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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 (