemia probe sets
1449835_at
2.55
2.86
5.05
0.30
0.38
0.35
Programmed cell death 1
Pdcd1
Hspa1b /// Hspa1a
Down-regulated B and T leukemia common probe sets

1427127_x_at
6.03
4.87
2.83
5.10
5.83
5.70
Heat shock protein 1B /// heat shock protein 1A
1452318_a_at
5.74
4.92
3.09
4.73
5.26
5.81
Heat shock protein 1B
Hspa1b
1427126_at
5.23
4.56
2.76
4.92
5.53
5.75
Heat shock protein 1B
Hspa1b
1448123_s_at
3.86
3.54
3.37
2.85
2.70
2.36
Transforming growth factor, induced
Tgfbi
1448162_at
3.84
3.92
3.30
4.15
3.86
4.01
Vascular cell adhesion molecule 1
Vcam1
1456250_x_at
3.57
3.18
3.79
2.29
2.18
2.24
Transforming growth factor, induced
Tgfbi
1460423_x_at
3.39
2.89
2.13
6.19
6.45
7.35
Ig chain
IgM
1418126_at
2.89
2.92
2.91
2.02
2.34
2.80
Chemokine (C-C motif) ligand 5
Ccl5
1417756_a_at
2.84
5.14
3.04
2.50
3.17
2.19
Lymphocyte specific 1
Lsp1
1449858_at
2.83
2.82
2.50
4.78
4.68
2.89
CD86 antigen
Cd86
1449164_at
2.76
3.00
3.03
2.83
2.94
2.67
CD68 antigen
Cd68
1418365_at
2.71
3.02
2.34
4.61
4.43
5.66
Cathepsin H
Ctsh
1415989_at
2.46
2.50
2.69
2.07
2.32
2.37
Vascular cell adhesion molecule 1
Vcam1
1434499_a_at
2.44
2.70
6.15
2.72
2.91
2.62
Lactate dehydrogenase 2, B chain
Ldh2
1448237_x_at
2.43
2.82
5.89
2.01
2.07
2.07
Lactate dehydrogenase 2, B chain
Ldh2
1450381_a_at
2.23
3.10
2.91
2.74
3.38
3.19
B-cell leukemia/lymphoma 6
Bcl6
1450381_a_at
2.23
3.10
2.91
2.74
3.38
3.19
B-cell leukemia/lymphoma 6
Bcl6


1905
Table 2. Probe sets already associated with B and T leukemias (continued)

Probe set IDs
T1-CT*
T2-CT*
T3-CT*
B1-CT
B2-CT
B3-CT
Gene title
Gene
symbol
Up-regulated B and T leukemia common probe sets

1422967_a_at
2.03
2.96
2.87
2.32
3.45
2.72
Transferrin receptor
Tfrc
1422198_a_at
2.03
2.21
2.54
2.39
3.05
2.17
Serine hydroxymethyl transferase 1 (soluble)
Shmt1
1425179_at
2.15
2.31
2.71
2.80
3.32
2.52
Serine hydroxymethyl transferase 1 (soluble)
Shmt1
1425923_at
6.04
5.92
5.57
4.66
4.87
4.19
v-myc myelocytomatosis viral-related oncogene,
Mycn
neuroblastoma derived (avian)

FMN2 gene overexpression in human pre-B leukemia
We measured the expression levels of human FMN2 in patients

with different types of B leukemias (Figure 4). No expression was
detected by RT-PCR in control samples, in mantle cell lymphoma,
follicular lymphoma, B-cell prolymphocytic leukemia, and chronic
lymphocytic leukemia. In contrast, FMN2 expression was easily
detected in almost all pre-B-ALL patients (L1 and L2; 18 of 19)
and in one of 2 Burkitt leukemias. Interestingly, the highest levels
of FMN2 were detected in all L1 pediatric pre-B-ALL samples
bearing a t(12;21) translocation (lanes 7, 9, 11, and 12). Another
tumor (lane 3) also showed high levels of FMN2 expression,
suggesting that it could harbor the translocation as well, although
this remained to be confirmed.
Discussion
Graffi MuLV is a good model to gain new insights on leukemia
development and progression and to identify new oncogenes. Gene
expression profiling of each type of leukemia (T lymphoid,
B lymphoid, myeloid, erythroid, and megakaryoblastic) served to
identify the cancer signatures of these specific leukemias.5 In
this paper, we determined the expression profile of 3 B and
3 T leukemias induced by this retrovirus compared with nonleukemic cell controls (CB and CT, Tables 2, 3; supplemental Table 3)
and to nonlymphoid leukemias induced by the same virus (Table 1;
www.biomed.uqam.ca/rassart/microarray2.html). Setting the minimal acceptable change in expression levels to 4-fold, we selected
388 probe sets corresponding to 305 genes: 48 genes specifically
modulated in T leukemias, 218 in B leukemias, and 40 in both types
compared with their respective controls.
Phenotypic properties and cancer signature of B leukemias
Our analysis suggests that B leukemias induced by Graffi MuLV

are arrested at the pre-B stage of differentiation based on the high
expression of pre-B specific markers (Table 1). As in human
pre-B-ALL, these leukemias overexpressed surface markers, such
as Cd79a and Cd79b, and lacked Cd20/Ms4a222 (Table 1). These
results suggest that data retrieved from the gene profiling analysis of B leukemias should be especially relevant for human
pre-B-ALL. Indeed, the results obtained for FMN2 expression in
human leukemias are in total agreement with the mouse data.
We selected 218 genes that were specifically deregulated in all
3 B leukemias; and as presented in Table 2, several of these genes
were already known to be involved in lymphoid leukemia. This not
only validates our microarray analysis but also further defines the
cancer signature of these B lymphoid leukemias.
Phenotypic properties and cancer signature of T leukemia
Three distinct T leukemias (T1, Cd4Cd8; T2, Cd4Cd8; and T3,

Cd4Cd8) were chosen for the microarray experiments. These
3 different phenotypes are frequently observed in Graffi-induced
Table 3. Genes selected for RT-PCR validation

Probe set IDs
T1-CT*
T2-CT*
T3-CT*
B1-CT*
B2-CT*
B3-CT*
Gene title
Gene
symbol
B leukemia probe sets

1450063_at
0.33
0.20
0.19
2.59
3.05
3.01
Formin 2
Fmn2
1429688_at
0.48
0.13
0.38
3.24
2.78
2.75
Aryl hydrocarbon receptor nuclear translocator-like 2
Arntl2
1434463_at
0.45
0.48
0.29
2.98
3.36
3.39
Beaded filament structural protein 2, phakinin
Bfsp2
1423007_a_at
0.41
0.04
0.01
3.12
3.22
2.68
Glial cell line derived neurotrophic factor family receptor
Gfra2
1426442_at
0.06
0.04
0.09
3.15
2.79
2.42
Glycoprotein m6a
Gpm6a
1425942_a_at
0.43
0.54
0.09
6.93
6.35
2.55
Glycoprotein m6b
Gpm6b
T leukemia probe sets

1419122_at
2.33
2.16
2.85
0.41
0.07
0.15
1451555_at
2.32
3.23
2.07
0.06
0.07
0.09
Methyltransferase-like 1
Mettl1
Neurolysin (metallopeptidase M3 family)
Nln
1432410_a_at
3.09
1.56
3.28
0.36
0.15
0.19
Bone morphogenetic protein 7
Bmp7
1451119_a_at
2.75
2.25
1.58
0.12
0.28
0.27
Fibulin 1
Fbln1
B and T leukemia common probe sets

1428142_at
2.68
2.24
3.42
4.04
3.53
3.66
ets variant gene 5
Etv5
1418925_at
2.38
1.50
1.20
2.99
2.99
2.41
Cadherin EGF LAG seven-pass G-type receptor 1
Celsr1
Results are presented in log base 2. A positive deviation of 4 and above and a negative deviation of 4 and below indicates a fold change of 4. Values between 0.585 and
0.585 are considered to be not significant.
*Ratio T-CT and B-CB: mean of the deviation of T1, T2, and T3/T control value and mean of the deviation of B1, B2, and B3/B control value.

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CHARFI et al
Figure 1. Analysis of selected genes differentially expressed in sorted lymphoid leukemia samples. Semiquantitative RT-PCR analysis in 5 T (T4, Cd4Cd8; T5,
Cd4Cd8; T6, Cd4Cd8; T7, Cd4Cd8; T8, Cd4Cd8) and 5 B (B4, Cd45Cd19Sca1; B5, Cd45RCd19Sca1; B6, Cd45RCd19Sca1; B7, Cd45RCd19Sca1;
B8, Cd45RCd19Sca1) leukemias. RT-PCRs were performed in triplicate for each gene. The actin gene was used as internal control in specific conditions described in
Semiquantitative RT-PCR and expression level in each leukemia is presented as a selected gene/actin density ratio. (A) B leukemia-specific genes. (B) T leukemia-specific
genes. (C) Genes common to both leukemias. Statistical analysis was performed using one-way analysis of variance, and P less than .05 was considered to be significant
(*P .05, **P .01, ***P .001) compared with the respective control (B cells from normal spleen [CB2] for the B leukemias and T cells from normal thymus [CT2] for the
T leukemias).
T leukemias and are also representative of normal T-cell types in

healthy mice.1 The retrovirus may have targeted slightly different
T-cell blasts or transformed the targeted cells through these
slightly different lineages. Based on the analysis, all T leukemias
expressed markers specific for the T lineage (Table 1) as well as
T-ALLassociated markers, such as Cd7 and Dntt (TdT).23 However, we also found that all 3 T leukemias expressed high levels of
Cd160. This marker, normally expressed on human NK and only on

a subtype of human CD8 cells,24 seems to be overexpressed in
almost all cases of B-cell chronic lymphocytic leukemia.25
The analysis reveals that a total of 48 genes were strictly
modulated in all T leukemias compared with control T cells (Tables
2-3; supplemental Table 3), including 9 probe sets already associated
with leukemias. The overall data certainly reveals the existence of a

1907
Figure 2. Effect of Fmn2 protein expression on anchorage of NIH/3T3

cells. Cells were transiently transfected with pCMV empty vector, with the
pCMV-Fmn2 vector, and the Ras (EJ 6.6) expression vector. Transfected
or control cells (104) were plated in soft agar as described in Colony
formation in soft agar. After 4 weeks, the number of colonies (at least
twice larger than colonies from controls) was scored (A). The results
represent the average of 3 independent experiments. Statistical analysis
was performed using one-way analysis of variance (P .05). Cells were
observed with an optical microscope (Ernst Leitz, 6MBH Wetzlar), and
representative fields were photographed using a numerical camera
(Nikon coolpix 4500; original magnification 40). Images were analyzed
using NIH ImageJ software Version 1.42l. Cells were left untransfected
(B) or transfected with Ras EJ 6.6 (C), empty vector (D), or pCMV-Fmn2
(E).
common leukemic gene signature between T leukemias induced by

the virus despite heterogeneity and phenotypic differences. We can
speculate that similar deregulation patterns observed in these
leukemias are associated with common characteristics of T leukemias or common oncogenic properties and are probably found in
human T leukemias as well.
Cancer signature common to both B and T leukemias
Because we simultaneously analyzed gene expression profiles of

B and T leukemias induced by Graffi MuLV, regardless of lineage
differences and tumor heterogeneity, we found that 57 others probe
sets modulated in T leukemias were similarly modulated in
B leukemias, including 21 previously reported genes (Table 2).
These results strongly reinforce the potential of the remaining
36 probe sets to be important for the development of B leukemias.
Among these, 23 probe sets had already been associated with either
T or B leukemias, with myeloid leukemia or other cancers
(supplemental Table 3). These genes are certainly part of the
common cancer signature of the T and B lymphoid leukemias and
may be relevant for human lymphoid leukemias.
New potential markers and candidate oncogenes from
B and T leukemias
The combination of our Graffi-induced tumor model and DNA

microarrays allowed us to identify potential new markers of B and
T leukemias that could also play an oncogenic role. Among the
selected 388 probe sets (at least 305 genes), we further identified
275 genes not yet associated with lymphoid leukemias (supplemental Table 3). Some were specific to B or T leukemias or common to
both types, and 124 were obvious oncogene candidates because
they had been already associated with nonlymphoid leukemia or
other cancers. More interestingly, we identified a total of 103 genes
that had never been associated with any type of cancer and 32 probe
sets corresponding to uncharacterized genes or genes with unknown function.
By RT-PCR, we have validated changes in the expression levels
of 10 of the 12 genes selected according to their specificity for
lymphoid leukemias (Figure 1; supplemental Figure 2). Our
analysis revealed deregulation of the expression of several genes
associated with Graffi MuLV-induced B leukemias (Table 1; Figure
1A; supplemental Figure 2). These genes are Fmm2, Arntl2 (from
the bHLH-PAS superfamily involved in regulating cell growth and
differentiation26), Bfsp2 (a member of the intermediate filament
family and component of cytoskeleton proteins in the lens cells,
maintaining their morphology and promoting their motility27),
Gfra2 (from the glial cell line-derived neurotrophic factor receptor- family28 and associated with primary neuroblastomas29 and
with some medullary thyroid carcinoma tumor cells30), and Gmp6a
and Gmp6b (members of the myelin proteolipid protein family and
neuronal homologues of PLP/DM2031).
We also identified several genes that were associated with
T leukemias (Figure 1B), namely, Nln (from the metallopeptidase
M3 family and involved in the metabolism of neurotensin32), Bmp7
(a cytokine from the transforming growth factor- superfamily
and expressed in various types of cancer, including prostate and
breast cancers and melanoma33-35), and Fbln1 (involved in heart
development and in cell signaling involving growth factors36 and
associated with human neoplasia, especially breast and ovarian
cancers37).
Etv5 (a member of the Ets family of transcription factors) was
already described in association with B leukemias38 but, according
to our data, it appears also overexpressed in T leukemias (Figure
1C). In contrast, the modulation of expression of Celsr1 (involved
in the regulation of cell polarity and in convergent extension39 and
expressed in gastrointestinal tumors40), expected to be specific to
both B and T leukemias based on the microarray analysis, was
validated in all T leukemic samples (Figure 1C; supplemental
Figure 2) but in only one B tumor (supplemental Figure 2).
Finally, the high level of expression of Mettl1 (highly expressed
in lung cancer41) observed by the microarray analysis was not
confirmed by RT-PCR (Table 3; supplemental Table 3; Figure 1B)
and, as for Celsr1, may reflect a certain degree of tumor heterogeneity as often observed in human leukemias as well.
We also compared gene expression between B tumors and
normal B cells from the bone marrow sorted with an anti-Cd45R
antibody. This control includes B cells at different stages of
maturation. The majority of the tested genes showed results similar
to those obtained with spleen the B cells as control except for
Fmn2, which was highly expressed in the bone marrow-derived
cells (data not shown). Although this had not yet been reported,
Fmn2 seems to be normally expressed at an early stage of B-cell

1908
CHARFI et al
Figure 3. Subcellular localization of Fmn2 and its

effect on cytoskeleton. The GFP-tagged Fmn2 protein
was localized in NIH/3T3 cells and (A) tested for its
effect shown on the shape of the cells. (B) Plasma
membrane labeling with CellMask. (C) Actin labeling
with AlexaFluor-555conjugated phalloidin. (D) tubulin labeling with anti-tubulin antibody. Images were
captured by a laser-scanning confocal microscope (BioRad MRC-1024 ES) mounted on a Nikon TE-300 using a
60/1.4 NA oil Plan Apo VC objective, digitally acquired
using Laser Sharp software Version 3.2 (Bio-Rad), and
analyzed using NIH ImageJ Version 1.42l software. Data
are representative of 3 independent experiments. The
GFP vector alone was used as a control. Ovals and
arrows indicate transfected and nontransfected cells,
respectively.
lymphopoiesis in the bone marrow. Its expression decreases when

B cells move from the bone marrow to the spleen for maturation.
This inverse correlation between the expression level of Fmn2
and B-cell maturation does not necessarily contradict its involvement in carcinogenesis. This is exemplified with GATA2, which
is essential for the maintenance and the proliferation of hematopoietic progenitors during normal hematopoiesis42 while having been
implicated in tumorigenesis.
Overall, these results strongly suggest that the majority of the
275 selected probe sets, in particular Fmn2, Arntl2, Gpm6a,
Gpm6b, Bfsp2, Gfra2, Nln, Bmp7, Fbln1, and Etv5, are potentially
new specific markers or oncogenes for B, T, or B and T leukemias.
Our analysis also identified several down-regulated genes, such as
Klk6 and TgfI (Table 2), which are already identified as tumors
suppressors. Further studies are required to determine whether the
modulation of expression also correlates with changes at the
protein level and most importantly to determine their potential role
in human leukemias. Regarding Fmn2, our attempts to measure
levels of this protein in mice tumors was hampered by the lack of
specific antibodies.
Fmn2 gene is a good candidate oncogene
Among the 10 genes validated by RT-PCR, we further characterized Fmn2, which was specifically associated with B leukemias.
Fmn2 is expressed in the developing and mature central nervous
system43 and in oocytes.16 It was identified as a formin homology
(FH) gene and the protein contains 2 characteristic FH protein
domains: FH1 (proline-rich region) and FH2. The latter is responsible for actin nucleation.44 The comparison of the mouse and
human Fmn2 showed 74.7% sequence homology. Members of the
formin family are implicated in cytokinesis, organogenesis, and
normal tissue homeostasis but are also involved in the invasive
potential of cancerous cells and metastasis.17 The implication of
Fmn2 in the development of tumors had not yet been demonstrated,
even though human FMN2 ESTs were found in several human
tumors (parathyroid tumor, glioblastoma, retinoblastoma, and
chondrosarcoma).17
In this paper, we report, for the first time, that Fmn2 is not only
specifically overexpressed in B leukemias induced by the Graffi
virus in mice (Figure 1; Table 3; supplemental Table 3) but more
importantly in human pre-B-ALL (Figure 4).

Figure 4. Expression analysis of human FMN2 gene in different types of B

leukemic samples. Semiquantitative RT-PCR analysis was performed on 12 pediatric
pre-B-ALL (lanes 1-12), 7 adult pre-B-ALL (lanes 13-19), 2 Burkitt leukemias (lanes
20 and 21), one mantle cell lymphoma (lane 22), one follicular lymphoma (lane 23),
one B-cell prolymphocytic leukemia (lane 24), and one chronic lymphocytic leukemia
(lane 25). RT-PCRs were performed in triplicate for each gene. The actin gene
was used as an internal control as described in Semiquantitative RT-PCR, and
expression level in each leukemia is presented as a selected gene/actin density ratio.
Statistical analysis was performed using one-way analysis of variance, and P less
than .05 was considered to be significant (*P .05, **P .01, ***P .001) compared with the respective control (CH).
Moreover, we demonstrate that ectopic expression of Fmn2

confers anchorage-independent growth to NIH3T3 cells (Figure 2).
This anchorage-independent growth conferred by Fmn2 is probably related to its ability to induce the disruption of the actin and
microtubule network and a reduction of the number of stress fibers.
The biologic role of Fmn2 in actin and microtubule network
disruption in B leukemia is not quite clear. However, we are
convinced that the up-regulation of FMN2 expression could disturb
the dynamic of the actin network of ALL cells accompanied by the
reorganization of their cytoskeleton, which in turn could contribute
to their abnormal behaviors. Some examples highlight the fact that
even B cells change form depending on their state of development
or their abnormal behaviors. Indeed, it was reported that during
spreading, the B lymphocyte cytoarchitecture is converted from a
semirigid (before migration) to a more flexible state (during
migration). This migration seems to involve up-regulation of CD44
adhesion molecule on activated B lymphocytes and to require the
rearrangement of many cytoskeleton components (actin, microtubules, and vimentin).45 In addition, Caligaris-Cappio et al demonstrated that cells from B-chronic lymphocytic leukemia or hairy
cell leukemia (HCL) showed an aberrant cytoskeleton organization.46 In addition, Schmitt-Graff et al observed changes in the
F-actin in B cells of patients with ALL.47
Implication of FMN2 in human pre-B-ALL
To determine the possible contribution of human FMN2 to

leukemogenesis, we measured its expression in 25 different
B leukemia samples. We showed that this gene was specifically
overexpressed in L1 and L2 pre-B-ALL (18 of 19 of cases; Figure
4), thereby agreeing with our microarray data (Table 1). More
importantly, we show that 4 pediatric pre-B-ALL samples with a
1909
t(12;21) translocation produced the strongest signals. Such t(12;21)

rearrangement involving the TEL/AML1 genes is more frequent in
childhood ALL (25%30%) with a B-precursor phenotype than in
adult ALL (3%5%).48 Although this translocation is associated
with a favorable outcome and a good response to conventional
chemotherapy, 25% of relapses occur off-therapy and require
additional therapeutic strategies. The strong expression of FMN2 in
pediatric pre-B-ALL with the t(12;21) translocation suggests that
the TEL/AML1 fusion protein could directly or indirectly upregulate FMN2 gene expression. Together, these results suggest
that very high expression of FMN2 in pre-B-ALL could be
correlated with a t(12;21) translocation and could be used as marker,
although this has to be confirmed with a larger panel of samples.
In conclusion, we identified a set of genes that are specific
markers for B and T leukemias induced by the Graffi MuLV and
thus may also serve as potential markers in human lymphoid
leukemias. Some of these genes may have oncogenic properties as
revealed with the mouse Fmn2 gene. For the first time, we show
that FMN2 is up-regulated in human pre-B-ALL and more specifically in pediatric pre-B-ALL with the t(12;21) translocation.
Additional investigations are necessary to further characterize its
function in tumor induction.
Acknowledgments
The authors thank Dr Daniel Sinnet for providing pediatric tumor
samples; Andre Ponton, Michal Blazejczyk, and Mathieu Miron
from Genome Quebec Innovation Center (Montreal, QC) for help
with the design and analyses of the microarray experiments; Dr
Benoit Barbeau for critical reading of the manuscript; and Denis
Flipo for help with the confocal microscopy analysis.
This work was supported by Canadian Institutes of Health
Research (grant MOP-37994; E.R.). C.C. is a recipient of a
studentship from the Tunisia Government and Fondation UQAM.
Authorship
Contribution: E.R. and V.V. designed the microarray experiments;
V.V. performed the microarray experiments; C.C. analyzed the
microarray data of the lymphoid leukemias and performed the
experiments; L.-C.L. contributed to some experiments and helpful
discussions; C.C., E.E., and E.R. wrote the manuscript; and E.E
and E.R. supervised the overall project.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Eric Rassart, Departement des Sciences Biologiques, Universite du Quebec a` Montreal, Case Postale 8888
Succursale Centre-ville, Montreal, QC, H3C-3P8, Canada; e-mail:
rassart.eric@uqam.ca; and Elsy Edouard, Departement des Sciences Biologiques, Universite du Quebec a` Montreal, Case Postale
8888 Succursale Centre-ville, Montreal, QC, H3C-3P8, Canada;
e-mail: edouard.elsy@uqam.ca.
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From bloodjournal.hematologylibrary.org by guest on October 5, 2012. For personal use only.

2011 117: 1899-1910

Gene profiling of Graffi murine leukemia virus-induced lymphoid

Updated information and services can be found at:

Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly

From bloodjournal.hematologylibrary.org by guest on October 5, 2012. For personal use only.

Gene profiling of Graffi murine leukemia virus-induced lymphoid leukemias:

The Graffi murine leukemia virus induces

found that human FMN2 is overexpressed

To understand the mechanism of induction of leukemia in humans,

Flow cytometry was performed as previously described.1 Cell populations

Submitted September 30, 2010; accepted November 16, 2010. Prepublished

The online version of this article contains a data supplement.

2011 by The American Society of Hematology

BLOOD, 10 FEBRUARY 2011 VOLUME 117, NUMBER 6

Human sample collection

From bloodjournal.hematologylibrary.org by guest on October 5, 2012. For personal use only.

BLOOD, 10 FEBRUARY 2011 VOLUME 117, NUMBER 6

RNA isolation, microarray hybridization, and

Colony formation in soft agar

We first analyzed the expression profile of genes that could

Table 1. B and T leukemia immunophenotype

BLOOD, 10 FEBRUARY 2011 VOLUME 117, NUMBER 6

From bloodjournal.hematologylibrary.org by guest on October 5, 2012. For personal use only.

From bloodjournal.hematologylibrary.org by guest on October 5, 2012. For personal use only.

Several of them had already been associated with B leukemias and

For all analyzed T leukemias, the expression levels of Cd3, Cd4,

To validate our microarray data, the expression levels of 12 genes

BLOOD, 10 FEBRUARY 2011 VOLUME 117, NUMBER 6

supplemental Figure 2). Significant overexpression of Fmm2,

We further focused our interest on Fmn2, a gene specifically

We next determined the subcellular localization of Fmn2. When

From bloodjournal.hematologylibrary.org by guest on October 5, 2012. For personal use only.

GENE SIGNATURES OF LYMPHOID LEUKEMIAS

Table 2. Probe sets already associated with B and T leukemias

Down-regulated B leukemia probe sets

Fc receptor, IgE, low affinity II, polypeptide

Fc receptor, IgE, low affinity II, polypeptide

RAS, guanyl releasing protein 3

Immunoglobulin heavy chain 4 (serum IgG1)

Immunoglobulin chain, variable 1

Tumor necrosis factor receptor superfamily, member 5

Histocompatibility 2, class II antigen A,

Urokinase plasminogen activator receptor

Tumor necrosis factor receptor superfamily, member 5

Ryanodine receptor 1, skeletal muscle

RAS, guanyl releasing protein 3

Regulator of G-protein signaling 13

Chemokine (C-C motif) receptor 1

Immunoglobulin heavy chain 4 (serum IgG1)

Immunoglobulin chain complex

Tnf receptor-associated factor 5

Nuclear factor of light polypeptide gene enhancer

Immunoglobulin chain, constant region

Membrane-spanning 4-domains, subfamily A, member 1

Membrane-spanning 4-domains, subfamily A, member 1

CD3 antigen, polypeptide

Programmed cell death 1 ligand 1

Up-regulated B leukemia probe sets

SMC4 structural maintenance of chromosomes 4-like 1 (yeast)

Kinesin family member 2A

Wolf-Hirschhorn syndrome candidate 1 (human)

NIMA (never in mitosis gene a)-related expressed kinase 2