From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Characterization
of a New
(PLB-985)
With
Granulocytic
By
A.
Kent
Tucker,
Human
Diploid
and
Michael
Myeloid
Monocytic
B. Lilly,
Louis
Heck,
Jr,
and
Thomas
ryl cyclic
adenosine
maturation
as
line
nine
into
the
in the presence
of inducing
agents.
By morphology,
the
analysis
of surface
antigens.
and cytochemical
staining
PLB-985
cells are myelomonoblasts.
Transmission
electron
microscopy
reveals
them
to be devoid
of neutrophilic
tase
and
cathepsin
primary
increased
capable
or
matin
of granulocytic
secondary
pattern
and
granules
with
monocytic
and
frequent
to
nucleoli.
maturation
have
The
an
open
modal
chro-
HE
ACUTE
mias
bone
HUMAN
(ANLL)
marrow
NONLYMPHOCYTIC
are
and
characterized
peripheral
morphologically
blood,
immature
myelopoiesis.
cells
In spite
tive
cells
nature
in vivo, however,
most fresh
are capable
of only a limited
number
exist.2
The
majority
The
research
are
in response
The
of their
capable
most
of some
attributes
of erythroid
degree
contributed
poietic
cell
cell line
cells5
induction.
is limited
by their
terminal
Thus,
study
surrounding
events
the
for example,
or megakaryocytes6
the
study
after
of
to achieve
each
these
their
line
Medicine,
has
hematousefulness
their
provides
stages
appro-
of complete,
from
myeloid
of
is
lines
of
a state
particular
Department
normal
a model
of
University
to
maturation.
of
Alabama
at
Birmingham.
Submitted
December
Supported
in part
CA 39042
Clinical
Oncology
Core Grant
Address
University
tion,
accepted
American
Career
reprint
Cancer
payment.
“advertisement”
Society
(NC!)
Grant
Award
this fact.
1 987 by Grune
Rado.
at Birmingham.
AL
Grant
No.
CH
319.
and
NC!
No.
(TAR.),
Department
unique
earlier
We
of new,
5/3
costs
ofthis
article
This
article
must
THT
in accordance
with
& Stratton,
Inc.
were defrayed
therefore
18
U.S.C.
positively
lose
the
Induction
is accompanied
inducible,
will
or later than has
have
established
line,
markedly
stain
ability
by a marked
thus
a
human
permit
myeloid
the
study
far been possible.
new
human,
diploid,
designated
PLB-985,
obtained
cell
myeloid
from
blood
of a patient
with refractory
myeloblastic
Phenotypically
these
cells are myelomonoblasts.
They have
macrophage
the capacity
maturation
report
for both
in the
documents
cell
the
lines
of events
peripheral
leukemia.
of the new
to
of differ-
Inc.
characteristics
cell
differof this
transcription.
& Stratton,
development
with
the
granulocytic
and monocyte/
presence
of inducing
agents.
establishment
and
characteriza-
line.
University
Sta-
in part
be hereby
§1734
by page
marked
solely
to
AND
METHODS
P.L.,
a 38-year-old
white
woman,
presented
in
1985 with acute myelogenous
leukemia.
Her peripheral
count was 12.9 x 10’/L. The differential
included
13%
1% promyelocytes,
1% myelocytes,
3% metamyeloband forms, 49% segmented
neutrophils,
30% lympho1% monocytes.
Her hemoglobin
concentration
was 6.7
her platelet
count was 20 x 10’2/L.
Bone marrow
revealed myeloid hyperplasia
with 43% myeloblasts,
5%
promyelocytes,
and decreased
levels of megakaryocytes.
Cytochemical studies indicated
that the leukemic
cell population
was positive
for Sudan black stain and myeloperoxidase
activity.
Approximately
a third of the cells displayed
nonspecific
acid esterase
activity,
and
no staining
with PAS was detected.
Surface
markers
were positive
for OKM1
(42%) but negative
for HLA-DR,
natural
killer cells
(NK),
and BI antigens.
NK antibodies
used included:
OKT3,
Leu- 19, and Leu- I 1 . Cytogenetics
revealed a normal 46,XX karyotype in all metaphases
available
for examination.
The patient initially was induced into a complete
remission
with
standard
chemotherapy.
Subsequently
she relapsed
and proved
refractory
to all further chemotherapy.
Cells obtained
at that time
were used to establish
the present cell line.
Case
report.
February
leukocyte
myeloblasts,
cytes, 2%
cytes,
and
g/dL,
and
examination
suspension
of Medicine,
35294.
0006-4971/87/7002-0005$3.OO/O
Grune
Establishment
to TA.
indicate
372
20, 1987.
Institute
Development
requests
of Alabama
The publication
(C)
March
Cancer
No. CA 13148.
Birmingham,
charge
16, 1 986;
by National
(M.B.L.)
by
to
display
and
pathway
presence
particles.
proteinases.
oncogene
cells
in the
ester
to plastic.
latex
esterase,
MATERIALS
HL-60,
indistinguishable
counterparts.
a 1987
either
elas-
phorbol
of maturation
to our understanding
and differentiation,
maturation
in myc
tion
to a variety
of agents.3’4
SimiK562 can be induced
to express
inability
along
decrease
This
to leuke-
or monocyte/macrophage
Although
significantly
proliferation
been
lines
differentiating
valuable
entiation
agents.
granulocytic
after exposure
primitive
line
has
acetate
35S-methio-
proteinases
PLB-985
grown
adherent
the neutrophilic
of
granule
induces
of
histochemical
incorporation
tumor-promoting
Cells
become
a-naphthyl
leukemia
prolifera-
isolates
of ANLL
of cell divisions
in
limited
proved
promyelocytic
characteristics
larly, the more
From
have
have
those
of displaying
priate
these
that
to differentiation-inducing
human
capable
of
lines
are
eventu-
The development
of myeloid
cell lines
and at present
only a few well-characterized
capacity.
in
that
proliferation
replaces
mia
presence,
of myeloid
whose
ally
culture.’
difficult
normal
and
the
acetate
in granulocytic
morphology,
primary
phagocytosis
synthesize
The
leuke-
by
and
as monocytes.
rapidly
results
by
G. The
myristate
entiate
Rado
monophosphate
determined
neutrophil
Line
Capacity
A.
characteristics,
phorbol
for
karyotype
of the line is 46,XX.
with
no consistent
marker
chromosomes or recognizable
translocations.
Myelomonoblasts
of
this line form colonies
in soft agar and induce
tumors
(chloromas)
in nude mice.
Growth
of the cells in the
presence
of dimethyl
sulfoxide.
cis-retinoic
acid. or dibuty-
T
staining
agent
Cell
Differentiating
A new human
diploid
cell line. designated
PLB-985,
has
been established
from the peripheral
blood of a patient
with acute nonlymphocytic
leukemia
(ANLL).
Cells of this
are
Leukemia
of
culture.
the
myelomonoblast
During
the
patient’s
cell
final
line
PLB-985
in
hospitalization
cells were isolated from a sample of peripheral
blood by
Ficoll-l-lypaque
density centrifugation.
The cells were washed once
and resuspended
in Iscove’s modified Dulbecco’s
medium (IMDM)/
RPMI
1640 (7:3) media containing
10% heat-inactivated
fetal calf
serum (FCS). A portion of the cells was resuspended
in RPMI with
10% FCS and 10% dimethyl
sulfoxide
(DMSO)
at a concentration
of 1 x l0 cells/mL
for cryopreservation
in liquid nitrogen
vapor.
Two weeks later a 1-mL aliquot of frozen cells was removed,
thawed,
mononuclear
Blood,
Vol 70,
No 2 (August),
1987:
pp 372-378
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PLB-985:
washed,
incubated
A HUMAN
MYELOID
LEUKEMIA
CELL LINE
resuspended
in IMDM/RPMI
(7:3) plus 10% FCS, and
at 37#{176}C
in a humidified
atmosphere
of 5% CO2 in air.
Cytochemical
staining.
The cells were observed
with Wright’s,
Sudan black, PAS, nonspecific
acid esterase,
naphthol
AS-D chloroacetate
esterase,
and neutrophil
alkaline
phosphatase
stain.
Supravital
staining
was performed
with acridine
orange (AO) and
examined
with fluorescence
microscopy.7’
Cell surface markers.
Cultured
PLB-985
cells were reacted
with a panel of mouse monoclonal
antibodies
against
B cell and
myelomonocytic
antigens9
and against the specific megakaryocytic
marker
PBM.’#{176}
After incubation
with the monoclonal
antibodies,
cells were stained with affinity-purified
goat antimouse
lgG antibodies labeled with fluorescein
isothiocyanate
(Southern
Biotechnology,
Birmingham,
AL) and examined
by fluorescence
microscopy
or flow
cytometry.
Ultrastructural
studies.
Preparation
of PLB-985
cells for transmission electron
microscopy
was performed
according
to Roth et
al”
Chromosome
analysis
Bone marrow
cells obtained
from the
patient at the time of diagnosis
were cultured
in RPMI 1640 media
with 10% FCS for 24 hours at 34#{176}C,
after which they were exposed
to colcemid
(0.06 ig/mL)
for 2.5 hours at 4#{176}C.
Cultured
PLB-985
cells were similarly
processed.
Routine
methods
were used for
culture
harvest, slide preparation,
and Giemsa banding
of chromosomes after trypsin
digestion.
Direct
examination
of metaphase
chromosome
preparations
was also performed.
Growth
in semisolid
medium.
PLB-985
cells from suspension
cultures
were suspended
in a medium
of IMDM
containing
20%
FCS, 20 mmol/L
glutamine,
1% penicillin/streptomycin,
and 0.3%
agar at a final cell density of 5 x l03/mL.
The plates were incubated
at 37#{176}C
in a 5% CO2 atmosphere
for ten to 14 days. Colonies (>40
cells) were evaluated
with an inverted-stage
microscope.
Metabolic
labeling of intact cells with 35S-methionine.
Metabolic
labeling
of PLB-985
cells was performed
as previously
described’2
with the following
modifications.
Preincubation
and
incubation
were carried out in medium
containing
5% Nu-Serum
(Collaborative
Bioresearch,
Bethesda,
MD) instead of FCS. Cells
were lysed in a buffer consisting
of 50 mmol/L
Tris, pH 7.5, 1%
sodium deoxycholate,
1% NP-40,
150 mmol/L
NaCI, and 0.02%
sodium
azide. Diisopropyl
fluorophosphate
was added to a final
concentration
of 20 mmol/L
immediately
before use.
Immunoprecipitation
of elastase and cathepsin
G from
cell
extracts.
Polyclonal and monoclonal
antisera to human neutrophil
cathepsin
G (HNCG)
and human neutrophil
elastase (HNE)
were
obtained
as previously
reported’3
by using purified HNE and HNCG
isolated
from normal
human
neutrophils.’4
Antisera
were stored
at -80#{176}C
at 1 mg/mL
in phosphate-buffered
saline (PBS). Staphylococcal
protein
A bound to sepharose
beads was obtained
from
Pharmacia
Fine Chemicals
(Piscataway,
NJ). ‘4C-protein
molecular
weight
markers
were from Amersham
Corp (Arlington
Heights,
IL).
Immunoprecipitations
were performed
as previously
described’5
with some modification.
For each reaction,
a volume
of lysate
containing
5 x l0 acid-precipitable
counts was diluted to 300 .tL
with
lysis buffer.
Antiserum
was added
at a 1:5 dilution
and
incubated
overnight
at 4#{176}C
with constant
agitation.
At the end of
this incubation,
50 L of protein A-Sepharose
(33% wt/vol suspension in PBS) was added and incubation
continued
for one additional
hour at 4#{176}C.
The protein
A-sepharose-antibody
complexes
were
recovered
by gentle centrifugation
and washed according
to Dunphy
Ct al.’6 The immunoprecipitated
proteins were solubilized
in 100 zL
treatment
buffer (4% sodium dodecyl sulfate, 20% glycerol, and I 25
mmol/L
Tris, pH 6.8). The samples were boiled for three minutes
and the Sepharose
beads removed
by centrifugation.
The supernatants were analyzed
by vertical electrophoresis
through
a linear 5%
373
to 20% SDS-polyacrylamide
gradient
gel prepared
according
to
Laemmli.”
The gels were stained with Coomassie
blue and treated
with En3Hance
(New England
Nuclear,
Boston).
Fluorography
was
carried out at 80#{176}C
on Kodak XAR-5 film for 48 to 96 hours.
Induction
of differentiation.
Induction
of granulocytic
differentiation was achieved
by using 1.25% DMSO (Fluka,
Ronkonkoma,
NY),”
I mol/L
cis-retinoic
acid (Sigma Chemical
Co. St Louis),’9
and 750 zmol/L
N6, 2’-dibutyryl
adenosine-3’,S’-cyclic
monophosphate (Bt2cAMP,
Sigma).#{176}
Induction
of monocytic
differentiation
was achieved
by using 50
nmol/L
phorbol- 12-myristateI 3-acetate
(PMA, Sigma).2’
Extraction
ofcellular
RNA and blot hybridization.
Total cellular RNA
was extracted
from PLB-985
cells by rapid lysis in
guanidinium
isothiocyanate22
followed by centrifugation
through 5.7
mol/L cesium chloride.23 The purity and molecular
weight of RNA
fractions
were
routinely
assessed
by electrophoresis
through
I .4%
agarose gels containing
10 mol/L methyl mercury
hydroxide,24
with
intact ribosomal
RNA (185 and 285) used as standards.
For blot
hybridization
experiments
formamide-denatured
RNA was separated in a formaldehyde/agarose
gel.25 After electrophoresis
the
nucleic acid was transferred
to nylon membrane
(GeneScreen
Plus,
New England Nuclear)
by capillary
blotting.26 Prehybridization
and
hybridization
conditions
were as recommended
by the manufacturer. Purified DNA probes for ‘y-actin27 and c-myc’
were labeled with
a-32P-dCTP
(deoxyeytidine
triphosphate)
by the random
oligonucleotide method of Feinberg
and Vogelstein.’
RESULTS
Cell culture.
3 to 4 weeks
thereafter.
FCS
RPMI
was
growth
The cells did not proliferate
in culture
but began
a gradual
1640
added
had
stabilized,
passages.
The
adapated
to growth
feeder
required
cells
have
been
with
appeared
been
efficiency
Auer’s
rods
black
a-naphthyl
cells
phatase
large,
are
but
acetate
display
is not
a slight,
present.
green-fluorescent,
to form
colonies
Tumors
taken
from
PLB-985
are
as
cells
the
original
growing
in susOcca-
pseudopodia.
There
is considerand the largest
cells are often
cytoplasm
azurophilic
seen.
All
Less
PAS
Supravital
irregular
a large,
round,
or
and one to three
is scanty
granules
of the
half
esterase.
diffuse
the same
myelomonoblasts.
the cells contain
fine chromatin,
than
tumors
display
characteristics
PLB-985
implanted
murine
and
time
in soft
(chloromas)
cells
growth
The
rare
less
a doubling
of 0.05.
malignant
culture
have
these
multinucleate.
Typically
slightly
irregular
nucleus,
(Fig
2A).
and only
No
was
The
when
resemble
rapidly
medium
of the culture.
mice
to in vitro
was
5% Nu-Serum.
suspension
They
are able
characteristics.
culture
Sudan
plus
in continuous
sional cells have blunt-ended
able variation
in cell size,
No
PLB-985,
1640
and antigenic
not shown).
nucleoli
basophilic,
cells
designated
I) and
Cells
(‘ytologic
pension
twice-weekly
(Fig
readapted
cytochemical
line (data
6 in culture,
required
14 months.
in nude
heat-inactivated
the
over
a plating
subcutaneously.
have
10%
By week
in RPMI
maintained
of 24 to 48 hours
have
with
initially.
or growth
factor-conditioned
initiation
or maintenance
passages
agar
and
new cell line,
layers
for
for >90
medium
as needed
during
the first
accceleration
cells
for
and
deeply
are present.
stain
positive
for
myeloperoxidase
than
positivity.
staining
nuclei
I 0%
of
and
uninduced
Alkaline
with
with
AO
phos-
reveals
prominent
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
TUCKER
374
ET AL
Ir
.
A
.
.
.
L9B5
9.SO
--
L8985±
R
LBT
CJHNG
‘H
‘
H
3
4
5
3
Fig 1 .
Growth
of PLB-985
cells in the presence
and absence
of
differentiation-inducing
agents.
Data points
represent
nonadherent cells in suspension.
The concentrations
of inducing
agents
are
as described
in Materials
and Methods.
nucleoli
and
only
rare
cytoplasmic
red-orange
granular
fluo-
rescence.
Transmission
the
PLB-985
cells,
with
electron
line
the
frequently
folded
prominent
nucleoli
nent
vast
consists
predominant
nucleus,
(Fig
U It rastructurally
microscopy.
of a heterogeneous
cell
fine
3). The
type
population
containing
dispersed
chromatin,
cytoplasm
contains
rough endoplasrnic
reticulum
and Golgi
majority
of the cells have no azurophilic
bodies,
granules.
a
of
large,
and
promi-
but
Fig 2.
(A) Wright’s
stained
preparation
of uninduced
PLB-985
cells in culture.
Typical open nuclei. frequent
nucleoli.
and agranular cytoplasm
are noted. A single mitotic figure is present.
(Original
magnification
x 1 .000; current
magnification
x400.)
(B) Wright’s
stained
preparation
of PLB-985
cells grown
for three days in RPMI
containing
5% Nu-Serum
and 750 zmol/L
Bt2 cAMP.
Neutrophilic
maturation
nucleoli,
cytoplasm.
x400.)
is
indicated
and the
(Original
by
increased
appearance
magnification
of
pyknosis
of
nuclei.
loss
of
azurophilic
granularity
in the
x 1 .000; current
magnification.
the
;
!
I
Fig 3.
Transmission
electron
micrograph
of
uninduced
PLB-985
cells resembles
that of a myeloblast.
Nucleoli
are present.
and there
is no
cytoplasmic
granularity.
(Original
magnification
x 19,000; current
magnification
x 12,730.)
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PLB-985:
A HUMAN
Fig 4.
(A)
MYELOID
After
three
LEUKEMIA
days
CELL
of growth
LINE
375
in the
presence
of granulocytic
inducing
agents.
cells
with typical
promyelocyte
morphology
are noted.
Nucleoli
are reduced
in number,
nuclear
indentstion is apparent,
and the cytoplasm
contains
abundant electron-dense
granules.
(Original
magnification
x 1 1 .000; current
magnification
x 7.81 0.) (B)
Cells
with
the morphology
of metamyelocytes
appear
after 72 to 96 hours in granulocytic
inducing medium.
Progressive
nuclear
maturation
with
disappearance
of nucleoli
is noted. Electron-lucent
and electron-dense
granules
are present
in the
cytoplasm.
(Original
magnification
x 1 8.000;
current magnification
x 12.780.)
Cell
surface
expressed
MY4,
markers.
1 , and
Leu-M
MY7,
and
uniformly
MY9
negative,
marker
PBM,
DR was also
HLA-Al,
all
half
as
was
that
-B4l,
of the
cultured
the
positive
Lymphoid
the
and
for
cells
0KM
markers
specific
differentiation
1,
were
from
The
whose
megakaryocytic
HLA
stain,
the
line
the
normal
patient’s
46,XX
a modal
chromosomal
Response
assess
bone
studies
marrow
karyotype.
karyotype
for the cell
To
Cytogenetic
The
of 46,XX.
Many
effects
None
cells
cells
revealed
also
displayed
metaphases,
however,
of these
are
specific
in all nuclei.
of PLB-985
the
on
at presentation
PLB-985
abnormalities.
line or present
performed
to differentiation-inducing
of
substances
known
agents.
to
stimulate
a
pyknotic,
Nucleoli
cells
and
supravital
stain
promyelocytic
were
with
of nuclear
Supravital
azurophilic
staining
and
Ultrastructural
induction
line
of
by Wright’s
electron
inducers
of the cells
and
demonstrated
in the nuclear/cytoplasmic
granulocytic
ratio,
indentation,
formation
of
(Fig
increase
I B).
in
and
fragmented
and there
the
nuclei
is an
granulation.
of cells
examination
revealed
cell
presence
by transmission
grown
in the
agents
is marked
by the appearance
cytoplasmic
granular
fluorescence.
cytic
the
of the granulocytic
or Bt2-cAMP,
many
multilobed,
and
tend
to disappear,
cytoplasmic
in
morphologically
AO,
the presence
acid (RA),
leukemia
cultured
examined
spindle
shaped
by a decrease
the appearance
Cytogenetics.
have
inducers
became
maturation
was
isolated.
from
in the human
PLB-985
microscopy.
In
DMSO,
retinoic
phenotype
blood
I-IL-60,
various
multinucleate
cells.
HLAHLA
antigens
included
-B60.
patient
of
were
antigens.
even in the large
negative.
Positive
-A2,
matches
Nearly
over
of
the
presence
of bright
PLB-985
presence
of
of these
red-orange
after
cells
granulo-
resembling
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TUCKER
376
metamyelocytes
and
band
populations
of granules,
lucent
are also
class,
were
consistently
any
of
presence
became
marked
with
either
with
phage
inducers
(Fig
exclusion
of
the
evaluated
the
the
inducer
HNCG
was
after
was
incubation
As
shown
Nu-Serum
in
cells
beads
compared
cells.
positive
culture
72 hours
staining
a
These
staining
effect
on
ofexposure,
however,
by
trypan
of granulocytic
the PLB-985
primary
cells
cell
neutrophil
and
PMA.
For the
of nonadherent
blue
The
or monoline we
granule
incorporation
HNE
and
in cells
exposed
to
latter
experiments
cells remaining
and the adherent
population
addition
of preincubation
and
to the culture
PLB-985
inducing
mark-
AO.
cathepsin
G.3’
immunoprecipitable
discarded
by the
was
monocyte/macro-
>90%
with
of the
6,
or
remained
directly
without
apparent
showed
or uninduced
in suspension
in cultured
Fig
30 mm-
readily
cells
strongly
1). After
inducers
or
number
(<5%)
media
PLB-985
within
latex
granulocytic
biosynthesis
induction
was
labeled
in situ
with
conditions
esterase.
metabolic
maturation
studied
changes
PMA,
This
was
PMA-treated
to engulf
proteinases
elastase3#{176}and
of 35S-methionine
into
granulocytic
the small
Two
cells
the
to plastic
cells
or supravital
To assess
cyte/macrophage
of the
adherent
by either
viability
All of these
under
induced
of PLB-985
inhibited
cells.
demonstrated
acetate
4B).
an electron-
Methods.
of exposure.
(Fig 5). The
also
and
and
monocytic
granulocytic
a-naphthyl
edly
and
in ability
cells
Growth
the
hour
4A
treatment
inducers
of the
increase
adherent
in these
after
irreversibly
utes to one
ultrastructurally
(Fig
electron-dense
granulocytic
in Materials
In the
cells
noted
present
the
described
forms
and
cells
agents
flask.
growing
display
in
Fig 6.
Fluorograph
of 35S-methionine-Iabeled
PLB-985
lysates
immunoprecipitated
with
antielastase
or anti-cathepsin
G as
described
in Materials
and Methods.
14C-labeled
proteins
were
used as molecular
weight
markers.
Lanes
1 and 2. lysate
of
uninduced
cells; lanes 3 and 4, lysate of cells induced
for 72 hours
with
DMSO;
lanes 5 and 6, lysate of adherent
cells treated
for 72
hours
with
PMA.
Each lane contains
5 x 1 0 cpm of acidprecipitable
radioactivity.
In each pair the first lane was immunoprecipitated
with
antiserum
to elastase
and the second
with
anti-cathepsin
G.
The
RPMI/
a constitutive
several
the complete
abolition
of detectable
elastase
and cathepsin
G
synthesis
(lanes
5 and 6). The viability
ofthe
cells under each
of these culture
conditions
was essentially
100% as assessed
amount
by supravital
ods.
(data
not shown).
expression
decrease
synthesis
of elastase
and cathepsin
G (lanes
I and 2). After
granulocytic
induction
the proportion
of acid-precipitable
radioactivity
incorporated
into these
proteins
is increased
(lanes
3 and 4). Induction
of the cells with PMA
results
in
staining
ET AL
of the
after
induction
other
cell
lines
sion
To determine
in PLB-985
and
from
cies.33
cells
of
transferred
radioactive
to
a
c-mvc
Differentiation
derived
the effect
we extracted
treated
RNA
c-myc
oncogene
has
ofdifferentiation
with
from
shown
hematologic
PMA
each
source
nylon
probe
membrane,
as described
the
or Bt2 cAMP.
was
size
to
in
exprescells
The
same
fractionated,
and
hybridized
in Materials
and
granulocytic
and
malignan-
of induction
of c-myc
RNA
from uninduced
from
in either
been
in HL-6032
or the
to a
Methmono-
Fig 5.
Monocytic
maturation
induced
by PMA
is characterized
by adherence
to plastic.
cytoplasmic vacuolization,
and the
absence
of granule
synthesis.
(Original
magnification
x 9,000;
current
magnification
x 5,580.)
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PLB-985:
A HUMAN
MYELOID
cyte/macrophage
the
direction
proportion
blots
of the
hybridized
decrease
in
RNA
reduction
(Fig
‘y-actin
7).
probe
granulocytic
RNA
377
LINE
in a marked
c-myc
purified
after
decrease
CELL
resulted
225
to
in signal
small
LEUKEMIA
displayed
maturation
from
cells
in
Duplicate
no
and
treated
only
with
a
phorbol
ester.
DISCUSSION
The
establishment
has
provided
tion
and
of human
much
differentiation.
culture
as
further
PL-2l
can
early
KG-
1 , ML-l
other
agents
,3, and
The
stimulated
along
which
the
is also
cells
differentiation
are
proceeds
cells
are
culture
renewal
these
electron
the
conditions,
with
of the
the opportunity
and
cell
marker
cell
HL-60
whether
terminal
less
Fig 7.
Hybridization
of PLB-985
RNA to c-myc
and ‘y-actin
probe. Each lane contains
30 .tg of total cellular
RNA. Sources
of
RNA: lane A, uninduced
cells; lane B, cells induced
with Bt2-cAMP
for 96 hours; lane C. adherent
cells induced with PMA for 48 hours.
18S and 28S molecular
weight
markers
are indicated.
Blot a,
hybridization
to c-myc;
blot b. hybridization
to ‘y-actin probe.
cyte/monocyte-macrophage
afforded
by HL-60.
commitment
earlier
than
that
PLB-985
in
liquid
ACKNOWLEDGMENT
for selfof the cells
stains
are
line.
to
or monocyte/
analysis
events
line
agent
conventional
to study
to
inducing
their
capacity
The morphology
they
has
or mono-
Growing
that
line
to respond
the granulocyte
suggests
promyelocytes
promoters
granulocytic
myelomonoblasts.
microscopy,
provide
whereas
determines
with
a serum
substitute,
appears
to be unlimited.
under
In partic-
The
by surface
malignant
influences.
myelomonoblastic
along
macrophage
pathway.
Phenotypically
and
in
of
maturation,34
of its capacity
bipotential.
exposed
grow
capable
leukemia
either
The
herein
which
the monocyte-macrophage
because
by manifesting
lines
prolifera-
are
to tumor
promyelocytic
characteristics.
described
lines,
the proper
cell
cell
cells,
l respond
study
agents
cytic
THP-
HL-60
intense
different
existing
granulocytic
by maturation
pathway.35
ANLL
myeloid
under
undergo
leukemia
about
Several
malignant
differentiation
ular,
myeloid
information
and
mature
PLB-985
may
involved
by
than
thus
in granulo-
The authors wish to express their gratitude
to Lawrence
Melson
for his assistance
with transmission
electron microscopy,
Dr Andrew
Carroll
for performance
of the cytogenetic
studies,
and Dr Ed
Prasthofer
for providing
the cell surface marker antibodies
and flow
cytometry.
We thank Dr Andrew
Lorincz for advice and assistance
with supravital
fluorescence
microscopy.
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1987 70: 372-378
Characterization of a new human diploid myeloid leukemia cell line (PLB985) with granulocytic and monocytic differentiating capacity
KA Tucker, MB Lilly, L Jr Heck and TA Rado
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