J Cancer Res Clin Oncol (1985) 109:103-106 C cer esearch Clinical @ncology �9 Springer-Verlag 1985

Cytostasis of Tumor Cell Lines by Promyelocytie Leukemia Cell Line HL60 Differentiated to Granulocyte Lineage*

T. Hara 1, T. Umeda 1, T. Niijima ~, and T. Okabe 2 1 Department of Urology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan

Department of Medicine, Faculty of Medicine, University of Tokyo, Hongo %3-I, Bunkyo-ku, Tokyo 113, Japan

Summary. The human promyelocytic leukemia cell line HL60, when cultured in medium containing di- methyl sulfoxide (DMSO) or granulocyte colony- stimulating factor (G-CSF), stopped dividing and dif- ferentiated into cells with granulocyte characteristics. We found that differentiated HL60 cells have no de- tectable cytolytic activity against cultured human bladder cell line (T24 cell), as measured by release of (3H) thymidine, or against K562 cells, as measured by release of chromium-51. Differentiated HL60 cells in- hibited incorporation of (~H) thymidine into the DNA of adherent T24 cells. Decreased incorporation was not caused by detachment of the target T24 cells from the culture wells. The degree of cytostasis was depen- dent on the E/T ratio, with a 70%-80% inhibition usually reached at the E/T ratio of 200:1. A wide variety of target cells was also shown to be sensitive to differentiated HL60 cell-mediated cytostasis.

Key words: HL60 cell line - Cytostasis - T24 cell line

Introduction

A human promyelocytic leukemia cell line (HL60) has been established from the blood of a patient with acute promyelocytic leukemia (Collins et al. 1977). This cell line continues to show distinct morphological and cytochemical myeloid characteristics (Gallagher et al. 1979). In response to various biological and chemical inducers, these immature cells develop many of the morphological, biochemical, and functional properties characteristic of mature granulocytes and macrophages (Collins et al. 1978). It has been reported that 12-O-tetradecanoylphorbol- 13-acetate differenti-

* This work was supported by Research Grant No. 58570650 from the Ministry of Education, Science and Culture, Japan

Offprint requests to: T. Umeda (adress see above)

ated HL60 cells to nondividing macrophagelike cells and that these differentiated cells were cytotoxic for tumor cells in vitro (Winberg 1981). However, infor- mations is limited about cytotoxic or cytostatic activ- ity of HL60 cells differentiated into cells with granulo- cyte characteristics. Human granulocytes obtained from peripheral blood are capable of mediating tumor cytostasis in vitro (Korec et al. 1980). Such granulo- cytes may act as the effector cells of a system capable of detecting and eliminating malignant cells. In this re- port, we show that HL60 cells treated with DMSO or G-CSF are cytostatic for human malignant cell lines.

Materials and Methods

Cell Culture

HL60 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 100 IU/ml penicillin. The human G-CSF was obtained from a T3M-1 cell line originally derived from human squamous cell carcinoma of the thy- roid gland in F10 medium (Okabe et al. 1982). In this study, we cul- tured HL60 cells in RPMI 1640 medium supplemented with G-CSF containing F10 medium at a concentration of 10%, which is the minimum concentration to obtain the maximal number of granulo- cyte colonies. The F 10 culture medium of T3M-1 cells showed only G-CSF activity and was free of stimulating macrophage-colony and eosinophil colony-formation activities. The HL60 cells were seeded in plastic flasks at the concentration of 2 • 10~/ml and cultured in the presence of 1.3% DMSO or in medium containing 10% F10 medium with crude G-CSF (10% G-CSF medium). Cells were harvested at indicated times and washed free of DMSO or G-CSF. Differential counts were performed on Wright-Giemsa-stained cytospin prepara- tions of the differentiated HL60 cell suspensions.

Cytolysis Assay

Two cytolysis assays were used. Chromium labeling was done by in- cubating 10 6 K562 cells in 1 ml of RPMI 1640 medium at 37 ~ for 60 min with 100 gCi 51Cr as sodium chromate (New Eng!and Nu- clear, Boston, Mass.). The SaCr-labeled K562 cells were washed three times with the medium and then resuspended in the RPMI 1640 medium at 105 cells/ml. Nine-tenths milliliter of the medium contain- ing 2 x 106 differentiated HL60 cells was added to 0.1 ml of the K562 cell suspension. The mixture was cultured for 18 h and centrifuged.

104 T. Hara et al.: Cytostasis by Differentiated HL60

The supernatant was counted in a Nuclear Chicago gamma counter. The percentage of isotope release was calculated as (A-C/B-C) • 100, where A was radioactivity released in the test sample and B was radioactivity released from a frozen thawed sample containing 104 SlCr-labeled K562 ceils. C was the radioactivity in the supernatant of samples containing 104 K562 cells alone. Spontaneous releases were usually lower than 15%. Tests in which the spontaneous release exceeded 20% were excluded from the present study.

A glass plate with U-shaped wells (diameter 15 mm, depth 4 mm) was placed in a Petri dish (diameter 9 cm, depth 1.5 cm). A human bladder-cancer-ceU line (T24 cells) was prelabeled with (methyl-3H)-thymidine[(3H)TdR] by incubating nonconfluent cells in 75 cm 2 tissue culture flasks (Falcon) for 24 h with 20 ml of RPMI 1640 medium containing 0.5 laCi/ml of (3H) TdR. Labelled T24 cells were detached from the flask after incubation in 0.25% trypsin with 0.1% ethylenediaminetetraacetate (EDTA; Gibco) for 20 min, and the washed three times with RPMI 1640 medium containing 10% FCS, Thus prepared, 104 T24 cells were seeded into each wells and incubated for 4 h for adhesion. HL60 cells differentiated with 1.3% DMSO or 10% G-CSF medium for 7 days were used as effector cells. These effector cells were added, with effector-to-target ratios of from 200:1 to 50:1. The suspensions were incubated for 24 h in a final colume of 0.3 ml of RPMI 1640 medium containing 10% FCS. After the incubation, the glass plate was washed in phosphate-buffered sa- line (PBS) and put in cold 5% trichloroacetic acid (TCA). Finally, the suspensions were washed with 95% ethanol and dried. The dried adherent T24 cells were dissolved by adding 0.5 ml of Soluen 100 (Packard). The Soluen-100 solution was transferred to 10 ml Aqua- sol. The radioactivity was measured in a Tri-Carb liquid scintillation counter. The percentage of cytolysis was calculated according to the EDTA (Gibco) and broken down to single cells. Ten thousand T24 cells co-cultured with the effector HL60 ceils differentiated with DMSO or G-CSF, and B was the radioactivity in T24 ceils cultured in the absence of differentiated HL60 cells. All assays were carried out in duplicate.

Cytostasis Assay-Adherent Cells

HL60 cells, differentiated with 1.3% DMSO or 10% G-CSF medium for 3 or 7 days, were used as effector cells. HL60 cells cultured with- out the addition of any inducers were used as controls. The T24 cells were detached from the plastic flasks by the addition of trypsin- EDTA (Gibco) and broken down to single cells. Ten thousand T24 cells in 0.2 ml medium were seeded in each well of a glass plate and incubated for 18 h. T24 cells were aspirated free of the medium and replaced by 0.2 ml of control or treated HL60 cell suspensions with effector-to-target ratios of from 200:1 to 5& 1. After 18 h culture, the control or differentiated HL60 cells and detached T24 ceils were re- moved by dipping the glass plate in 1,000 ml of RPMI 1640 medium (pH 7.4). The T24 cells adhering to the glass wells were exposed to 0.1 ml of medium containing 1 pCi of (3H)-TdR for 3 h at 37 ~ After the labeling, the glass plates were washed by dipping in PBS and put in cold 5% TCA. Finally, they were washed in cold 95% ethanol and dried. The percentage inhibition of (3H)TdR uptake was calculated according to the formula (1-A/B) x 100, where A is the radioactivity incorporated in T24 cells in the presence of effector HL60 cells and B is the isotope uptake of T24 ceils cultured without effector HL60 cells,

Cytostasis Assay-Leukemia Cell Lines

The DMSO or G-CSF differentiated HL60 cells were cultured with 10 4 various human leukemia cells for 18 h in 1.0 ml of the RPMI 1640 medium, and labeled with 0.5 ~tCi (3H)TdR for 3 h at the end of 18 h's culture. The incubation medium was poured on Millipore filter. The filter was washed by PBS, 5% cold TCA and 95% cold ethanol, and dried. The radioactivity was measured in Aquasol 2. Si-

multaneously, the same number of DMSO or G-CSF treated HL60 cells were labeled with 0.5 I~Ci (3H)TdR. The radioactivity incor- porated in the different leukemia cell lines in the presence of effector HL60 cells was determined by subtracting the radioactivity incorpo- rated in effector cells (treated HL60 cells) alone; this was 30% of that incorporated in cultures with both leukemia cells and effector cells.

Results

When HL60 cells were cultured with either 1.3% or 10% G-CSF medium, their growth was suppressed as culture time progressed. With G-CSF, the growth was markedly suppressed until day 3 and then stopped after day 4. When the cells were treated with 1.3% DMSO, the cells ceased to proliferate after day 6. The addition of DMSO or G-CSF to the culture medium induced HL60 cells to differentiate into myelocytes, metamyelocytes, and banded or segmented neutro- phils (Table 1). The HL60 cells differentiated with DMSO or G-CSF for 7 days were stained for alpha- naphthyl butyrate esterase or naphthol chloroacetate esterase. More than 97% of these differentiated cells showed positive reactions to naphthol chloroacetate esterase staining. Less than 3% of the populations of differentiated HL60 cells were positive for a non- specific esterase staining using alpha naphthyl butyrate and negative with the addition of sodium fluoride. This finding shows that these HL60 cells differentiated into cells with morphological properties of granulocyte lineage.

Representative results of the 18-hSXCr-release as- say and the 24-h (3H) TdR release assay are summa- rized in Table 2. The HL60 cells differentiated by DMSO or by G-CSF for 7 days showed no detectable cytolytic activity against T24 cells at an effector-to- target ratio of 200:1 during incubation for up to 24 h. We were also unable to show any cytolytic activity against K562 cells.

As shown in Fig. 1 A und B, untreated HL60 cells showed no detectable cytostatic activity against T24

Table 1. Differential counts of HL60 cells after incubation with inducers

Inducer Time of culture

(Myeloid cell type, percent of total cells)

Myelobl Promyelo Myelo Meta Band Seg

None 2 96 2 0 0 0 DMSO 3days 0 82 18 0 0 0 G-CFS 4 91 5 0 0 0

None 3 89 9 0 0 0 DMSO 7days 0 6 25 23 15 31 G-CSF 3 11 25 31 21 10

Myelobl, myeloblasts; Promyelo, promyelocytes; Myelo, Myelocytes; Meta, metamyelocytes; Band, banded neutrophils; Seg, segmented neutrophils

T. Hara et al.: Cytostasis by Differentiated HL60

Table 2. Evaluation of cytolytic activity of differentiated HL60 cells

Inducer Target cells Percentage cytotoxicity at various E/T ratio

200:1 100:1 50:1

DMSO T24 - 1.8 a -4.8 - 10 G-CSF T24 -0.9 a 2.0 - 13 DMSO K562 0 b NT NT

" Cytolysis by HL60 cells differentiated at day 7 as measured by release of (SH)TdR in 24-h assay

b Percentage of SlCr release from the target by HL60 cells dif- ferentiated at day 7 in 18 h assay HL60

NT: not tested. HL60 cells were cultured in the presence of 1.3% DMSO or 10% G-CSF medium

i00 A

m

5(1 ul

o

E/T ratio 50 i00 200

u n t r e a t e d

50 i00 200

3 days

+ ?

50 100 2(10

7 days

100 B

m cq

*~ 50

,J

o

E / T r a t i o 50 1 0 0 2 0 0

u n t r e a t e d

50 i00 200

3 days

+

l 50 i00 200

7 days

Fig. 1. A Cytostatic activity during the differentiation of HL60 cells against T24 cells. HL60 cells were cultured in the presence or absence of 1.3% DMSO for the indicated culture time. The cytostatic activity was assayed as described in Materials and Methods. Verticalbars rep- resent the mean, __+ one standard deviation, of three different experi- ments. B HL60 cells were cultured in the presence or absence of 10% G-CSF medium

105

Table 3. Cytostatic activity of HL60 cells differentiated at day 7 against various human leukemia cell lines

Inducer Target cells Percentage of growth inhibition at various E/T ratios

200:1 100:1 50:1

DMSO K562 78 a 43 30 Raji 74 62 33 Molt 3B 56 44 34 Molt 4B 67 57 14 Molt 4F 53 24 20

G-CSF Raji 59 39 20 Molt 3B 32 32 11 Molt 4B 51 32 10 Molt 4F 51 38 10

a Percentage of decrease in (SH) TdR uptake in 18-h assay. Mean of two or three experiments

cells. Af t e r cu l tur ing for 3 days in the presence o f D M S O or G - G S F , cy tos ta t ic ac t iv i ty deve loped aga ins t T24 cells. The effects var ied wi th effector- to- t a rge t ra t io in a dose -dependen t manner . A f t e r 7 day ' s cul ture , the cy tos ta t ic ac t iv i ty o f the cells became m o r e p r o n o u n c e d . The D M S O or G - C S F d i f fe ren t ia ted H L 6 0 cells also showed subs tan t ia l cy tos ta t ic ac t iv i ty aga ins t a wide range o f h u m a n l eukemia cell l ines tes ted (Table 3). A n a lmos t para l le l r e la t ionsh ip was obse rved be tween the d e v e l o p m e n t o f cy tos ta t ic act iv- i ty and the extent o f d i f fe ren t ia t ion o f H L 6 0 cells. By mic roscop ic examina t ion , it was obse rved tha t the n u m b e r o f t a rge t T24 cells adher ing to the wells af ter i ncuba t ion - wi th or w i thou t the effectors - was es- sent ia l ly ident ical . This resul t con f i rmed tha t the de- crease in i n c o r p o r a t i o n was no t caused by d e t a c h m e n t o f the ta rge t cells f r om the cul ture wells du r ing the as- say. Supe rna t an t s f rom d i f fe ren t ia ted H L 6 0 cells cul- tu red for 18 h showed no cy tos ta t ic ac t iv i ty (da ta no t shown). This resul t indica tes tha t cytos tas is was no t m e d i a t e d by soluble m e d i a t o r s p r o d u c e d by the effec- tor cells and tha t the decrease o f i n c o r p o r a t i o n o f ( 3H )T dR was no t a resul t o f d i lu t ion o f the labe led subs tance by the non labe l ed thymid ine re leased by the effectors.

D i s c u s s i o n

I t has been r e p o r t e d tha t the h u m a n p romye locy t i c l eukemia cell line H L 6 0 was d i f fe ren t ia ted into m a c r o - phage l ike cells by 12 -O- t e t r adecanony l -pho rbo l - 13- ace ta te and tha t these cells expressed cy to tox ic effects on t u m o r cells - inc luding paren t , un t r ea t ed H L 6 0 cells in v i t ro (Winbe rg 1981). The a d d i t i o n o f D M S O to the cul ture induced the cells to d i f ferent ia te in to cells wi th the m o r p h o l o g i c a l charac ter i s t ics o f g ranu- locytes (Col l ins et al. 1978). They exhibi t phagocy t i c

106 T. Hara et al.: Cytostasis by Differentiated HL60

activity (Collins et al. 1978) und develop formyl pep- tide binding with the appearance of chemotaxis (Wood and Douglas t982) or enhancement of siali- dase activity (Nojiri et al. 1982).

By Wright-Giemsa staining of the cytospin prepa- ration of HL60 cell suspensions, it was shown that the HL60 cells treated with 1.3% DMSO or 10% G-CSF medium were differentiated into cells with the mor- phological characteristics of granulocytes. By staining chloroacetate esterase or nonspecific esterase, it was demonstrated that more than 97% of differentiated HL60 cells have the characteristics of granulocyte lineage and that less than 3% of these treated HL60 cells differentiated into macrophagelike cells. These findings led us to assume that the cytostatic activity expressed by these differentiated HL60 cells was at- tributable to cell populations with the morphological characteristics of granulocytes. We have also shown that HL60 cells treated with 1.3% DMSO or 10% G- CSF medium were able to induce cytolysis for a wide variety of human tumor cell lines. It has been reported that human peripheral blood granulocytes are ca- pable of inducing cytostasis for a wide variety of tu- mor cell lines, although these granulocytes do not me- diate cytolysis. The peak of cytostasis observed after 24h was delayed, compared to that induced by natural killer cells (Koree et al. 1980). These cytostatic patterns resemble those of HL60 cells differentiated into granulocyte lineages. In conclusion, the HL60 cells that differentiate into cells with morphological granulocyte characteristics have also expressed the functional characteristics of granulocytes, as exempli-

fled by the development of cytostatic activity during the process of differentiation into granulocyte line- ages.

References

Collins SJ0 Gallo RC, Gallagher RE (1977) Continuous growth and differentiation of human myeloid leukemia cells in suspension culture. Nature 270:347-349

Collins SJ, Rucetti FW, Gallagher RE, Gallo RC (1978) Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci 75:2458-2462

Gallagher RE, Collins S J, Trujillo J, McCredie K, Ahearn M, Tsai S, Metzgar R, Aulack G, Ting R, Rucetti FW, Gallo RC (1979) Characterization of the continuous differentiation myeloid cell line (HL60) from a patient with acute promyelocytic leukemia. Blood 54:713-733

Korec S, Herberman RB, Dean JH, Cannon GB (1980) Cytostasis of tumor cell lines by human granulocytes. Cell Immunol 53:104-115

Nojiri H, Takaku F, Tetsuka T, Saito M (1982) Stimulation of siali- dase activity during cell differentiation of human promyelocytic leukemia cell line HL-60. Biochem Biophys Res Commun 104:1239-1246

Okabe T, Nomura H, Ohsawa N (1982) Establishment and charac- terization of a human colony-stimulating factor-producing cell line from a squamous cell carcinoma of the thyroid gland. J Natl Cancer Inst 69:1235-I243

Winberg JB (1981) Tumor cell killing by phorbol ester-differentiated human leukemia cells. Nature 213:655-657

Wood PA, Douglas SD (1982) Development of chemotaxis and for- myl peptide binding in human promyelocytic leukemia cell line (HL60). Proc Soc Expt Biol Med 169:421426

Received July 30, 1984/Accepted October 24, 1984