Jan 12, 1989 - can Cancer. Society grant ... and are transmitted to cells that ..... clonal lines were more sensitive than the KM 101 clonal lines to the highest.
Journal
of Leukocyte
Biology
46:221-229
(1989)
Differentiation of Human Hematopoietic Cells Increases Expression of a Gene Transferred by a Retroviral Vector Christie
A. Holland,
PEN.),
Sunil Chada, Jocyndra Joel S. Greenberger,
Departments University
expression
established
human
of a retroviral hematopoietic
cell line (KMIOI)
transferred
Constance
and Peter
Whitney,
Kenichi
Harigaya,
E. Newburger
of Radiation Oncology (C.A.H., J.S.G.) and Pediatrics (S.C., J.W., C.W., of Massachusetts Medical School, Worcester, and Department of Pathology, Chiba University School of Medicine, Chiba City, Japan (K.H.)
To study stromal
Wright,
neomycin
vector
cell
lines
were infected
resistance
in human (HL6O
and
with the vector
gene (nec’)
of pZlP-SV(X)
hematopoietic K562)
and
lineages,
two adherent
a human
pZlP-SV(X).
Expression
of the
as the ability of of the neomycin
was evaluated in the presence
the cells to form colonies (>50 cells) in an agar assay analogue, G418. After infection, all three cell lines produced colonies resistant to G418. The level of neor mRNA in separate colonies was analyzed by Northern blot analysis. The neor gene transferred by the vector pZlP-SV(X) was expressed in both human hematopoietic and stromal cell lines. In addition, primary adherent human stromal cells infected with pZlP-SV(X) grew in the presence of G418. To determine if differentiation of hematopoietic cells affects expression of the retroviral vector, HL6O cells infected with pZIP-SV(X) were induced to differentiate, and the level of neor mRNA measured. The amount of neor mRNA increased when HL6O cells were induced to differentiate along the granulocytic differentiation
pathway. (TPA),
Conversely, when HL6O cells were induced toward the level of neor mRNA did not significantly increase.
monocytoid
We conclude that the neor gene transferred by a retroviral vector, pZIP-SV(X), is functionally expressed. In addition, expression of the transferred neo’ gene is regulated during myeloid differentiation of HL6O cells. Key words:
gene therapy,
human marrow
INTRODUCTION Retroviral gene
vectors
therapy
18,27,42].
of Using
transferred lineages.
been
proposed
disorders
vectors,
genes
are transmitted to cells that system of lethally irradiated
it is clear
that
application
for [15,
have
been
efficiency to murine hematopoietic and genes transferred are stably
retroviral
vectors
transfer to murine hematopoietic 15,18,23,24,27,41,42]. The
as vehicles
hematopoietic
retroviral
with high The vectors
inherited and hematopoietic fore,
have
heritable
of
retroviral
can
reconstitute the mice. Therebe used
progenitors vectors
for gene [1 1 2, 13,
into
marrow
human cultures
as
hematopoietic progenitors has been accomplished
tion-competent virus in the presence defective vector carrying the gene [18,36]. In addition, a HPRT-negative has been converted to a HPRT-positive a retroviral
©
vector
[34,43].
1989 Alan R. Liss, Inc.
Gene
transfer
gene expression
man hematopoietic replication-defective The
present
cells has been vectors as well study
of the neomycin retroviral vector
was
initiated
resistance into human
accomplished (20,21 ,28].
to investigate
(neo’) gene hematopoietic
with
expression
transferred cells.
by a A hu-
man stromal cell line (KM1O1) and two human leukemic cell lines (K562 and HL6O) were used. The KMlOl cell line was chosen as a model of a differentiated human hematopoietic stromal cell [19]. The line is fibrocytic, without
preadipocyte
Received
January
or endothelial
cell
characteristics.
,
vehicles
for
gene transfer to human hemapotoietic progenitors has been less extensive. Transfer of the v-ras oncogene and the hypoxanthine phosphoribosyl transferase (HPRT) gene
stroma,
in long-term using a replica-
of a replicationto be transferred human B cell line phenotype using to primary
hu-
This
work
CA38325, can
supported
CA39851,
Cancer
PEN.
was
12, 1989;
Society
accepted
March
by
Public
U.S.
CA40818, grant
is an Established
17, 1989. Health
and POl-ND-l9767
Service
and
grants
by Ameri-
IN- I 29.
Investigator
of The American
Heart
Associ-
ation.
Reprint requests: Christie A. Holland, cology, University of Massachusetts nue, North. Worcester, MA 01605.
Department of Radiation Medical Center. 55 Lake
OnAye-
222
Holland
It has
et al.
been demonstrated proliferation and
ports
that the differentiation
KM1O1 cell line of GM-CFU-C,
supG-
number
CFU-C, and M-CFU-C [19]. The HL6O lines were chosen as models of hematopoietic lineages. HL6O cells are predominantly
K562 cell progenitor promyelocytes
and
and
the
chosen human
are
capable
monocytoid
of differentiation
pathways
a multipotential of erythroid,
[8]
along
The
.
and
K562
cell
myeloid
line
stem cell capable of induced megakaryocyte, or myeloid
or
represents
.
transcription
transferred of retroviral
of retroviral
and
MuLV)
to be affected
determine
in the
case
of
Lines
HL6O
and
cells
medium (vol/vol)
To
hematopoietic
cells
of a gene transferred by a compared the level of neor cells
to that
in HL6O
in Dulbecco’s
(Gibco) fetal
ml penicillin in a 7% CO2
and 5 gIml atmosphere
twice
to maintain
streptomycin, at 37#{176}C.Cell cell
l0 cellslml. The cells inducers of differentiation
modified
supplemented calf serum
were
with (FCS),
and stocks
concentrations
cells
x and (60
cells to exclude 0. 1% trypan blue dye. For morphological assessment of the cells, slides were prepared by centrifugation of the cells in a ShandonlSouthern Cytospin, with Wright-Giemsa microscopy under
stages
of granulocytic
promylocytes,
differentiation
myelocytes,
and polymorphonuclear KM 101 cells were
‘I’ am 10
were
distinguished: band
(PMNs) [26]. as described
marrow
cultures
by Five
.
were
cells,
plasmid
and by
DNA
of
SV(X) as described [5]. Cells and individual G418-resistant
the
were retroviral
K562 the
were
1 x
l0
vector
per
infected
HL6O
After
units
milliliter
with
line
of
pZIP-SV(X)
described
( 1 x l0
cells
to 4 ml.
of 2 pgIml
2 hours,
above. cellslml)
or
at a multiplicity per cell in the 48 hours, HL6O
of polybrene
the volume
Forty-eight
hours
in 0. 1 ml of
of media
later,
1 x
was l0
expanded cells
were
plated in a CFU-F assay in the presence or absence of G41 8 [14]. The media was changed every 3 days, and colonies (>50 cells) were scored on day 10. Stromal cells of a primary human long-term bone marrow culture were infected as described previously and plated in a CFU-F assay as described for KMlOl [2].
Nucleic Acid Preparation Southern Blot Analysis High pared
molecular
weight
as described
and
ysis [38]. drochloride
RNA was procedure
genes
(>
pZIP-
The probe in pBR322.
G4 18 The
Kathleen versity
Dr.
DNA
from
analyzed
by
[2,32]. probe
Gail
for the c-myc The clone
Kelly and was of Massachusetts
[32]. for
Bruns
cell
lines
was blot
preanal-
guanidinum hyand analyzed
The blots either the washed,
(800
or
Southern
using the glyoxylated,
cpm/g),
108
as described catalase cDNA from
for Northern
prepared [23],
catalase
were selected with colonies isolated.
weight
pSV(X)-7
maintained
with
as
cells PA12
by Nothern blot analysis with a 32P-labeled probe
transfected
forming
cell line (PA12 CFU/ml and was
virus capable of infecting in culture. All G4l8 con-
calculated
in the presence
graphed The [9,31,35]
l0
One G4l8
cells (1 x l0) or K562 cells (5 x l0) were plated in the presence or absence of G4 1 8 in a CFU-C assay as described [26]. Colonies (>50 cells) were scored at day 14. Approximately 1 x l0 adherent KM 101 cells were infected at the same multiplicity as the HL6O and K562
obtained
PA 12 cells
as G41 8r colony [9].
by the
on NIH3T3
of pure defective virus to infect cells. PA12 pSV(X)-7 did not pro-
K562 and [19,26,33].
Vectors and
of
and examined at 100 x
metamyelocytes,
cells passaged
Human long-term bone as described [36].
Retroviral
(Sigma), immersion
oil
were
produced
determined
K562 (1 x l0 cellslml) were infected of two infectious particles of pZIP-SV(X) presence of 2 igIml of polybrene. After
media.
of 2-20
scored
replication-competent cells during 6 months
Approximately
cells
seeded at 2.5 X lO5Iml, were added 24 hours later
was
Infection of HL6O, K562, and KM1 01 Cells and Hematopoietic Cell Colony-Forming Assays
10% 5.0 UI
maintained were split
and
particles
lines
as the source hematopoietic
centrations volume.
Eagles
mM DMF). Cell counts were performed in a hemacytometer, and viability was assessed by the ability of the
stained phase
cells
HL6O
Conditions
grown
or in RPMI heat-inactivated
weekly
[22,29].
HeLa
retroviral
or ‘I’ am
(G4l8 CFU) as described pSV(X)-7) produced 6 X
produced
(Friend
METHODS
Culture
were
activity tissue
retroviruses
of human
HL6O
AND
as genes
to be
differentiation
of expression we have
in untreated to differentiate.
MATERIALS
certain
by cell
if differentiation
affects the level retroviral vector,
Cell
as well
by retroviral vectors. Transcriptional LTRs appears in some cases
specific
mRNA induced
genes
PA12
duce HeLa
expression phenotypes
[30] We chose these three cell lines to compare the levels of expression of a gene transferred by a retroviral vector to different human hematopoietic lineages. Elements in the retroviral long terminal repeat (LTR) control
of infectious
G4l8
were neor,
hybridized c-myc,
and
autoradio-
bp)
in PKY2
(Harvard
Medical
or
1 8 [4] was School).
gene was the third exon of c-myc was originally obtained from provided Medical
by Susan School).
Schiavi
(Uni-
200
B f-I)
a,
U)
C
a,
0
C
0
0
U
0 0
0
100
-
a, 0)
a, .0
C
E
0
z
a,
50
0
0
0 G418 100
1 G418
(pg/mI) 100
C
2
3
(mg/mI)
D
U)
a,
U)
a,
C
0
C
0
0
0
0
0
0
50
a,
50
0
0)
Cs
a, .0
C
E
z
0
a, 0
0
0
o
5o0 G4 18
1000
a
1500
(pg/mi)
G418
100
100
(mg/mI)
F
U)
a,
U)
C
a,
0
C
0
0
0
0 0
0
a,
50
0
50
0) Cs
a, .0
C
E
a, 0
z
a,
0
0
a G418
(pg/mI)
Fig. I . Effect of G418 concentration on colony formation of KM1O1 (A,B), HL6O (C,D), and K562 (E,F) cells either infected with pZIP-SV(X) (o-o) or not infected (a-.) The cells in A, C, and E were plated in a CFU-C assay at 1-5 x i0 cells/dish. Colonies (>50 cells) were scored at day 14 and the percentage of colonies formed at each drug concentration calculated as a
1 G418
percentage
2
3
(mg/mI)
of the colonies formed when no drug was present. Colonies that grew in 750 tg/mI of G418 were removed and expanded into cell lines. These lines (KMIO1, B; HL6O, D; K562, F) were replated in higher G418 concentrations, and colonies (>50 cells) were scored at day 14.
224
Holland
et al. >
>
xa
C/)
>
a
I
ISDISDISDI
,
OO (0(0
E
CJ
-J-..i
II-l.
23.6-
9.66.64.3-
28S2.32.0-
18S-
A
B
Fig. 2. Expression of pZIP-SV(X) in hematopoietic cells. A: Southern blot analysis of K562, HL6O, and KMIO1 DNA isolated from cells infected with pZlP-SV(X) and able to form colonies in 750 &g/ml of G418 in the CFU-C assay shown in Figure 1A,C,E. Colonies were expanded, and DNA was isolated and digested with either SAC I (S) or Dra I (D), electrophoresed on a 0.7% agarose gel, blotted, and hybridized with a 32P-labeled probe
(>108 cpm/ig) to the nect gene. B: A Northern blot analysis of total RNA isolated from control or infected HL6O cells, infected KM1O1 cells, and a line [‘I’2pSV(X)7] packaging the vector, pZIPSV(X). The RNA was treated with glyoxal and analyzed on a 1% agarose gel, blofted, and hybridized with a 32P-labeled probe (108 cpm/tg).
RESULTS
the retroviral vector N2 [2 1] The centage of G41 8 colonies formed
differences in the with the different
lines
in the
.
Infection of Human a Retroviral Vector The
retroviral
vector
three
established
fected (G4l8 were
at a multiplicity CFU) per cell. plated in a CFU-C
Hematopoietic pZIP-SV(X)
human
plated in a CFU-F cells were plated
cell
lines.
of two Infected assay,
Cell
Lines
was
used
All
lines
With
to infect were
in-
infectious viral particles HL6O and K562 cells and the KM1O1 line was
assay. In parallel, the same number of in control assays and in increasing con-
might
be due
to differences
cols, assays, and vectors the infectability of each demonstrate hematopoietic
infection
Stability of the Vector Genome Expression of the neo” mRNA
be expressed cell lines.
and
Level
To determine when transferred SV(X), individual
cells,
C or CR3-F assay in increasing concentrations (Fig. 1B,D,F). All three cell lines were resistant
,
formed
of
G4l8
the report infection
that with
ferent
protocols
nearly
100%
K562
cells,
colonies. HL6O cells the retroviral
and
These
50 cells) were observed with each line in a concentration of G41 8 that was toxic to control cells plated in similar conditions (Fig. 1 A,C,E). Approximately 10% of infected KM 101 -3%
proto-
used as well as to differences line. Nevertheless, these
that pZIP-SV(X) can as well as stromal
percell
in each
The
and
HL6O
replated
and
in a CFUof G4l8 to high
K562
than the KM 101 clonal of G41 8. The number infected
cell
line
was
clonal lines to of copdeter-
Gene Transfer presence
to Human
of G418
(Fig.
grew viable
in the presence for 20 days,
pressed
in primary
KMlOl
stromal
Hematopoietic
3).
Infected
human
human
225
stromal
(100
of G4l8 indicating
cell
Cells
pg/ml) and that pZIP-SV(X)
stromal
cells
cells
remained is cx-
as well
as in the
line.
E
Eftect mRNA
U)
a) 0 a)
of Differentiation Levels
of HL6O
Cells
on Neo”
.0
The
Cs >
Days
G418
after
effect
granulocytic formamide). contained
differentiation with DMF Each of these expanded a single copy of the retroviral
Fig. 3. Effect of G418 concentrations on the survival of adherent stromal cells from day 45 human LTBM cultures. Cells from control (s-.-..) or pZIP-SV(X) infected (00) cultures were plated in 60 mm dishes at 5 x 10 cells/dish without G418. At 5-day intervals, they were trypsinized, and the number of viable cells/mI was determined. Control adherent stromal cells were plated with 100 tg/ml (A-A) and 500 pg/mI (#{149}-U) of G418. Stromal cells from cultures infected with pZIP-SV(X) were also plated in 100 pig/mI (-t) and 500 g/ml (D-LJ) of G418.
As
in G4l8 colonies
by Southern of each cell
cuts (Fig.
blot line
analysis. High molecular was digested with Sac
in the LTRs of pZIP-SV(X). 2A) was detected when
DNA
A single 4.7 from each
DMF
predicted,
neor
age with Dra I produced single (Fig. 2A). Therefore, all three
(Fig.
metric
phages
kb band line was
showed (DMF)
bands greater than cell lines contained
gle copy of pZIP-SV(X) integrated sites. These data demonstrate that through
many
tic and
stromal
cell
divisions
at different pZIP-SV(X)
in both
human
4.7 kb a singenomic is stable
hematopoie-
cells.
The levels of neor gene transcripts in infected cells were compared to these in KM1O1 cells by em blot isolated
analysis. from one
compared to RNA
to RNA isolated
single
copy
Figure 2B representative
isolated from from infected
of pZIP-SV(X)
ilar levels of neor man hematopoietic To mal
determine cells
are
to express the man long-term pZIP-SV(X) adherent
cells
is a Northern infected
was
mRNA were and stromal whether
simlar
to the
blot HL6O
control KmlOl
HL6O cells.
present
HL6O Northof RNA cell line
cells and When a
in the cells,
sim-
detected in infected cell lines.
hu-
primary KmlOl
neor gene transferred bone marrow cultures
adherent
human
cell
in the ability
line
by pZIP-SV(X), were infected
on days 5 and 19 of culture. were tested for the ability
strohuwith
On day 45, the to grow in the
mRNA
4A,
Lanes
1 ,3).
DMF
increased the level of neor mRNA (Fig. 4A, Lanes 2,4). The level creased approximately threefold
weight I, which
probed with the neor gene. Therefore, all three cell lines contain one or more full-length copies of the pZIPSV(X) genome. In addition, the DNAs were cut with Dra I, which does not cut in the pZIP-SV(X) genome. Cleav-
levels
of the
neor
HL#{212}Ocells by expanding ten in liquid culture and inducing (N, N-dimethylHL6O cell lines vector (data not was
detected
in all
ten HL6O lines as an unspliced (4.2 kb) as well spliced (3.7 kb) messenger RNA. The levels of mRNA varied among the lines that were not treated
scanning
of the blots
cells were induced radeconoyl-phorbolmined DNA
on the
tested G4l8
shown).
Addition
of differentiation
gene was individual
(Fig.
nificantly
4B)
the
level
that
(data
not
shown).
When
with to generate
the
1 2-O-tetmacro-
of neor
mRNA
was
not
treated
with
the
inducers
changes typical (TPA) differentiation.
of
Cells
morphological or monocytoid
consistently
in each individual line of the neor mRNA inas assessed by densito-
to differentiate 1 3-acetate (TPA)
increased.
demonstrate
treatment
as a neor with
expression
of the
neo’
gene
sig-
myeloid These data transferred
by pZIP-SV(X) is regulated during differentiation of human hematopoietic cells to myeloid but not monocytoid lineages. The c-myc gene is amplified and developmentally regulated during differentiation of HL6O cells [17,28,39]. The level of c-myc RNA rapidly decreases upon induction of differentiation because of a reported block in the elongation
of
the catalase
expression
differentiation pression
c-mvc [3].
of
clones, the rehybridized
c-mvc Northern with
transcription varies
[28,35] little,
To characterize and
catalase
blots probes
the in
Conversely,
during
regulation
the
infected
HL-60 of cxHL6O
shown in Figure 4A,B were to the c-myc and catalase
genes. Consistent with previously control HL6O cells, DMF treatment cells changed the amount of catalase (Fig. 4C). Levels of c-myc mRNA approximately infected with To determine
.
if any,
reported results in of infected HL6O mRNA only slightly were depressed by
the same amount in differentiated cells the vector as in control cells (Fig. 4D). if gene transfer affected the differentia-
tion of HL6O cells, control HL6O cells and two infected HL6O cell lines were induced to differentiate and characterized by Wright’s-Giemsa staining on day 4 after differentiation.
As
shown
in Table
1 most
of the
HL6O
226
Holland
et al.
123
234
1
28S28S-
18S18S-
B
A
1234 1
28S
2
34
28S-
18S-
18S-
C
D
Fig. 4. Northern blot analysis of mRNA isolated from before and after induction of differentiation of HL6O cells. Migration of 28S and 18S rRNA is indicated. RNA from two G41& HL6O clones was analyzed. RNA from one clone was isolated before (A, lane 1) and after (A, lane 2) treatment with DMF (day 4), analyzed by Northern blot analysis and hybridized with the neor mRNA probe. RNA from a second clone before (A, lane 3) and after (A, lane 4) treatment with DMF was also analyzed. The results with these two HL6O populations are representative of ten independent clones analyzed. The Northern blot shown in B Is of RNA from uninduced HL6O cells infected with pZIP-SV(X)
(B, Lane 1) or from the same cells treated with TPA (B, Lane 2) or DMF (B, Lane 3). The Northern blot shown in A was rehybridized with a probe complementary to the catalase gene (C) and then stripped (100#{176}, 0.01 x SSC) and rehybridized with the cmyc gene (D). The neor mRNA band can still be visualized on each blot because of its abundance in the cellular mRNA. The lower band in C and D (-19S) hybridizes with the catalase and c-myc probes, respectively. RNA was isolated from untreated pZIP-SV(X)-infected HL6O cells (C, lanes 1 ,3; D, lanes 1 ,3) and DMF-treated cells (C, lanes 2,4; D, lanes 2,4).
cells (>70%) that were differentiated into mature The pZIP-SV(X)-infected
myelocytes into myelocytes and only rarely (
