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Characterization of a Factor-Dependent Acute Leukemia Cell Line With
Translocation (3;3)(q21;q26)
By John Oval, O.W. Jones, Mark Montoya, and Raymond Taetle
A strictly factor-dependent cell line (UCSD/AMLI ) was
established from a patient with the syndrome of multilineage acute leukemia with high platelets. The patient's cells
and the cell line karyotype were 45,XX. - 7,t(3;3)(q21 :q26),
typical of the syndrome of acute leukemia with high
platelets. The cell line expresses CD34, CD7, TdT, and
myeloid (CDI 3, CD14, CD33) and megakaryocytelplatelet
(CD36, CD41, CD42b. CDw49b) antigens. In short-term
culture, UCSD/AMLI cells proliferate in response to interleukin-3 (IL-3). IL-4, IL-6, macrophage colony-stimulating
factor (M-CSF), and granulocyte-macrophage CSF (GMCSF), but not IL-I, IL-2, IL-5, or G-CSF. In long-term culture,
proliferation can be sustained by GM-CSF. IL-6, or M-CSF.
When maintained in GM-CSF, a small percentage of cells
form multinucleated megakaryocyte-like giant cells. Culture with GM-CSF combined with IL-6, but not with IL-6
alone, increased giant cell formation fourfold to sevenfold.
IL-6 alone or in combination with GM-CSF increased
expression of platelet-related antigens. In contrast, culture
with phorbol ester induced formation of macrophage-like
cells. UCSD/AMLI is the first human acute nonlymphocytic
leukemia cell line established from a patient with an acute
leukemia syndrome associated with a specific chromosome
abnormality.
0 1990 by The American Society of Hematology.
C
multiple separate nuclei. Blasts constituted 58% of the nonerythroid
elements and ranged in size from small cells with a high nuclear/
cytoplasmic ratio, and indistinct nucleoli to cells of medium size with
a moderate nuclear/cytoplasmic ratio and 1 to 3 nucleoli. There
were no granules or Auer rods. Occasional dysplastic erythrocyte
precursors showed nuclear/cytoplasmic dyssynchrony and multiple
nuclei. A karyotype performed at an outside laboratory was
45,XX,-7,t(3;3)(q21;q26). Surface marker analysis, also performed at an outside laboratory,showed a mixture of cells expressing
T-cell and granulocyte/monocyterelated antigens (Diagnosis,Table
1). A stain for nuclear TdT was negative.
The patient developed progressive anemia over the next month,
but her platelet count remained elevated. She was treated with
daunorubicin, cytosine arabinoside, and high-dose cytosine arabinoside, but achieved only a brief remission. A repeat bone marrow was
performed at relapse and showed greater than 70% small blasts with
a T-cell phenotype (Relapse, Table 1). A karyotype performed at
University of California, San Diego was again 45,XX,-7,t(3;3)(q21;
q26) but, in addition, 4 of 12 metaphases analyzed demonstrated
45,XX,- 7,t(3;3)(q21;q26), t( 12;22)(p13;ql2).She started on adriamycin/vincristine/prednisone therapy, but developed refractory
leukemic meningitisand died.
ERTAIN SUBTYPES of human acute leukemias are
characterized by specific clinical features and karyotype abnormalities,'.* suggesting that genetic lesions determine the pathogenesis or phenotypic manifestations of these
syndromes. One such syndrome is characterized by acute
nonlymphocytic leukemia (ANLL) presenting with high or
normal platelet counts and chromosome deletions or translocations involving 3q2 1 and/or 3q26.'-7 Clinical findings in
these patients are consistent with increased and abnormal
megakaryo~ytopoiesis.~-~
Because genes involved in Fe metabolism (ie, transferrin and its receptor) are located in this area
of chromosome 3, and Fe deficiency is characterized by
increased platelet production, it has been suggested that
these genes may somehow determine the clinical features of
this syndrome.'.'
In the present studies, we describe a new, strictly factordependent cell line (UCSD/AMLl) established from a
patient with the syndrome of acute leukemia with high
platelets and t(3;3)(q21;q26). These cells express megakaryocytic/platelet antigens and undergo primary proliferative
responses and abnormal megakaryocytic differentiation in
response to interleukin-6 (IL-6). Because IL-6 was recently
shown to be a n in vitro megakaryocyte maturation factor and
an in vivo thrombopoietin?-" our findings suggest the A N L L
syndrome associated with t(3;3)(q21;q26) involves cells with
the ability to express megakaryocytic differentiation programs, and suggest IL-6 may play a role in the phenotypic
manifestations of this disease. UCSD/AMLl cells also
express antigens associated with multilineage hematopoietic
progenitor cells, and display some characteristics of a second
acute leukemia syndrome, CD7 positive acute leukemia with
multilineage d i f f e r e n t a t i ~ n . ' ~ . ' ~
CASE HISTORY
The patient was a 73-year-old woman who presented to an outside
hospital with progressive fatigue. She had no known exposure to
cytotoxic drugs or radiation, and no prior history of a blood cell
disorder. Her plasma Hb was 9.5 g/dL, platelet count 450,000/mm3,
and white blood cell (WBC) count 6,800/mm3. The leukocyte
differential showed 26% PMN, 9% bands, 9% metamyelocytes, 11%
myelocytes, 28% lymphocytes, and 26 NRBCs/100 WBC. A bone
marrow performed at an outlying hospital was hypercellular and
showed normal to slightly increased megakaryocytes. Many of the
megakaryocytes were small in size with a single large nucleus, or had
Blood, Vol 76, No 7 (October 1), 1990: pp 1369-1374
MATERIALS AND METHODS
Cell culture. Bone marrow cells were obtained at relapse from
induction therapy for ANLL. The cells were separated on Ficoll/
Hypaque and cultured at 5 x 105/mL in RPMI 1640 medium with
10% fetal bovine serum (FBS; Hyclone, Logan, UT) and 30 U/mL
recombinant granulocyte-macrophage colony-stimulating factor
From the Divisions of Hematology/Oncology, Genetics, and
Laboratory Medicine, Departments of Medicine and Pathology,
University of California, San Diego: and Cancer Biology Program,
University of CaliforniaSan Diego Cancer Center.
Submitted January 30,1990; accepted June 4,1990.
Supported by National Institutes of HealthlNational Cancer
Institute Grants CA32094 and CA23100.
Address reprint requests to Raymond Taetle, MD. University of
California Medical Center (H720T). 225 W Dickinson St, San
Diego. CA 921 03.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7607-0018$3.00/0
1369
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1370
OVAL ET AL
Table 1. Surface Marker Analysis of Leukemic Cells
Diaanosis
RelaDse
92
CD5
65.
34
52
CD7
CD8
UCSD/AMLl
T-cell -associated
CD2
CD4
2
1
1
NT
99
25
2
44
25
0
0
30
31
3
0
0
66
NT
NT
48
Myeloid
CD13
CD14
CD33
39
20
58
Primitive
CD34
Platelet-associated
NT
NT
21
16
2
NT
NT
NT
NT
55
22
1
7
6
0
0
TdT
0
91
100
Glycophorin
4
1
1
CD36
CD4 1
CD42b
CDW49b
17
B cell
CD19
CD20
5
Other
Abbreviation: NT, not tested.
*Percent of cells positive by immunofluorescence.
(GM-CSF; kindly provided by Dr Stephen Gillis, Immunex, Inc,
Seattle, WA). At weekly intervals or when viable cell counts reached
1 x 106/mL, half the medium and cells were removed and the
culture refed with fresh medium containing GM-CSF. After approximately 10 weeks, consistent proliferation of cells was obtained with
a doubling time of approximately 72 hours. The cells were expanded
in GM-CSF and characterized after approximately 14 weeks of
culture. The resulting cell line (UCSD/AMLl) was maintained in
RPMI 1640 with 10% FBS and 30 U/mL GM-CSF.
Characterization of UCSDIAMLI cells. Surface antigens on
Ficoll/Hypaque separated bone marrow or the UCSD/AMLl cell
line were detected by immunofluorescencestaining using monoclonal
antibodies (MoAbs) to the CD2, CD3, CD4, CD5, CD7, CD13,
CD14, CD33, CD34, CD45 antigens (nomenclature is per reference
14), and erythrocyte glycophorins as previously described." The
platelet-associated CD41 (glycoprotein [GP] IIb/IIIa) antigen was
detected using antiplatelet MoAb (DAKO Diagnostics, Santa Barbara, CA) or antibody 4F10 (reference 16; kindly provided by Dr
Virgil Woods, University of California, San Diego). MoAbs to
VLA-2 (12F1; reference 17) (platelet GPIa; CDw49b); platelet
GPIb (CD42b) (P3; reference 18); and to platelet GPIV (CD36)
(5F1; reference 19) were also provided by Dr Woods. Histochemical
stains were performed using standard techniques as described.20
Nuclear TdT was detected by immunofluorescence staining using a
specific heteroantiserum (Supertechs, Bethesda, MD). Southern
blots were also performed using standard techniques,2' and immunoglobulin (Ig) and T-cell j3 receptor gene rearrangements assessed
using probes to the Ig J, and T-cell j3 receptor constant regions
(Oncor, Gaithersburg, MD). Placental DNA (Oncor) served as a
germline control.
Giemsa-banded karyotypes were obtained as previously described?2
Chromosomes from 12 separate metaphase cells from the relapsed
marrow and 19 cells from the cell line were analyzed in detail.
Growth factors. Recombinant erythropoietin and G-CSF were
purchased from Amgen, Inc (Thousand Oaks, CA). Recombinant
GM-CSF, IL-3, and IL-4 were kindly provided by Dr Stephen Gillis.
Recombinant M-CSF (CSF-l), IL-5, and IL-6 were kindly provided
by Dr Steven Clark (Genetics Institute, Cambridge, MA). Ultrapure IL-1 was purchased from Genzyme Corp (Boston, MA) and
recombinant IL-2 from Cellular Products (Buffalo, NY). Erythropoietin, CSFs, and ILs were studied over a concentration range from 0.1
to 30 or 100 U/mL. COS cell supernatant containing recombinant
IL-5 was also provided by Dr Clark, and studied at dilutions from
1:lO to 1:500.
Growth factor responses. To determine responses to growth
factors, cells were washed three times and plated into triplicate
microtiter wells at 4 x 10S/mL in RPMI 1640 with 10% FBS and
various concentrations of recombinant growth factors. At the fourth
day of culture, the cells were pulsed with 'H-TdR (Amersham, Inc,
Arlington Heights, IL) for 14 hours and harvested as previously
described.23
To screen for synergy between growth factors, cells were washed
and plated with the highest concentration of growth factors that
failed to stimulate cell growth used alone, and complete GM-CSF,
IL-3, or IL-6 dose-response curves ranging from 0.1 to 100 U/mL
obtained. The following combinations were assessed: IL-3 doseresponse with 1 or 10 U/mL IL-4; IL-3 with 100 U/mL G-CSF;
GM-CSF dose-response with 1 or 10 U/mL IL-4; IL-6 doseresponse with 1 or 10 U/mL IL-4. Cells were pulsed with 'H-TdR
and harvested as described above.
To assess the abilities of growth factors to maintain continuous
UCSD/AMLl cell growth, cells were washed three times and grown
with FBS and 30 U/mL GM-CSF, 30 U/mL IL-3,30 U/mL IL-6,
or 10 U/mL M-CSF. Viable cell counts were performed every 2 to 3
days for 3 weeks. To determine whether UCSD/AMLl cells
survived in the absence of growth factors, cells were washed and
plated at 4 x 10S/mLin RPMI 1640 with 10% FBS and observed for
2 weeks. At the 7th or 14th day of culture with media and FBS, an
aliquot of cells was washed and resuspended in RPMI/FBS with 30
U/mL GM-CSF. These subcultures were then maintained for 2
weeks and viable cell counts determined every 2 to 3 days.
Induction of cellular differentiation. To assess macrophage or
megakaryocytic differentiation, UCSD/AMLl cells were grown
with GM-CSF, GM-CSF with 30 U/mL IL-6,30 U/mL IL-6 alone,
lo-' mol/L tetradeconylphorbol-acetate (TPA; Sigma Chemicals,
St Louis, MO), or TPA with 30 U/mL GM-CSF for 5 to 7 days.
Morphology was assessed by an observer blinded to the incubation
conditions using Wright-Giemsa-stained cytospins. Expression of
the CD13, CD14, CD36, CD41, CD42b, and CD49b antigens was
assessed after 5 days.
RESULTS
After establishing the cell line in culture, the dominant
cells were large blastlike cells with minimal granularity (Fig
1A). However, rare giant cells with multiple, distinct nuclei
were also present (Fig 1C). Histochemical stains were
strongly positive for acid phosphatase and nonspecific esterase, but negative for chloroacetate esterase. Surface
marker analysis (Table 1) showed that UCSD/AMLl cells
continued to express the CD7 T-cell-associated and CD34
primitive hematopoietic cell antigens, and remained TdT'.
The cells also expressed the CD13, CD14, and CD33 myeloid
antigens, and the platelet GPIIb/IIIa complex (CD41).
Subpopulations expressed platelet GPIb (CD42b), the VLA2
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1371
ANLL CELL LINE W T H ~(3:3)(021:0261
The cell line karyotype was 45,XX,-7.t(3;3)(q21;
q26),t( 12;22)(p13:ql2) in all metaphases examined. The
t(3;3)(q21;q26) was apparently identical to that of the
patient’s cells at diagnosis and relapse, and the t( 12:22)(pI 3;
q12) was identical to a subgroup of cells present at relapse
only (Fig 2).
To assess growth factor responses by UCSD/AMLI cells,
log-phase cells were washed three times, plated with various
concentrations of growth factors and FBS, and proliferation
assessed by TdR uptake. As shown in Fig 3, UCSD/AMLl
cells proliferated in response to IL-3, IL-4. IL-6, GM-CSF,
and M-CSF, but showed no response to IL-I, IL-2, IL-5, or
G-CSF (data not shown). The cells could be maintained as a
factor-dependent cell line in GM-CSF, IL-6, or M-CSF with
a doubling time of 48 to 72 hours, but could be continuously
grown with IL-3 only with markedly reduced cellular viability. When GM-CSF was withdrawn, the cells ceased proliferating and 50% to 75% of cells died within 2 weeks. However,
when the remaining cells were replated in GM-CSF. they
rapidly recovered to their baseline growth rate (three experimen ts).
In other factor-dependent cell lines, growth factors synergize in stimulating cell g r o ~ t h . ’ ~ .Because
’~
the cell line
expressed the primitive CD34 antigen. we screened several
growth factor combinations, which showed synergism in
culture systems for putative early (“blast”)
by combining the highest concentration of a growth factor
that did not stimulate cell growth with a complete doseresponse curve of a second growth factor. Combinations of 1
or IO U/mL IL-4 and GM-CSF or IL-6 showed no interaction. Similarly, G-CSF (100 U/mL) and IL-4 had no effect
on IL-3-stimulated cell growth (not shown).
I
B
3
C’
I
12
22
V
T-
Fig 1. Morphology of UCSDlAMLl cell line grown with FBS
and 30 UlmL GM-CSF (A), or with lo-’ mollL TPA and GM-CSF
(B). Multinucleatedgiant cells (C) were observed under all culture
conditions, but increased in number when cells were grown with
30 UlmL IL-6 and GM-CSF b“text).
or platelet GPla (CDw49b). and the platelet GPlV (CD36)
antigens. Southern blots showed Ig and T-cell /3 receptor
genes to be in germline configuration (data not shown). Thus,
the cell line retained characteristics of primitive hematopoietic cells expressing the CD7 and CD34 antigens and TdT,
but also had characteristics consistent with GM and megakaryocytic cells.
Fig 2. Detail of chromosome translocations in UCSDlAMLl
showing t(3:3)(q21:q281 and t(12=1(p13:q12).
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1372
OVAL ET AL
I
DISCUSSION
0 11-6
300
200
100
0.03 0.1 0.3 1
3
10 30 100
U/ml
Fig 3. Proliferation of UCSD/AMLl cells in response to hematopoietic growth factors. Cells grown with FBS and OM-CSF
were washed and grown with no addition or various concentrations of the growth factors shown for 4 days. Results shown are
the means of three to five experiments.
The expression of primitive hematopoietic cell, myeloid,
and megakaryocytic antigens by UCSD/AMLl cells suggested these cells could undergo multilineage differentiation.
When UCSD/AMLl cells were cultured with GM-CSF and
TPA, they rapidly became adherent, and a few showed
distinct spreading on plastic. The morphology of the cells
became macrophage-like (Fig lB), and expression of the
CD14 antigen increased from 25% to 52% of cells (means of
two experiments). Increased cellular adherence and spreading were also observed after long-term (2 weeks) culture with
M-CSF. In the presence of GM-CSF and TPA, the number
of multinucleated giant cells also increased slightly over
cultures maintained in GM-CSF alone from 3%
1% of
3% (means f SE; three experiments).
total cells to 8%
Identical results were obtained when cells were grown for 5
days with TPA alone, but cell viabilities were decreased.
Because IL-6 was recently shown to be a maturation factor
for megakaryocytes: surface antigen expression and morphology of UCSD/AMLl cells were also examined after culture
with IL-6. After 5 days of culture with GM-CSF and 30
U/mL IL-6, the number of multinucleated giant cells
increased fourfold to sevenfold (18% f 6%; N = 3), but did
not increase in the presence of IL-6 alone. In contrast,
immunofluorescence staining after exposure to IL-6 (not
shown) or IL-6 with GM-CSF showed increased expression
of three platelet-associated antigens (platelet GPIIb/IIIa
complex, platelet GPIa, and platelet GPIV) (Fig 4). When
cells were gated according to low-angle light scatter, increased platelet antigen expression was observed in cells of
both intermediate and large size, suggesting the increase in
platelet antigen expression was not restricted to multinucleated giant cells. Thus, UCSD/AMLl cells showed evidence
for both macrophage and megakaryocytic differentiation.
The new cell line described in the current studies retains
properties of the acute leukemia with high-platelets
The cell line has a 3q21 translocation characteristic of this syndrome. Monosomy 7, present in the patient’s
cells and the cell line, and pre-existing myelodysplasia were
also previously described as components of this ~yndrome.~.~.’
Expression of G P IIb/IIIa, CD13, and CD14 by UCSD/
A M L l cells is similar to expression of platelet antigens by
cells from a patient with ANLL and t( 1;3)(p36;q21).6 Thus,
UCSD/AMLl cells appear to be the first cell line to preserve
properties of an ANLL syndrome associated with a specific
chromosomal translocation.
The original tumor cells and the cell line also share
properties with multipotent CD7+ acute leukemias.12~13
The
cell line retains CD7 expression and TdT positivity. In vitro,
UCSD/AMLl cells form multinucleated giant cells consistent with abnormal megakaryocytes. Virtually identical cells
were observed in short-term cultures of CD7+ acute leukemia cells.12 Several patients with CD7+ acute leukemia
described in a previous report also had normal or high
platelet counts,12 but lacked chromosome 3 abnormalities.
Although UCSD/AMLl cells differentiate into both macrophage and megakaryocyte-like cells, thus far we have been
unable to induce the broad range of lymphocyte and myeloid
differentiation observed in short-term cultures of CD7+
acute leukemia cells.’’
The ability of UCSD/AMLl cells to undergo aberrant
megakaryocyte differentiation and proliferate in response to
IL-6 may explain some features of the ANLL with highplatelets syndrome. The appearance of increased multinucleated giant cells after incubation with IL-6 and GM-CSF, but
not IL-6 alone, suggests that UCSD/AMLl cells will be
useful for studying cytokine interactions in this syndrome. In
loor
GPIIWIIIa
VLA-2
loor
Fluorescence Intensity
Fig 4. Expression of platelet-related antigens by UCSD/AMLl
cells detected by immunofluorescence after culture with FBS and
GM-CSF (--------I
or with GM-CSF and IL-6 (----I.
Representative results from one of three experiments are shown. Staining
with irrelevant control antibody (-1.
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1373
ANLL CELL LINE WITH T(3;3)(o21;a26)
future studies, it will also be important to examine effects of
IL-6 on proliferation and differentiation of primary tumor
cells from patients with t(3;3)(q21;q26).
Whether genes involved in the t(3;3)(q21;q26) induce
leukemic transformation or alter its phenotypic manifestations is a t present unclear. Although it has been postulated
that genes involved in cellular Fe metabolism may be
involved in these translocations,’ Southern blots of D N A
from the UCSD/AMLl cells showed no gross rearrangements of the transferrin receptor gene (data not shown). The
establishment of the UCSD/AMLl cell line should allow
cloning and characterization of the genes involved in this
translocation.
In short-term culture, UCSD/AMLI cells respond to a
wide variety of growth factors, including IL-6, IL-4, and
M-CSF, which may stimulate primitive normal marrow
(“blast”) progenitor^,^^^^^ but do not directly stimulate growth
of normal G M progenitors2’ or most ANLL cells in vitro.29
The extent to which human factor-dependent leukemia cell
lines reflect growth factor responses by primary ANLL cells
is unclear. It is possible that continuous exposure to growth
factors in vitro selects for cells or mutations allowing
factor-dependent growth in the same way culture with FBS
and media alone selects for relatively factor-independent
cells. UCSD/AMLl cells contain a t(12;22) present in only a
subpopulation of the patients’ cells at relapse, indicating
selection in culture for this tumor cell population. The broad
range of growth factor responses displayed by these cells
could give these cells a proliferative advantage in vivo over
their normal counterparts.
A subpopulation of UCSD/AMLl cells survives growth
factor deprivation for extended periods. These results differ
from studies using murine factor-dependent cells that require
growth factors, such as IL-3, for survival and rapidly die
when growth factors are ~ i t h d r a w n . ~We
’ are currently
evaluating whether growth factors contained in FBS may
partially maintain UCSD/AMLl cell viability. This property of the cell line may allow detailed study of the nonproliferating (Go)state in factor-dependent human myeloid leukemia cells.
UCSD/AMLI represent a valuable addition to the limited
roster of available factor-dependent, human A N L L cell
lines. Establishment of cell lines from patients with other
acute leukemia syndromes may provide similar insights into
the pathophysiology of these acute leukemia syndromes, and
the eventual definition of the roles chromosome translocations play in the establishment and maintenance of acute
leukemia.
ACKNOWLEDGMENT
We thank Dr Ignacio Iturbe, without whom these studies would
not have been possible; and Mary Rutherford and Catherine Stanish
for their technical assistance.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1990 76: 1369-1374
Characterization of a factor-dependent acute leukemia cell line with
translocation (3;3)(q21;q26)
J Oval, OW Jones, M Montoya and R Taetle
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