[CANCER RESEARCH
45, 3262-3267,
July 1985]
Oncogene Activation in Human Myeloid Leukemia1
Johannes W. G. Janssen,2 Ada C. M. Steenvoorden, John G. Collard, and Roel Nusse
Divisions of Molecular Biology [J. W. G. J., A. C. M. S., P. N.] and Cell Biology [J. G. C.], The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, 121
Plesmanlaan, 1066 CX Amsterdam, The Netherlands
ABSTRACT
We have studied by means of DMA-mediated gene transfer
the activation of protooncogenes in human myeloid leukemias
that represent various stages of myeloid differentiation. DMA
from three cell lines, HL-60 (promyelocytic leukemia), Rc2a (myelomonocytic leukemia), and KG-1 (acute myeloblastic leukemia),
was capable of transforming NIH/3T3 cells. Hybridization analy
sis indicated that, in all three tumor cell lines, the N-ras oncogene
was activated. The cell lines U-937 ("histiocytic lymphoma") and
K-562 (erythroblastic leukemia) yielded no transforming DMA.
Fresh leukemia cells derived from an acute myelomonocytic
leukemia patient and from a juvenile chronic myelogenous leu
kemia patient contained an activated N-ras and c-Ki-ras onco
gene, respectively. DMA from some other myelogenous leukemia
patients was not able to transform NIH/3T3 cells.
Our results indicate that hematopoietic tumors of the myeloid
lineage may contain oncogenes active in NIH/3T3 cell transfor
mation and that, in particular, the N-ras oncogene may be
INTRODUCTION
DMA-mediated gene transfer has allowed the detection and
isolation of transforming genes from a variety of neoplastic cells,
including chemically transformed cells, a large number of human
tumor cell lines, and primary human tumors (reviewed in Refs.
10 and 27). Restriction enzyme analysis and molecular cloning
have shown that most transforming genes belong to one of the
3 members of the ras oncogene family: c-Ha-ras; c-Ki-ras; or Nras (10, 27). Comparison of activated ras oncogenes with their
nontransforming homologous DMA sequences from normal cells
has shown that a single point mutation in the protein-encoding
sequences is responsible for their activation (reviewed in Ref.
27). Nevertheless, some oncogenes detected by transfection do
not belong to the ras gene family or to the group of oncogenes
carried by known transforming retroviruses. These include on
cogenes of chicken lymphomas (11 ), rat neuro- and glioblastomas (35), various mammary carcinomas (28), a chemically trans
formed human cell line (9), human melanoma cell lines (36), and
tumors representing a specific stage of lymphoid differentiation
(29). In the latter study, it has been suggested that the pattern
of oncogene activation in mouse lymphoid cells is specific for the
differentiation state of the leukemia (29). Whether similar mech
anisms of oncogene activation occur in myeloid leukemias is
unknown. This prompted us to investigate oncogene activation
in human hematopoietic tumors of the myeloid lineage repreby the Netherlands Cancer Foundation
(Koningin Wilhelmina Fonds).
2 To whom requests for reprints should be addressed, at The Netherlands
Cancer Institute, Antoni van Leeuwenhoek Huis, Dept. Experimental Cytology (H6), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Received 11/6/84; revised 2/27/85; accepted 3/20/85.
CANCER
MATERIALS
AND
METHODS
Tumor Material from Acute Nonlymphocytic Leukemia Patients.
Leukemia cells were obtained from peripheral blood or bone marrow
specimens and kindly provided by Dr. W. P. van Beek (Division of Cell
Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands)
and Dr. K. J. Roozendaal (Department of Hematology, Onze Lieve
Vrouwe Gasthuis, Amsterdam, The Netherlands). DNAs of the following
patients with their respective diagnoses were investigated: Patient 1,
AMMoL3 (FAB type M4) (2); Patient 2, CML (Philadelphia chromosomepositive, clinically stable CML); Patient 3, juvenile CML (Philadelphia
chromosome-negative, clinically stable CML); Patient 4, acute myeloge
nous leukemia (FAB type M2); and Patient 5, acute monoblastic leukemia
(FAB type M5b).
Cell Lines and Culture Conditions. The human hematopoietic cell
lines HL-60 (8), U-937 (44), and K-562 (30) were kindly provided by Dr.
K. Nilsson (Uppsala, Sweden). The KG-1 cell line (25) was obtained from
activated in tumors representing various stages of maturation.
1This research has been supported
senting specific stages of differentiation.
We have tested DNAs of fresh human donor material and
DNAs of established myeloid tumor cell lines in the NIH/3T3
morphological transformation assay. Some of the DNAs were
able to induce foci, and the transformed cells were further
analyzed to identify the human transforming genes.
Dr. H. Koeffler (UCLA School of Medicine, Los Angeles, CA). The Rc2a
cell line (5) was kindly provided by Dr. P. Tetteroo (Central Laboratory
of the Netherlands, Red Cross blood transfusion service, Amsterdam,
The Netherlands). The different cell lines and their FAB type (2) are
indicated in Table 1. Cells were grown in RPMI 1640:20% fetal calf
serum. The NIH/3T3 fibroblasts were originally obtained from Dr. M.
Wigler (22). The cells utilized in transfection assays (see below) were
derived from a single clone (B25) selected by us on the basis of flat
morphology, low incidence of spontaneous overgrowth, and ability to
transform efficiently with T24 bladder carcinoma DNA (6). Stocks of B25
cells were stored under liquid nitrogen, and cells to be transfected were
either used directly after thawing or passaged once at subconfluent
densities. Foci of morphologically transformed NIH/3T3 cells were iso
lated using cloning cylinders. Transformed cells were selectively grown
by "shaking off" the less attached transformed cells. The cells were
grown in Dulbecco's modified Eagle's medium supplemented with newbom calf serum (5%, v/v) and antibiotics in a humidified CO2 incubator
at 37°C.
Transfection Assays. Leukemic blast cells from bone marrow or
peripheral blood were isolated by standard FicolUsopaque centrifugation.
Trypsinized monolayer cells or cells growing in suspension were pelleted
and washed with phosphate-buffered saline [CaCI2 (100 mg/ml):KCI (200
mg/ml):KH2PO4 (200 mg/ml):MgCI2-2H2O (100 mg/ml):NaCI (8 g/liter):Na2HPO4 (1.15 g/ml), pH 7.2]. To isolate DNA, cells were resuspended in 50 mw Tris-HCI (pH 7.5):100 DIM NaCI:5 rnw EDTA:2%
SDS:proteinase K (200 ^g/ml) and incubated overnight at 37°C. NaCIO4
was added to 1 M, and lysates were subsequently extracted twice with
phenol:chloroform:isoamylalcohol
(25:24:1), precipitated with 0.6 volume
3The abbreviations used are: AMMoL, acute myelomonocytic
leukemia; CML,
chronic myelogenous leukemia; kb, kilobase; SDS, sodium dodecyl sulfate; LTR,
long terminal repeat; SSC, sodium chloride:sodium citrate; FAB, French-AmericanBritish.
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ONCOGENE
ACTIVATION
isopropanol, and washed with cold 70% ethanol. DNA pellets were
resuspended in 5 mw Tris-HCI (pH 7.5):10 mw NaCI:0.5 mw EDTA and
treated with 100 ^g of RNase per ml for 1 h at 37°C, followed by a
proteinase K incubation in 50 mw Tris-HCI (pH 7.5):100 mw NaCI:5 mw
EDTA:0.5% SDS for 1 h at 37°C. The DNA was extracted once with
phenol:chloroform:isoamylalcohol
and once with chloroform:isoamylalcohol, followed by isopropanol precipitation. The precipitate was resus
pended in 10 mM Tris-HCI (pH 7.5):0.1 mw EDTA. The average size of
all DNAs was larger than 50 kb as estimated by electrophoresis in 0.3%
agarose gels. DNA transfections were performed by using small modifi
cations of established procedures (1, 20, 46). Eighty f/g (2 ml) of cellular
DNA in CaCI2 (250 mw) were slowly added to an equal volume of 2x
buffer [50 mw 4-{2-hydroxyethyl)-1 -piperazineethanesulfonic acid: 1.4 mw
sodium phosphate:274 mM NaCI:10 mw KCI:12 mM dextrose (pH 7.02
to 7.05)], which was bubbled through with a stream of sterile air. The
mixture was incubated for 30 min at room temperature to allow the
precipitate to form. Twenty /¿gof DNA precipitate (1 ml) were added to
each 90-mm culture dish in which 150,000 NIH/3T3 cells were seeded
in 10 ml of Dulbecco's modified Eagle's medium supplemented with
newborn calf serum (10%, v/v) 24 h prior to transfection. After an
overnight incubation, the DNA precipitate was removed, and 10 ml of
fresh medium plus 10% newborn calf serum were added. After an
additional 24 h, this medium was replaced by medium containing 5%
newborn calf serum. Medium was changed every 3 to 4 days. Dishes
were examined for foci of morphologically transformed cells 12 to 15
days after transfection.
In one experiment, we used a modified protocol, which should lead to
more efficient transfection and expression of donor DNA (19, 23). Genomic DNA (20 ng) was cotransfected with murine leukemia virus LTR
sequences (23), and during transfection, the cells were treated with 7
mM sodium butyrate for 12 h (19).
Tumorigenicity Assay. This bioassay uses in vivo selection of trans
formed cells as described by Blair et al. (4). In addition, we selected for
cells that have acquired exogenously added DNA by cotransferring the
tumor DNA with a dominant drug-resistant selectable marker pRSVneo
(18), followed by G418 selection. Transfection of 20 ng of tumor DNA
with 300 ng of pRSVneo onto a dish (9 cm) seeded 1 day previously
with 2.5 x 105 NIH/3T3 cells gave approximately 5 x 102 G418 resistant
colonies. Splitting, selection, and injection of the transfected cells were
performed as described by Rasano ef al. (15).
Digestion of DNA by Restriction Endonucleases. Cellular DNAs
were digested with restriction endonucleases (Boehringer Mannheim
Biochemicals) using a 4-fold excess of enzyme as recommended by the
supplier. The extent of digestion of all DNAs was monitored by adding
bacteriophage A to portions of the digestion mixture and subsequent
analysis for the complete digestion.
Detection of Repeated Human DNA Sequences. Digested DNA (10
iig/lane) was subjected to electrophoresis in 0.6% agarose gels and was
transferred to nitrocellulose filters by the method of Southern (41 ). Human
repetitive sequences were detected with nick-translated BLUR-8 plasmid
(39) (specific activity, 5x10"
dpm/Mg), which contains a copy of the alu
family of highly repeated sequences.
Nitrocellulose filters were prehybridized for at least 2 h at 63°Cin 3x
SSC (450 mM sodium chloride:45 mM sodium citrate):5x Denhardt's
solution
[bovine
serum albumin
(1 mg/ml):Ficoll
400 (1 mg/
ml):polyvinylpyrrolidine (1 mg/ml):0.1% SDSidenatured salmon testes
DNA (50 ¿ig/ml):dextran sulfate (10%)] and hybridized for 16 h at 63°C
in hybridization mix with a ^P-labeled probe (5 x 105 dpm/ml). Filters
were washed 5 times in 3x SSC:0.1% SDS at 63°C. Hybridization was
detected by autoradiography for 1 to 7 days at -70°C using Kodak XS1 film and an llford intensifying screen.
Detection of DNA Sequences Homologous to the Oncogenes of
the Human Harvey-, Kirsten-, and Neuroblastoma-transf orming Gene.
A plasmid containing the human Harvey-transforming gene of the T24
bladder carcinoma, pEJ, was kindly provided by Dr. M. Wigler (17). A
plasmid, p640, containing part of an intron of the human c-Ki-ras-
CANCER
IN HUMAN LEUKEMIA
transforming sequence was obtained from Dr. R. Weinberg (31). An Nras subclone (0.9-kb Pvull fragment designated Probe B) lacking repeti
tive sequences was obtained from Dr. A. Hall (21). Plasmid DNAs were
labeled with [^PJdCTP by nick translation. Hybridizations were per
formed as described above except for the last washings. N-ras filters
were finally washed in 1x SSC:0.1% SDS, c-Ki-ras filters were washed
in 1x SSC:0.1 % SDS, and c-Ha-ras filters were washed in 3x SSC:0.1 %
SDS at 63°C. The filters shown in Fig. 5 were hybridized with N-ras
DNA, purified from the plasmid by low-melting agarose (45).
RESULTS
Transforming Capacity of DNA from Human Myeloid Leu
kemia Cells. High-molecular-weight DNA was isolated from
established human myeloid hematopoietic tumor cell lines and
primary tumor tissues of myeloid origin and transfected onto
NIH/3T3 cells. The results of the transfection experiments are
summarized in Table 1. DNAs of 3 of 5 established myeloid
leukemia cell lines and DNAs of 2 of 5 myeloid leukemia patients
were positive in the transformation assay. In addition to the
previously described transforming activity of DNA isolated from
the HL-60 promyelocytic leukemia cell line (32), we observed
transformation with DNA derived from the Rc2a cell line (AMMoL
cell line), with DNA derived from the KG-1 cell line (acute myeloblastic leukemia cell line), with DNA from a primary AMMoL
(Patient 1), and with DNA from a primary juvenile CML (Patient
3).
Transfection experiments performed with DNA of the KG-1
cell line resulted in only a single focus (0.0125 foci/Mg; Table 1)
in the NIH/3T3 focus-forming assay using an alternative trans
fection protocol (19, 23). This was confirmed by means of the
tumorigenicity assay as described by Rasano ef al. (15). This
bioassay is based on the tumorigenicity of cotransfected NIH/
3T3 cells in nude mice. Tumor formation of KG-1 DNA-cotransfected NIH/3T3 cells was observed 2 months after injection.
Control NIH/3T3 cells cotransfected with normal mouse DNA did
not develop a tumor in 6 months.
Foci were isolated from different plates, and transformed cells
were grown up into cell lines. The cell lines retained a distinct
transformed morphology. In order to demonstrate that the trans
forming activity was due to acquisition of human sequences,
DNA from all foci obtained in primary transfection was analyzed
by filter hybridization with the human a/u-repeat probe, BLUR-8
(39). All primary transfectants showed numerous bands of human
repetitive sequences. High-molecular-weight DNA prepared from
different primary transfectants was used for a second cycle of
transformation. Analysis of secondary transfectants by blot hy
bridization to the human alu probe showed only a few fragments
harboring an a/u-repeat sequence. We conclude that only the
a/u-containing fragments closely linked to the transforming gene
itself are transferred. Fig. 1 shows a representative autoradi
ogram of digested DNAs from primary and secondary transfec
tants of the AMMoL patient (Patient 1) and the acute promyelo
cytic leukemia cell line (HL-60) after blot hybridization to the
human a/t/-repeat probe, BLUR-8. The lane containing a primary
AMMoL transfectant (P AMMoL-5) showed a smear of hybridi
zation, whereas different secondary AMMoL transfectants
(AMMoL 5/1 to AMMoL 5/7), secondary HL-60 (S HL-60/1), and
tertiary HL-60 transfectants (T HL-60/1.1 to T HL-60/1.3) con
tained only a few a/u-hybridizing fragments. The a/u-repeat band
ing pattern of secondary transfectants of the AMMoL patient
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ONCOGENE
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IN HUMAN LEUKEMIA
Table 1
Transformingefficiencies of cellular ONAs
activated^Ha-rasN-rasN-rasN
classM3M6M4M4Stage
cellsPromyelocyticMyeloblasticMyelomonocyticMonocytoidEarly
of differentiation of
fromBladder
Cell patientT24HL-60KG-1Rc2aU-937K-562Patient
line +
carcinomaAcute
leukemiaErythroleukemiaAMMoL"Histiocytic
promyelocytic
lymphomaPhiladelphia
chromosome-positiveCML
crisesAMMoLPhiladelphia
in blast
erythroblasticMyelomonoblasticAll
blastic and/or
1Patient
2Derived
chromosome-positiveFAB
stages of differentiationFoci/xg80.50.25+c0.15——0.12—Oncogene
CML in chronic phase
Patient 3
Philadelphiachromosome-negative
All stages of differentiation
0.075
Kkas
CML in chronic phase
Myeloblastic
M2
Patient 4
Acute myeioid leukemia
Monoblastic and atypical monocytic
M5b
Patient 5
Acute monocytic leukemia
8 High-molecular-weightDNA (20 ng) was utilized to transfert 1.5 x 108NIH/3T3 cells, seeded 24 h before transfection as described in "Materials and Methods."
Focus formation was scored at 12 to 18 days. Eight recipient cultures were transfected. Minus sign means that no foci were detected in 8 plates, meaning that the
transforming
is below
6 Differentefficiency
transfectants
were0.006
testedfoci/^g.
for the presence of human activated ras oncogenes as described in "Materials and Methods" and "Results."
c DNA was negative in the standard NIH/3T3 "focus forming" assay, but positive with a modified transfection protocol (0.0125 foci/pig)and in the tumorigenicity assay.
d Originally classified as "histiocytic lymphoma" (44), but generally consideredto be of monocytoid origin (24).
fc
s
u
ï
z
ifÃ-
(/)
s
u
kb.
t/l
kb.
— 23J
if
.23.7
ft
H
-9.5
-6.7
—6.7
.4.3
—2.0
.22
-2jO
Fig. 1. Detection of human DNA sequences in NIH/3T3 primary (P), secondary
(S), and tertiary (T) transfectants derived from the AMMoL patient and the HL-60
cell line. DNA (10 fig/lane) was digested with EcoRI. subjected to electrophoresis
in 0.6% agarose gels, and transferred to nitrocellulosefilters. The filter was probed
with the BLUR-8 plasmid. The positions of migration and sizes of the X H/ndllldigested DNA markers are indicated.
and the HL-60 cell line was quite similar, indicating activation of
the same oncogene, N-ras (33) in both DMAs. An identical
analysis of secondary transfectants from the AMMoL cell line,
Rc2a, showed that in this DNA N-ras activation also had occurred
(see below).
Secondary transfectants of DNA from the CML patient (Patient
3) show a completely different a/u-banding pattern. Many more
bands are detected which did not comigrate with those of HL60, AMMoL, or Rc2a secondary transfectants.
Hybridization Analysis of the Transforming Oncogenes. In
order to determine whether the transforming genes of these
CANCER
RESEARCH
12345
6
7
8
9
1011
12
Fig. 2. Detection of c-Ha-ras sequences in NIH/3T3 primary transfectants. A
Southern blot of BamHI-digested DNAs was probed using plasmid pEJ, that
contained the complete human c-Ha-ras gene. DNAs were from NIH/3T3 cells
(Lane 7),human control cells; Rc2a, BM (bone marrow from a healthy person), and
T24 (Lanes 2 to 4); and different primary (P) transfectants (Lanes 5 to 12). The
positions of migration and sizes of H/ndlli-digested X DNA markers are indicated.
myeioid leukemias are related to previously described transform
ing genes (27), we hybridized the Southern blots with DNA from
different primary and secondary transfectants of the myeioid
leukemias to probes for the 3 ras oncogenes (c-Ha-fas, c-Ki-ras,
and N-ras). Fig. 2 shows SamHI-digested DNA of different trans
fectants hybridized to the c-Ha-ras oncogene. A faint endoge
nous mouse c-Ha-ras fragment with a molecular weight of 3.5
kb is observed in Lanes 1 and 5 to 12. The human control DNAs
(Lanes 2 to 4) show a strong hybridization signal at 6.6 kb. Only
one transfectant contained a hybridizing fragment corresponding
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ONCOGENE
ACTIVATION
IN HUMAN LEUKEMIA
B
to the human c-Ha-ras gene: a primary transfectant of T24 DNA,
P T24/1 (Lane 12) which was included as a control. Fig. 3 shows
EcoRI digests of the same transfectants as presented in Fig. 2
that were hybridized to a human c-Ki-ras oncogene probe (31).
kb.
—23.7
Under the conditions used by us, this probe did not hybridize to
mouse sequences. Lanes 2 to 4 containing the human control
DNAs show hybridization to a 2.8-kb fragment. An identical
kb.
— 23.7
—9.5
— 9.5
—6.7
— 6.7
—4.3
— 4.3
kb.
•22
-2.0
—23.7
—9.5
—6.7
—22
—4.3
123456
1
2
3
Fig. 5. Detection of N-ras sequences in secondary transfectants derived from
KG-1 DNA (A) and in the primary tumor derived from KG-1 DNA (B). A Southern
blot of EcoRI-digested DNAs was probed using a human N-ras exon probe. A,
DNAs from the KG-1 primary transfectant KG-1-3 (Lane 1); control NIH/3T3 cells
(Lane 2); and secondary transfectants derived from KG-1-3 DNA, KG-1-3F, KG-1
3E, KG-1-3C, and KG-1-3B (Lanes 3 to 6). respectively. B, DNAs from the primary
tumor derived from KG-1 DNA, KG-1 control cells, and NIH/3T3 control cells (Lanes
i to 3), respectively. The position of migration and sizes of the H/'nolll-digested X
—2.2
—2.0
DNA markers are indicated.
1 2
3
4
5
6
7
8
9 10 11 12
Fig. 3. Detection of c-Ki-ras sequences in NIH/3T3 primary transfectants. A
Southern blot of EcoRI-digested DNAs was probed using plasmid p640 that
contained a portion of an intron of the human c-Ki-ras gene. DNAs were identical
as in Fig. 2 and are indicated at the (op. Positions of migration and sizes of H/ndllldigested \ DNA markers are indicated.
ss
m
a s i j
ää3 5
H
...
o.
o.
o.
o.
S
<
5
<
CL
O.
O.
kb.
j —23.7
— 9.5
— 6.7
(A) and an analysis of DNA of the tumor obtained after injection
of KG-1-cotransfected NIH/3T3 cells in nude mice (B). Fig. 5A
shows an endogenous mouse N-ras fragment of approximately
—4.3
2.2
2.0
1234
5678
fragment is observed in the 2 lanes containing DNA from 2
different primary transfectants of the CIVIL patient. Analysis of 3
other primary transfectants of this CML patient showed similar
results (not shown), confirming that the c-Ki-ras oncogene has
been activated and was transferred to the 3T3 cells. Fig. 4
shows a filter with DNA of the same transfectants hybridized to
an N-ras probe (21). Lanes 1 and 5 to 12 show a faint endoge
nous mouse N-ras fragment of approximately 7.5 kb. A human
N-ras hybridizing fragment is detected in primary transfectants
of DNA from the HL-60 cell line, the Rc2a cell line, and leukemic
cells of the AMMoL patient. Some size heterogeneity was ob
served, probably due to loss and gain of restriction enzyme sites
occurring during transfection, a phenomenon also described by
others. Additional independent Rc2a and AMMoL secondary
transfectants had also acquired human N-ras sequences (data
not shown).
Fig. 5 shows a hybridization analysis of DNA from different
secondary transfectants of the only KG-1-induced primary focus
9 10 11 12
Fig. 4. Detection of N-ras sequences in NIH/3T3 primary transfectants. A Southem blot of EcoRI-digested DNAs was probed using a human N-ras exon probe.
DNAs were identical as in Fig. 2 and are indicated at the (op. Positions of migration
and sizes of H/'ndlll-digested X DNA markers are indicated.
CANCER RESEARCH
7.5 kb in all 6 lanes. All lanes except Lane 2, which represents
a mouse control DNA lane, show an additional human N-ras
fragment. The results of the analysis of the KG-1 DNA-induced
tumor DNA (B) are comparable to that in A. An additional human
N-ras hybridizing fragment is observed in Lane 1, representing
the KG-1-induced tumor. Fig. 5>4,as well as Fig. 5fl, shows again
heterogeneity in size and amplification of the human N-ras gene
in the different secondary transfectants and tumor DNA.
Murray ef a/. (33) have already described the activation and
isolation of the human N-ras oncogene from the HL-60 cell line.
In addition, we observed an activation of the same oncogene in
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ONCOGENE
ACTIVATION
the Rc2a cell line, the KG-1 Å“il line, and in tumor cells of an
AMMoL patient.
DISCUSSION
DMAs from a number of human tumors and tumor cell lines
have been found to induce malignant transformation of NIH/3T3
cells (10, 27), including tumors of hematopoietic cells. Most of
these last-mentioned studies concerned tumors of the lymphoid
lineage. Lane ef al. (29) have reported that specific transforming
genes distinct from the ras oncogenes had been activated in
mouse neoplasms representing specific stages of B- or T-cell
differentiation. Ozanne ef al. (34) have shown transforming activ
ity in the human leukemia cell line SMS-SB which was derived
from a patient with an unusual pre-B-acute lymphoblastic leu
kemia. Several other groups have reported activation of the Nras oncogene in human leukemia cell lines and tumors of T-cell
origin (14, 42); an activation of the c-Ki-ras oncogene in a human
T-cell leukemia cell line has also been reported (14).
Information concerning oncogene activation in tumors of the
myeloid lineage is limited. This prompted us to investigate cell
lines and fresh cell samples of patients with hematopoietic tu
mors of the myeloid lineage arrested at specific stages of mat
uration. We have found activation of the N-ras oncogene in DNA
from an AMMoL cell line (Rc2a), in DNA of an acute myeloblastic
leukemia cell line (KG-1), and in DNA of a patient with AMMoL
(FAB type M4). We have also confirmed the activation of the Nras oncogene in the promyelocytic leukemia cell line, HL-60 (33).
DNA from a juvenile CML patient contained an activated c-Ki-ras
oncogene.
In the case of the KG-1 cell line, we have detected only one
focus in an experiment in which we were testing the enhance
ment of DNA transfection efficiency by the addition of LTR
sequences to the precipitate (23) and treatment of the cells with
sodium butyrate (19). It may therefore be argued that linkage of
an LTR sequence to an unmutated N-ras gene has led to a high
transcription rate of the gene and thus to transformation of the
3T3 cells (3, 7). Since no exogenous LTR sequences were
detected in the different secondary transfectants derived from
DNA of this single focus (data not shown), this explanation is not
likely to be valid.
Other reports concerning oncogene activation in hemato
poietic human tumors of the myeloid lineage have demonstrated
N-ras activation in DNA of a patient with acute myeloblastic
leukemia (16) but also N-ras activation in a CML cell line (IM9)
(14).
Our results, combined with the findings of others (mentioned
above), indicate that the transforming oncogenes present in
various myeloid leukemias are not closely linked to specific
stages of differentiation. This apparent lack of specificity does
not preclude the possibility that activation of additional onco
genes in the same leukemia cells might correlate with the differ
entiation state.
Carcinogenesis is considered to be a multistep process. Al
though Spandidos and Wilkie (43) have shown that high tran
scription rates of a mutated c-Ha-ras gene can transform early
passage cells, other reports have shown that a single oncogene
is not capable of transforming primary cells, while certain com
binations of oncogenes do (13, 26, 37,40). On the basis of these
experiments, oncogenes may be divided in 2 groups: Class I
CANCER
IN HUMAN LEUKEMIA
oncogenes (myc-like oncogenes, including myc, E1A, p53, and
polyoma large-T) and Class II oncogenes (ras-like oncogenes,
including N-ras, H-ras, K-ras, and polyoma middle-T). Indeed,
certain tumors show the involvement of multiple oncogenes (27),
e.g., c-myc amplification and N-ras activation in HL-60 cells (33)
and activation of c-myc and B/ym-1 in various Burkitt lymphomas
(12).
Recent studies have shown that nonrandom chromosomal
aberrations can be demonstrated in most or all patients with
myeloid leukemia (38). These include t(8;21 ) in acute myeloblastic
M2 leukemia, t(15;17) in acute promyelocytic M3 leukemia,
inv(16) in AMMoL M4 leukemia, and 11q aberrations in acute
monocytic M5 leukemia. It has been suggested that the chro
mosomal break points involved in these translocations are situ
ated in the proximity of cellular oncogenes that have not yet
been identified. It is conceivable that these genes might play
some role in the arrest of differentiation. Cloning the specific
chromosome translocation junction fragments may answer this
question.
ACKNOWLEDGMENTS
We thank Dr. A. Hall for the generous gift of the Nvas clone.
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Oncogene Activation in Human Myeloid Leukemia
Johannes W. G. Janssen, Ada C. M. Steenvoorden, John G. Collard, et al.
Cancer Res 1985;45:3262-3267.
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