Leukemia Research Vol. 15, No. 5, pp. 341-350, 1991. 0145-2126/91 $3.00 + .00 Printed in Great Britain. Pergamon Press plc

INDUCTION OF GRANULOCYTIC MATURATION IN ACUTE MYELOID LEUKEMIA BY G-CSF AND RETINOIC ACID*

V. SANTINI, PH. COLOMBAT, R. DELWEL, R. VAN GURP, I. TOUW and B. LOWENBERG

Dr Daniel den Hoed Cancer Center, Rotterdam, The Netherlands

(Received 22 December 1989. Revision accepted 8 September 1990)

Abstract--AML cells were cultured free of serum with G-CSF in combination with ail-trans-retinoic acid (RA), prostaglandin E2 or 8-bromocyclic AMP to see whether the maturation blockade of these cells could be overcome. The combination G-CSF + RA was most effective in inducing morphologic maturation, i.e. in 7/10 cases. Morphological alterations in response to G-CSF + RA indicated progression of the cells along the granulocytic pathway towards metamyeiocytes and granulocytes. However, morphologically mature AML cells remained negative for myeloperoxidase and Sudan black stainings, indicators of granuiocytic maturation. Chloracetate esterase positivity and CD15 membrane antigens became expressed on cultured AML cells, i.e. on unstimulated and G-CSF/RA exposed blasts. Ingestion of latex beads and reduction of nitroblue tetrazolium salt occurred in cultured AML cells regardless of the presence of inducers. In almost all cases clonogenic cells persisted after exposure to G-CSF + RA suggesting that subpopulations of immature cells escaped the action of these inducers. Thus although G-CSF + RA were capable of inducing maturation of AML cells along the granulocytic lineage, maturation was incomplete and the effect was evident in a subfraction of the cells only.

Key words: Acute myeloid leukemia, maturation, G-CSF.

INTRODUCTION

IN VITRO cultures have shown that proliferation of acute myeloid leukemia (AML) cells is dependent on hematopoietic growth factors (HGFs) [1, 2]. HGFs activate cell cycling following binding to specific receptors present on the surface of A M L cells [3- 7]. Although AML blasts share this dependence on HGFs with normal hematopoietic precursors, they are unable to mature to granulocytes following stimu- lation with granulocytic colony stimulating factor (G- CSF) or even a complete mixture of granulocytic- macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and G-CSF supplied at optimal concentrations [7, 8]. G-CSF as a single stimulus induces terminal granulocytic maturation in normal hematopoietic progenitor cells [9-11]. Retinoids may promote granulocytic maturation in the human cell line HL-60 [12], and in hematopoietic cells from patients with myelodysplasia [13] and acute pro- myelocytic leukemia in vitro [14] or in vivo [15, 16]. However, generally retinoids do not promote granu- locytic maturation in other subtypes of primary AML

* This work was supported by the Dutch Cancer Society "Koningin Wilhelmina Fonds".

Correspondence to: Dr B. L6wenberg, Dr Daniel den Hoed Cancer Center, P.O. Box 5201,3008 AE Rotterdam, The Netherlands.

[14]. Prostaglandin E2 (PGE2) and cyclic adenosine 3'-5' monophosphate (cAMP) analogs may act syn- ergistically with retinoic acid in inducing differenti- ation in cell lines [17-19]. In addition, cAMP analogs are capable of inhibiting proliferation, and inducing maturation in certain human leukemic cell lines [20- 21]. Similar effects have been reported for PGE2 when combined to interferon-3, and vitamin D 3 [23].

In an attempt to overcome the maturation block- ade of AML cells towards granulocytes we have examined the effects of G-CSF in combination with retinoic acid, 8-Br cAMP, PGE2 in serum-free culture.

341

MATERIALS AND METHODS

Patients and cells AML was classified cytologically according to the criteria

of the French-American-British Committee (FAB) (see Table 1) [24, 25]. AML cells were obtained after informed consent from bone marrow and peripheral blood of 10 patients with AML, separated following bovine serum albumin (BSA) density gradient [26, 27] or Ficoll-Isopaque centrifugation [28] and used for experiments irrespective of their source. T iymphocytes were removed by sheep red blood cell rosetting and the AML cells were subsequently cryopreserved as described [26, 27, 29]. The cells were washed twice after thawing and depleted of adherent cells following incubation in serum-free medium [30] (1 h, 37°C,

342 V. SA~'a~I et al.

TABLE 1. PERCENTAGES OF MATURE GRANULOCYTIC CELLS IN AML CELL SUSPENSIONS BEFORE CULTURE AND FOLLOWING CULTURE WITH G-CSF, RETINOIC ACID OR G-CSF + RETINOIC ACID

Patient # 1 2 3 4 5 6 7 8 9 10 FAB* M4 M1 M5 M4 M4 M2 M2 M2 M3 M3

Prior to incubation: Metam. 0 0 0 0 0 7 9 0 0 0 Gran. 0 0 0 0 0 0 0 0 0 0

After incubation with: No supplement Metam. 9 (10) 0 0 12 8 0 16 (30) 0 (0) 4 0

Gran. 0 (9) 0 0 0 12 0 0 (I) 0 (0) 0 0 G-CSF Metam. 22 (6) 0 0 3 28 24 37 (63) 5 (14) 51 0 (10ng/ml) Gran. 6 (8) 0 0 3 19 i 6 (0) 1 (18) 18 0 Retinoic acid Metam. 44 (28) 27t 0 24 31 9 50 (31) 22 (26)t 29 10 (1 ~tM) Gran. 5 (50) 0 0 0 38 2 0 (1) 17 (13) 29 15 G-CSF (10ng/ml) + Metam. 15 (16)t 24 0 38t 42t 17t 53 (36)t 23 (20) 17t 20t Retinoic acid (1 p.M) Gran. 32 (77) 0 0 27 40 6 19 (30) 12 (47) 69 26

AML cells (4 x 105/ml) were cultured for 7 days with G-CSF, retinoic acid or G-CSF + retinoic acid and examined morphologically as described in Material and Methods.

* Indicates classification of AML according to FAB nomenclature. Metam. = denotes cytologically identifiable metamyelocytes. Gran. = denotes cytologically identifiable banded and segmented neutrophils. t Indicates the culture conditions per experiment in which maximal granulocytic maturation was obtained after 7 days

of culture. Between brackets the results are given of AML cells analysed morphologically after 14 days of culture (case 4~ 1, 7, 8).

4 x 106 cells/ml) in plastic culture flasks (250 ml; Greiner, Nurtigen, F.R.G.).

Chemicals All-trans-retinoic acid (RA), Prostaglandin E2 (PGE2)

(Sigma Chemicals, St Louis, MO) were dissolved in ethanol (100%), stored in the dark at -20°C, and diluted immedi- ately before use in HBSS. 8-Bromoadenosine 3'-5' cyclic monophosphate (8-Br cAMP; Sigma Chemicals) was dis- solved in HBSS and kept in the dark at 2--4°C.

Culture conditions Four x 105 cells per ml were seeded in suspension in

polystyrene culture tubes (Greiner, Nurtigen, F.R.G.). Cultures were run serum-free [30] with or without the addition of IL-3 10 U/ml (1 U giving 50% maximal DNA synthesis stimulation) (gift from L. Dorssers) [31], GM- CSF 25 ng/ml (Biogen, Geneva) [32], G-CSF 10 ng/ml (Immunex, Seattle, WA) [3, 10], RA 1 .uM, PGE2 10 ~M. 8-Br cAMP 100 ~M as single compounds and/or in various combinations.

AML cell maturation in culture: cytology and functional tesL~"

Prior to and after 7 and 14 days of culture, cytospins were prepared (Shandon, Cheshire, U.K.), stained and examined by light microscopy using 100 x lens and immer- sion oil. Following staining with May-Griinwald-Giemsa (MGG) at least 100 cells were counted and the percentages of blasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, monoblasts and monocytes scored. The cyto- chemical stains performed on cultured cells included mye- Ioperoxydase (MPO), Sudan black, periodic acid schiff (PAS), ac-naphtylacetate esterase (NAE) and naphtyl AS- D chloracetate esterase [33-34]. Positive cells were scored according to the numbers of granules; 0 ( - ) , less than 10 (+), between 10 and 50 ( + + ) and more than 50 ( + + + ; ) .

Further, the ability of the cells to reduce nitroblue tetra- zolium salt (NBT) [35] and ingest latex beads (Sigma, St Louis, MO) [36] were assessed as functional markers of maturation. The latter methods were applied by incubating (30 min at 37°C and in addition 15 min at room tempera- ture) 4 × 105 cells in 150 ~tl of phosphate-buffered saline (PBS) with 5% human serum albumin (HSA), and an equal volume of 0.2% NBT solution and latex beads. Cells were subsequently adhered onto glass slides, fixated in methanol (1 min) and stained with nuclear fast red or Giemsa. Cells containing more than five latex particles surrounded by a ring of blue-black formazan deposits were scored as positive.

Immunofluorescence and FA CS analysis The granulocytic differentiation surface antigen CD15

was analysed as an additional marker to assess maturation. For this purpose, cells pre- and postculture were labeled with the murine monoclonal antibody CD15 (B 4.3; reacting with sugars present on mature granulocytes; IgM) [37, 38] and then with the second step reagent goat anti- mouse Ig (GAM-FITC) (Nordic, Tilburg, The Nether- lands). Following incubation, cells were washed twice and resuspended in PBS (1 × 106/ml). Flow cytometric analysis was performed on a FACS 440 (Beckton Dickinson, Moun- tain View, CA) calibrated with 1.0 and 2.38 ~tm diameter fluorescent standard beads (Polyscience Inc.), as described [39].

AML colony formation in vitro and AML-CFU recooery from suspension cultures

AML cells were incubated in suspension for 7 days in the absence or presence of G-CSF and/or RA and the number of clonogenic cells were determined. The cells from 8 patients (# 1, 2, 3, 4, 5, 6, 7, 9) were cultured, then washed and re-seeded in methylcellulose cultures containing IL-3 10 U/ml, GM-CSF 25 ng/ml and G-CSF

Granulocytic maturation in AML 343

10 ng/ml to assess recovery of AML-CFU. AML colony formation was assessed in methylcellulose cultures exactly as described [3]. Cells were plated (1 × l05) in 3 cm dia- meter Petri dishes (Greiner) containing 1.0 ml serum-free medium [30] in 0.9% methylcellulose and cultured in fully humidified atmosphere 5% COz at 37°C. Colony numbers represent means of duplicate experiments. Colonies of more than 50 cells and clusters of more than 10 cells were scored at day 14 and the cytology of cultured cells examined in cytospin preparations stained with MGG.

STATISTICAL ANALYSIS

Student's t-test (level of conf. 95%; d.f. = 9) for paired data was applied to verify significant dif- ferences in the presence of mature granulocytic cells between G-CSF, RA and G-CSF + RA treated AML cells.

RESULTS

Morphological assessment of A M L maturation in vitro

G-CSF, RA, 8-Br cAMP, PGE2 alone and various combinations of these molecules were supplemented to the cultures of 10 specimens of primary human AML and morphological granulocytic maturation was assessed (Table 1). Occasionally, small numbers of metamyelocytes and segmented granulocytes were apparent among the precultured AML cell sus- pensions or among AML cells incubated in medium without inducers. Greater percentages of granu- iocytic ceils were recovered from cultures sup- plemented with G-CSF alone or RA alone. The combination of G-CSF + RA appeared compara- tively most active (7/10 cases) in inducing mor- phologically mature granulocytic cells. The overall proportion of mature cells found after 7-day G-CSF or RA treatment differed significantly (p < 0.05) from that of cultures carried in the presence of the combination of G-CSF + RA. The increased pro- portions of mature cells in G-CSF + RA cultures resulted from cell production and not from selective survival of metamyelocytes and granulocytic cells. In most cases the absolute cell numbers in G-CSF + RA stimulated samples had increased following culture, i.e. from 4 x 105cells/ml preculture towards 4 x l(15-10~'/ml postculture.

Cultures were also analysed following 14 days of incubation whenever cell growth was maintained. In those instances, the percentages of mature gra- nulocytes (Table 1 and Fig. 1) had further increased.

The addition of PGE2 and 8-Br cAMP alone or combined (with or without G-CSF) did not provoke morphologic maturation of AML cells in any of thc

cultures. Cells from case ~ 3 (M5b) could not be induced to mature with any of the molecules.

Cytochemical stainings and functional assays AML cells were negative for myeloperoxidase and

Sudan black after 7 days of culture in the absence of stimuli. In one case only (~ 7) myeloperoxidase and Sudan black positive cells were observed after G- CSF + RA stimulation. G-CSF or RA alone were not tested in this experiment. Similarly, NAE pos- itivity of the AML blasts was less than 10% in all cases, with the exception of cells from the M5 case (~ 3) which had been positive before and remained positive after culture.

Chloracetate esterase positive cells were discerned in control (non-stimulated) cultures i.e. about 100% positivity of the cells in 6 cases (~ 1 ,3 .4 , 5, 6, 8), and approximately 50% chloracetate esterase positivity in cases # 2 and 7 (Fig. 2). In these latter two cases the addition of RA, G-CSF or RA + G-CSF further elevated the number of chloracetatc esterase positive ceils. In the other cases addition of the latter factors did not lead to greater chioracetate esterase posi- tivity.

PAS positive cells appeared in the control cultures of only one case of A ML (~: 3). In 4 cases, however, (~ 1, 4, 6, 7) cells became PAS positivc following stimulation of the cells with G-CSF and/or RA. In those cases G-CSF + RA appeared more active than either inducer alone in stimulating PAS positivity of the cells (Fig. 2).

The efficacy of G-CSF plus RA to induce func- tional maturation in AML cells was also investigated (cases ~ 1, 3, 4, 5, 6, 7, 9). Cells capable of reducing nitroblue tetrazolium salt and ingesting latex beads appeared in non-supplemented culturcs as well as in cultures supplemented with G-CSF, RA or G- CSF + RA (Table 2).

Maturation of A M L in vitro assessed with CDI5 surface antigen analysis"

in 5 cases (# 1, 4, 5, 6, 8) CD15 surface antigens bccamc apparent on the cells following 7 days of culture either in the absence or in the presence of G- CSF and/or RA. AML cells from case ~ 9 became CD15 positive with RA (59% of the cells) and G- CSF + RA (64%) whereas in unstimulated or G-CSF induced AML samples only up to 12% of the cells were CD15 positive. Cells from case # 7 depended on G-CSF and/or RA for the appearance of CD15 positive cells (Fig. 3). Hence, the combination of G- CSF + RA appeared more effective in promoting CDI5 expression than the single stimuli, as the num- ber of CD15 positive cells as well as the mean fluor- csccncc intensity (i.e. antigen density) incrcased.

344 V. SANTINI et al.

% p o s i t i v i t y 100 r

80

60

40

20

A

abed abcd abed abd abed abcd abed abed abed 1 2 3 4 5 6 7 8 9 10

% p o s i t i v i t y 100 B

80

60

, o 1 a b e d a b c d a b e d abed abed a b e d a b c d ad

1 2 3 4 5 6 7 8 9 10

FIG. 2. Cytochemical staining of cultured AML cells. A = periodic acid Schiff; B = AS-D chloracetate esterase. AML cells were cultured without a stimulus (a), G-CSF (b), RA (c) or G-CSF + RA (d). Cytospins were prepared after 7 days of culture. The following discriminations were made: negative, <10 granules: k~, 10--50 granules: D, and >50

granules: II.

In case ~: 3 among the cohort of 10 patients, a significant proport ion of the cells had been CD15 positive before culture and in two other cases (4~ 2 and 10) CD15 antigens were neither expressed before nor after culture of the cells.

A M L - C F U recovery

To investigate whether induction of maturation

with G-CSF + RA would exhaust the A M L clon- ogenic cell population, the cells after 7 day sus- pension culture were replated in colony culture with IL-3, GM-CSF and G-CSF and the recovery of A M L clonogenic Cells was assessed. The effects of G-CSF and/or RA upon AML-CFU survival were variable among the patients (Table 3). Clonogenic cells per- sisted or increased following incubation of the cells

(a)

!

(b)

FIG. I (a, b).

FIG. 1. Morphology (May-Gri.inwald-Giemsa staining) of AML cells obtained from case # 7 after incubation (14 days) with IL-3 + GM-CSF (a); G-CSF (b). RA (c) and

G-CSF + RA (d).

345

, ~ ~. ," , : ~ ~.

(c) :;!'

L

i . ° •

3 4 6

Granulocytic maturation in AML 347

TABLE 2. PERCENTAGE OF A M L CELLS POSITIVE FOR NBT REDUCTION/LATEX BEADS INGESTION ASSAY (DAY 7)

Patient ~: 1 3 4 5 6 7 9

After incubation with: No supplement 67 41 35 64 30 4 9 G-CSF(10ng /ml ) 44 44 33 44 49 20 14 Re t ino i cac id ( lp tM) 35 0 56 39 39 8 24 G-CSF(10ng /ml ) 42 0 35 65 50 26 23 Retinoic acid (1 ~tM)

AML cells were scored as positive when containing more than 5 latex particles surrounded by a blue formazan ring.

of most cases with G - C S F a n d / o r R A . G r e a t e r num- bers of A M L c lonogen ic cells were r e c o v e r e d f rom cul tures s u p p l e m e n t e d with G - C S F + R A than f rom con t ro l cu l tu res in cases ~: 4 and 6. In cases ~ 3, 5 and 9 the n u m b e r s of A M L - C F U r e c o v e r e d f rom G- C S F + R A s t imu la t ed suspens ion cu l tu res had dec rea sed as c o m p a r e d to the n o n - s t i m u l a t e d A M L cul tures .

D I S C U S S I O N

H u m a n A M L blasts genera l ly express a n o r m a l

E =:

n-

I j t s

, i ; q

_a, °

lOO

no factors

200

f=l d ,o ,

i

e

loo

G-CSF

2~0

retlnolc arid G-CSF *

/: f ~ i |

10o 2()0 10o Log fluorescence int~slty

FIG. 3. CD]5 antigen expression of AML cells after 14- day culture. Distribution of AML blast cells as a function of the fluorescence intensity following CD15 labeling (unbroken line); GAM-FITC labeled control cells are indi- cated by the dotted line. A = cells of patient ~ 7 recovered from non-stimulated culture; B = cells cultured with G- CSF; C = cells cultured with RA; D = cells cultured with

G-CSF + RA.

tetlnolc acid

200

TABLE 3. RECOVERY OF AML-CFU AFTER 7-DAY CULTURE IN THE PRESENCE OF G-CSF AND/OR RETINOIC ACID

Patient *~ 1" 2 3* 4 5* 6* 7 9*

Without pre-incubation: 14 0 28 4 751 280 () 0 After pre-incubation with: No supplement 310 0 726 66 965 57 >500 158 G-CSF (10 ng/ml) 226 0 168 99 769 35 >500 88 Retinoic acid (1 ~tM) 355 0 5 597 353 89 >500 12 G-CSF (10 ng/ml) + 370 0 5 >1000 255 188 >500 46 Retinoic acid (1 IxM)

* Colonies (more than 50 cells). * 4 and 7 clusters (more than 10 cells). AML colony formation was determined in methylcellulose cultures containing IL-3, GM-CSF and

G-CSF.

348 V. SANTINI et al.

susceptibility to G-CSF. This is evident from effective stimulation of AML colony formation and activation of DNA synthesis of AML blasts in vitro by G-CSF [3] and also from G-CSF receptor analysis indicating physiological numbers and dissociation constants of G-CSF receptors on the cell surface of AML blasts [6]. Thus, although the ligand binds to a functional receptor, G-CSF is unable to mediate the response that leads to granulocytic maturation of the ceils [7].

Morphological evaluation revealed in vitro matu- ration of AML blasts in particular following stimu- lation with the combination of G-CSF + RA. Our results suggest that the combination G-CSF + RA is more active than RA or G-CSF alone in inducing morphologic granulocytic maturation. According to a comprehensive set of criteria maturation was usually incomplete, i.e. (1) AML cells remained myelo- peroxidase, Sudan black and cr-naphtyl esterase negative after culture, (2) the cultured cells often became chloracetate esterase positive in non-sup- plemented and in cultures with G-CSF + RA, (3) AML cells with the capacity to ingest latex beads and reduce NBT were frequently observed in cultures of non-stimulated and G-CSF + RA activated cells, (4) CDI5 surface markers were frequently expressed on AML cells irrespective whether the cells had been cultured with or without stimuli, (5) in 4 of 10 cases G-CSF + RA stimulated cells became PAS positive. These results are in agreement with earlier studies showing that certain molecules, e.g. RA, DMSO or dihydroxy vitamin D3 may infrequently induce morphologic granulocytic maturation of human AML blasts [4(~-44]. However, in most of these studies morphologically differentiated cells remained negative for myeloperoxidase, Sudan black, NAE or chloracetate esterase as well. Likewise, morphologic granulocytic maturation was not accompanied by acquisition of functional properties such as latex beads ingestion and NBT reduction. From these results one might conclude that A M L blast cells are intrinsically unable to mature to functional end cells. It is also possible that the proper stimuli were not provided and that the cells require other stimuli in addition to G-CSF + RA to accomplish complete maturation.

The mechanism by which retinoic acid as a co- stimulus of G-CSF induces more mature granulocytic cells from AML blast cells in vitro is unclear. The inability of AML cells to mature in response to G- CSF alone may be attributed to second messenger abnormalities. In this study we analysed the role of cAMP as second messenger, considering the possi- bility that it might be involved in granulocytic matu- ration. However, cAMP analogs could not abrogate

the block of maturation in the 10 specimens of clinical AML. Hence, it is rather unlikely that a failure in cAMP elevation upon G-CSF stimulation is respon- sible for the maturation arrest. At the present time it remains uncertain whether specific pathway dys- functions exist in acute myeloid leukemia blasts, thereby determining inappropriate maturation.

Acknowledgement--The excellent technical assistance of Joke Krefft and Carin van Buitenen is deeply acknowl- edged and we thank Mariska Drinkwaard for typing the manuscript.

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