cell line Establishment and characterization of a new ...

From bloodjournal.hematologylibrary.org by guest on June 11, 2013. For personal use only.

1985 66: 1233-1240

Establishment and characterization of a new human eosinophilic leukemia cell line H Saito, A Bourinbaiar, M Ginsburg, K Minato, E Ceresi, K Yamada, D Machover, J Breard and G Mathe

Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml

Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.


Establishment

and

Characterization

of a New

Leukemia By Hiroshi

Aldar

Saito,

Bourinbaiar, David

Cell

Mich#{234}leGinsburg,

Machover,

Jacqueline

A human eosinophilic leukemia cell line, designated as EoL. was established from the peripheral blood of a patient with Philadelphia chromosome-negative eosinophilic leukemia (EL). The EoL cell line grows in single cell suspension with a doubling

time

of 48

hours

for

about

one

year.

The

reactivity

of these cells was tested with a panel of monoclonal antibodies; they were found to express surface Ia antigen, myeloid antigen (IF1 0, MY9) and membrane receptors for interleukin 2 (IL-2, Tac antigen). Under standard culture conditions, a small percentage of cells having more typical eosinophilic characteristics was present. These cells had cytoplasmic granules and were positive for Luxol-fast-blue

E

OSINOPHILIC 1912

disease present

leukemia

by Stillman.’

Since

(EL)

was

then,

diagnosis

remains a problem, since large numbers of immature

putative not yet lines

characteristic been clearly

have

been

features defined.25

established

granulocytic,7’#{176}

and

first

described

blasts leukemic

patients with leukemias,”2

monocytic

rare

most cases of EL do not eosinophilic cells, and

of eosinophilic Several human

from

in

of this

Keisuke

Minato,

lymphocytic,6 and they have

Enzo

Br#{233}ard, and Georges

Eosinophilic

Ceresi,

Kazuyoshi

Yamada,

Math#{233}

and eosinophil peroxidase. Under culture conditions to induce the maturation of myeloid cells, such as alkaline medium or addition of dimethyl sulfoxide (DMSO). the frequency of cells with typical eosinophilic features increased to about 40%. In addition, cytogenetic studies showed that cultured cells and original leukemic blasts presented similar chromosome abnormalities. EoL seems to be a unique leukemic line committed to the eosinophilic lineage and can provide a useful in vitro model for the study of malignant eosinophilic properties. S 1985 by Grune & Stratton. Inc.

Cell culture and cloning. On Feb 24, 1984, the proportion of WBCs rose to 16.9 x 104/,.L, 43% eosinophils, and 54% blasts. After obtainment of the patient’s informed consent, according to institutional

have cell

Human

Line

guidelines,

30 mL

of peripheral

blood

the blast-rich fraction was purified gradient separation (50%-60%--68%

by Percoll Percoll

from

and

the

48-well

top

layer

tissue

1640

were

culture

(GIBCO,

collected

plates

Grand

(Costar,

Island,

was

cultured

at

Cambridge,

NY)

obtained,

and

discontinuous density solution). The blasts 1 x

NY)

supplemented

106/mL

using

with

in

RPMI

10%

fetal

proven to be very useful in in vitro models for the study of leukemic cell properties. To date, however, no eosinophilic cell line has been reported. Recently we were able to estab-

calf serum (FCS), 2 mmol/L of glutamine, I mmol/L of pyruvate and 1% nonessential aminoacids (Seromed, Mulhouse, France). The cultures were incubated at 37 #{176}C in a humidified atmosphere of 5%

lish a continuous

CO2 and

and characterization

culture

line ofeosinophilic

we describe of this

patient,

cells

from

an EL

in this report the establishment unique cell line, EoL.

ability

and

MATERIALS

the

D.T.,

report.

Paul-Brousse

Hospital,

of hyperleukocytosis ciency.

The

increasing

upper

history

His

that

past he

and

an

examination

In I 979,

fibrosis

was

was

out.

with

immunology,

and

(WBC:14.8

also

x

showed

hematopoietic

allergic

negative. syndrome

edema.

replacement

1984 and laboratory findings were as follows: g/dL, hematocrit 41%, RBC count 402 x l04/zL, 16.6 x 104/,.L with 50% blasts, 12% myelocytic

hemoglobin and WBC eosinophils,

12.8 count I 1%

neutrophils,

1%

basophils,

and

marked

increase

phils,

10%

elocytic

metamyelocytic

consisting obtained, lation

cerebral

Blood,

4%

and

of

steroids the

(prothrombin

and

patient time

33%

treated

blasts.

77 sec/

An

No

of disseminated

4%

EL

promy-

diagnosis

remission

1 3 5cc, fibrinogen

was

chemotherapy could

intravascular

hemorrhage.

Vol 66. No

a

eosino-

2% metamyelocytic

by combination

vincristine.

died

showed

2% eosinophils, neutrophils,

and

was

examination

8% promyelocytic

eosinophils,

neutrophils,

5%

marrow

precursors,

I % myelocytic

the patient and

Bone

in eosinophil

neutrophils,

neutrophils,

made,

3% lymphocytes.

eosinophils,

50

leukemic

EoL-1

granules

Leukem

Chromosome

No. of Cells Analyzed

of Cells

Name

is

ET AL

SAITO

to

In

did

basophils,

not

9

(2%) which

stain

the

I cells. cell

to induce alkaline

for seven

identify

1

7

differentiation. the

Several

maturation

medium

to nine days

culture

of myeloid

buffered

with

(pH 7.8 to 8.0),

condicells

were

7

2

1

6

2

5

18

6

7

29

1

31

22

2

1

immature

and

mature

(Fig

2E).

The

differentiated

with large

peroxidase spherical

phils.

eosinophils

EoL-l

cells

cultured

did

not

number

of

cells

increased

HEPPS

or

increased

in a similar

of

noic

(0.2

(Fig to those

with

differentiate

the number

2

cells showed

and Luxol-fast-blue granules similar

to 1.2%)

acid

1

DMSO into

positive

for

fashion

to 1.0 jzmol/L)

to about

a strong

40%

reaction

2F), and contained of normal eosinofor nine

neutrophils,

days

(0.8%

whereas

Luxol-fast-blue

the

staining

as in alkaline

medium.

Reti-

had

on EoL-1

cells.

no effect

I! 1

2

3

m,

u 6

t

5

I,’

U 8

9

t2

X

7

VI

4

‘a

I

, 10

11

ft 13

14

15

16

17

18

1

U 19 ..

21

20

‘.a’

-I-

22

V

‘2 ‘J

Fig 3. yotype

+6.

Giemsa-banded of

+8.

EoL-1

and 9q-.

cells,

stained showing

kar48

XV.


HUMAN EOSINOPHILIC LEUKEMIA CELL LINE These

results

seem

to

are

analysis

cells

were

MY9), same

summarized positive for

and surface

IL-2

that

indicate

committed to eosinophilic Cell surface markers.

The

results

in Table Ia antigen,

T4.

The

of surface

EoL-l

of the original

cells

EoL-1

did

is already

the

marker

not

cells

46

EBNA

and

EBNA

TdT.

No

staining

the same patient was EoL-1 cells by indirect Cytogenetic

EBNA

of Eo-B

cells

was

cells.

analysis.

+6, +8, 9q-

(Fig

3). Fifty-three

cellular

cells.

A few

mality

was

population

cells

were

anti-T

have

any

phagocytic

additional

in the

present somes

and representing of original cells

modal

number

detected lines)

was

from

detected

of As

EoL

derived

EoL-l,

in the

percent

of EoL-2

analyzed

8 in all

together

had

mitochondria,

1

other rough

6

II 10

7

$1

original

GTG

of

the

and abnormal

banding.

Abnor-

of

9q-

and

extra

leukemic

cells

(Fig

4),

with

other

5, 9,

1 2, and

I 3 sometimes

being

various subclones. The chromoranged from 43 to 50 per cell, with a

of cored general ER,

Electron microscopy had round or deep folded present, it was small and

vesicles

and

cytoplasmic and

Golgi

a few

components apparatus

were

vacuoles, such seen

as (Fig

II

2

Is

18

of 48.

A number with

normal

for

the

on

presence

chromosomes

compact.

3, 48

of EoL-3

heterogeneity

containing

by

I’

It

the

Electron microscopy findings. studies revealed that EoL-l cells nuclei. When the nucleolus was

EoL-2,

shown in Table male karyotype

performed

revealed

expressed

chromosome

+ 12, + 13; 62%

9q-,

analysis cells

the

Chromosomes

xY,

blood

with

positive. No TdT immunofluorescence.

EoL-3 cell lines were analyzed. of EoL-l cells had a hyperdiploid

Cytogenetic

membrane cell

+8,

49 XY, +4, +8, 9q-, + 13 (data not cells showed mainly a normal karyotype

to express

(B cell

and 61%

XY.

original

activity.

lines.

49 XY,

karyotype The Eo-B

peripheral

had the

leukemic

karyotype

had the shown).

2. The original leukemic myeloid antigen (IF1O,

(anti-Tac).

as those

line

pathways.

A fraction of the cells was also found Fc ‘y receptors and to react weakly antibody

cell

differentiation

receptors

markers

the

1237

3

4

Iu

5

UI’ \

8

U

11

12

13

14

15

16

17

18

1$-

I.

19

20

9

-1X

_O_,

Fig 4. Giemsa-banded karyotype of fresh original philic leukemia cells. showing +8.

+9.

and 9q-.

stained eosino48 XV.

-4

B-

-4-

21

22

V

4


SAITO

1238

5A).

No viral

structure

cells

induced

by DMSO

was

asynchrony

characterized

and

mature

a more

spherical, normal lacked

(Fig

differentiated immature

5B).

mic

and

were

often

mem-

to granules

The granules, (Fig 5C).

of

however,

derived

here

from

the

suffering nating

in

surface

markers,

of the

EoL

C

cells

stained

for

eosinophils,

with

toluidine

stained

peripheral

of

man

for

and

EoL cells originated

cytogenetic

analysis

from the patient’s had

a blastic

appearance

demonstrated

leukemic without

blasts.

we

with

IL-2 cells

that

positive specific results

receptors. had

a battery

of

cell types.

The

some

myeloid

In addition, anti-T4

antibodies are consistent

not

which

metachromatic

is

when

of the EoL cells were studied. As marker for mature or immature

Fc ‘y receptors, with

Luxol-fast-blue,

were

with

granules.

blue.

used and

with

but

various

Ia antigen

Most

cytoplas-

stained

granules

reacting

case termistaining,

when

specific

were

to myelo-

(2%) of the cells,

These

eosinophils, a 33-year-old

similar

percentage which,

size and color of eosinophilic

EoL,

hypereosinophilia Cytochemical

morphologically

A small granules,

had the typical

of a EL cell line,

blood

were

MGG,

the establishment

from a six-year-long blastic transformation.

and

The surface markers there is yet no specific

DISCUSSION

We report

granules

blasts or monoblasts. however, contained

nucleus

The granules

in appearance

precursors. crystalloids

EoL-1

nuclear-cytoplasmic

electron-dense

and were similar

eosinophilic characteristic

The

showed

by a relatively

cytoplasm

homogenously

brane-bound,

found.

( 1 .0%)

ET AL

a small and

antibody. were with

some

monoclonal

EoL cells antigens,

and

positive

expressed

fraction (I 1%) of the ( I 6%) were weakly

cells

All other

negative. the notion

antibodies

were

T, B, or monocyte-

Taken together, these that EoL cells belong to

\

Fig 5. cultured

(A) Ultrastructural appearance of EoL-1 cells under usual condition (original magnification x4.800; current magnification x3.120). (B) Ultrastructural appearance of EoL-1 cells cultured in dimethyl sulfoxide (1 .0%) for seven days (original magnification x 11.000; current magnification x 7.1 50). (C) High magnification of granules in EoL-1 cells (original magnification x25.000; current magnification x 16.250).


EOSINOPHILIC

HUMAN

the

granulocytic

EoL

cells

LEUKEMIA

lineage.

The

is interesting.

addition

to activated

hairy cell cell lines,

presence

Recent

of IL-2

studies

T cells,

leukemia, and on

1239

LINE

CELL

these

the

presence

abnormal AMMoL

were

found

in

cases

of EL that

B

such

as the

data

indicated

on

virus-transformed therefore, the

receptors are not specific of the T cell lineage thought.2224 We are not aware, however, demonstrating

in

shown

receptors

some Epstein-Barr activated B cells;

that,

receptors

have

IL-2

IL-2

receptors

on

myeloid

of the

EoL

cell

showed

cells.

Cytogenetic abnormal

analysis karyotype,

also found chromosome

and

to have an 8 and 9q-

.

the

original

lines

leukemic

abnormal In contrast,

an

cells

karyotype of the karyotype

an extra of the B

were

to 8.0)

increased

features

We

gave

established

the

of the

a leukemic tried

to induce

40%.

frequency

of cells

granules,

Luxol-fast-blue

The

addition

comparable

have

abnormalities

any

line

the

differin 7.0

staining) in the culture

In contrast,

It is interesting

culture (pH

of

eosinophilic

of DMSO

results.

effect.

with

above

cell

cells by various techniques. The buffered with HEPPS or HEPES

2% to about not

showed

of I 6.2533 All

therefore

(cytoplasmic

medium

reported

we had

nature. of these medium

did

already

or deletion

that

entiation alkaline

from

were

lacking crystalloid. The eosinophils of to be part of the leukemic process, but no

inversion

eosinophilic

as previously of any report

of

granules appeared

retinoic

acid

to compare

these

cell line obtained from the same blood sample was normal. We suggested that, during the evolution of leukemic cells, dominant clones in EoL-l (48 XY, +6, +8, 9q- , EoL-2 (49

results

xY,

the

DM50.’3 The uniform maturation of EoL-l cells in eosinophils under various culture conditions is thus in favor of a commitment of these blasts to the eosinophilic lineage. Such features have not been described in any previously reported myeloid leukemia cell lines. In this respect, EoL lines seem to

dif-

have

9q

+8,

9q-,

, + 1 3) might

-

tageously 9q

EL

extra

and

EoL-3

be derived

from

more

original

had

the

a variety

XY,

+8,

or advan-

while

keeping

of the

cases

of cytogenetic

reported

abnormalities,

chromosome.25

abnormalities

+4,

expansive

Several

Philadelphia

chromosomal

(49

subclones,

8 chromosome.

have

including ferent

+13),

modified

and

-

as

+12,

Among

described

the

in EL,

on other

sively

found

acute

nonlymphocytic

that

in

patients

MoL)

EL.

9q

anomaly

-

myeloproliferative

with

acute

is almost

disorders,

leukemias.3#{176} It

and abundant

sion

The

myelomonocytic

bone

marrow

of 16 chromosome.3t’32

The

shown

eosinophils

unique

into

neutrophils

under

influence

of

characteristics.

disease,

and therefore

few fresh

blasts can be obtained. These cell lines in vitro model for the study of malignant ties and

the

eosinophilic

differentiation

eosinophilic

could thus be a useful eosinophilic proper-

pathways.

excluin

ACKNOWLEDGMENT

recently

leukemia

eosinophils

and

EL is a rare

especially

was

medium

trisomy

of group C chromosomes is a relatively frequent finding,229 but interstitial deletions of the long arm of chromosome #9 (9q - ), which were found on EoL cell lines, have not been reported

with those with the promyelocytic leukemia cell line, which differentiates into eosinophils in alkaline

HL-60,

The authors

(AM-

Elisabeth

had an inverof AMMoL

had

Daniel helpful

gratefully

appreciate

Couv#{233}. The authors and Professor comments.

the editorial

also gratefully

G. Flandrin

(Hbpital

assistance

of Mrs

acknowledge

Dr MT.

Saint-Louis)

for their

REFERENCES I . Stillman

RG:

ofeosinophile

A case

cells.

Med

of myeloid Rec

2. Benvenisti DS, Ultman iE: five cases and review ofliterature. 3.

Antenius

Acute

A, Audebert

eosinophilic

leukemia

81:594,

human

predominance

leukemia.

Report

of

Ann Intern

Med 71:731, 1969 M, Debray i, Robineaux

Krulic

Virchows

Arch

B Cell

Pathol

4. Stavem

P, Ly B, Blichfeld

Scand

leukemia.

i Haematol

21:355,

5. NIH Conference: Clinical,

R:

the

The idiopathic and

Intern Med 97:78, 1982 6. Minowada i, Tatsumi K, Zgoda

of

study,

in

Schlossman

Bernard

leukemias.”

L,

A,

Boumsell

Ann

7.

Lozzio line

CB, with

Reber Ti: Eosinophilic

of

leukemia

L,

on the marker profiles of The results of workDausset

i,

Berlin,

Springer

Milstein

differentiation

K, Sonobe

of a new

Blood

63:254,

H,

Ohtsuki

Y,

peroxidase-positive

1984

C,

Verlag,

and characterization

of a human

differentiation

acute

26:171, 1980 JR. Misawa 5,

of the human

promyelocytic

eosinophil

colony

by Luxol-fast-blue

staining.

Exp

Hematol

8:549,

I980 Lozzio positive

Si,

Science

cell line, HL-60. i Exp Med 160:179, 1984 14. Aglietta M, Camussi G, Piacibello W: Detection of basophils growing in semisolid agar culture. Exp Hematol 9:95, 1981 I 5. iohnson GR, Metcalf D: Detection of a new type of mouse

BB:

Human

chronic

Philadelphia

myelogenous

chromosome.

leukemia Blood

16.

45:321,

Gallo of human

RC, Gallagher myeloid

ture. Nature 270:347, 1977 9. Koeffier HP, Golde DW:

RE: Continuous

leukemic

cells

growth

in suspension

Yamada

Myeloid IF1O.

Collins

P.21.

K: Establishment

1,

Typing.

activity.

monocytic leukemia cell line (THP-1). Int i Cancer I 3. Fischkoff SA, Pollak A, Gleich Gi, Testa

A scheme

197S 8.

line,

Sugimoyo

1984, pp 519-527 cell

cell

L, Gould

D:

K, Niiya

I: Establishment

K, Lok MS.

Kover

Leukocyte

I, Machida

myeloid

T, Tada

syndrome:

considerations.

E, Sagawa

L, Prestine

SF (eds):

eosinophilo-

hypereosinophilic

therapeutic

human hematopoietic differentiation based the cultured and fresh leukemia-lymphomas: shop

P: Acute

“in-between

Kubonishi

K, Miyoshi

human

to colony-stimulating

1978

I I . Ben Bassat H, Korkesh A, Voss R, Leizerowitz R, Polliack A: Establishment and characterization of a new permanent cell line (GDM-l) from a patient with myelomonoblastic leukemia. Leuk Res 6:743, 1982 I 2. Tsuchiya 5, Yamabe M, Tamaguchi Y, Kobayashi Y, Konno

27:237,

1978

pathophysiologic

Minato

P. Andreassen One

10.

Iwata

I978 myelomonocytic

cell line responsive

200:1153,

Eosinophilic

AA,

leukemia.

with

1912

and cul-

Am

17. mia

and

i Hematol

Foon

myelogenous

leukemia.

A

Okabe 15:181,

cells.

Y, Uchiyama

i: Characterization

N, antigen

KA, Schroff

lymphoma

18. Wano Acute

K,

differentiation

RW, Recent

Saito

H,

defined

Suzuki

R,

Kumagai

by a monoclonal

K:

antibody

1983

Gale

RP: Surface

advances.

T, Fukui

K, Maeda

of human interleukin

markers

Blood

60:1,

on leuke1982

M, Uchino

2 receptor

H, Yodoi

(Tac antigen)

in


1240

SAITO

normal and leukemic receptors on HUT-102 19.

A

Griffin

iD,

monoclonal

myeloid 20.

T cells: Co-expression of normal and aberrant cells. J Immunol 132:3005, 1984

Linch

D, Sabbath

antibody

progenitor

reactive

cells.

Reedman

BM,

Leuk

Klein

K, Larcom with

normal

Res 8:521,

P, Schlossman and

SF:

leukemic

human

1984

G: Cellular

of an

Epstein-

antigen in producer Int J Cancer 1 1:499,

I973 21

.

Humains.

Dutrillaux B: Sur Ia Nature Monographie des Annales

Scientifique 22.

Francaise,

LM, Marshall

Si, Jensen

iP, Bakhshi

TA: Rearrangement expression

mia.

Proc

23.

Robb

Immunol 24.

Natl

Ri:

Today Tsudo

Leukemia.

Sci

USA

5:203,

normal

Sandberg New

York,

The

Depper

antigen

JM,

of immu-

in hairy

cell

leuke-

The

F, Beckman

G, Laurell

H, Nordenson

I:

of the eosinophilic bone marrow pool in and eosinophilic leukemia. Acta Med Scand

1977

Huang

CS,

Chromosomes

Gomez

and

GA,

Kohno

causation

5, Sokal

of human

iE,

Sandberg

cancer

and

AA:

leukemia.

XXXIV, a case of “hypereosinophilic syndromes” with unusual cytogenetic findings in a chloroma, terminating in blastic transformation and CNS leukemia. Cancer 44:1284, 1979 29.

Goldman

chromosome

JM,

analysis

Najfeld

V,

Th’ng

ofeosinophilic

KH:

leukemia.

Agar

culture

i Clin Pathol

and

28:956,

30.

Mecucci

12:309,

molecule

and

its

function.

B cells.

H: Expression i Exp

Chromosomes

Elsevier,

1981,

Med in

160:612, Human

ofTac

antigen

Cancer

and

pp 227-230

26. Weinfeld A, Westin i, Swolin B: Ph’-negative eosinophilic leukemia with trisomy 8. Case report and review of cytogenetic studies. Scand i Haematol 18:413, 1977

DC, Bloomfield

of chromosome 32.

Griffin

1984

K, Kulling

G, Michaux

iL,

Noens

L,

1984

phocytic T, Uchino

C, Vermaelen

Hove WV, Tricot G, Louwagie A, Berghe HVD: Interstitial 9qdeletions in hematologic malignancies. Cancer Genet Cytogenet 3 I . Arthur

1983

1984

human AA:

2:

WJ,

and expression

of Tac 80:4522,

Interleukin

M, Uchiyama

on activated 25.

Acad

i, Hsu SM, Neckers

A, Leonard

noglobulin

and

L, Mitelman

composition eosinophilia

197S

WC, Cossman

Jaffe ES, Waldmann genes

des Chromosomes Paris, Expansion

1975

Si, Greene

Korsemeyer

et l’Origine de Genetique.

Brandt

201:177, 28.

localization

Barr virus (EBV)-associated complement-fixing and non-producer lymphoblastoid cell lines.

27.

Different reactive

ET AL

16 and

leukemia:

A new

Tantravahi with

CD: Partial marrow M,

Blood Henkle

Hi: A pericentric

dysplastic

marrow

deletion

of the long arm

eosinophilia

association.

R, Schwenn

iD, Weinstein

is associated

bone

in acute

61 :994, C, Nell

inversion eosinophils

nonlym-

1983 M,

Leavitt

of chromosome in acute

ocytic leukemia. Blood 63:800, 1984 33. Rowley iD: Biological implications ofconsistent rearrangements in leukemia and lymphoma. Cancer I 984

PR, I6

myelomon-

chromosome Res 44:3 159,