h i t . J . Cancer: 24, 87-91 (1979)

ACTINOMYCIN D RESTORES IN VIVO SENSITIVITY TO

MOUSE MYELOID LEUKEMTA CELLS DTFFERENTIATION INDUCTION OF NON-DIFFERENTIATING

Junko OKABE, Yoshio HONMA, Moriaki HAYASHI and Motoo HOZUMI Department of Chemotherapy, Saitama Cancer Centpr Research Institute, Ina-machi, Saitama-362, Japan

Resistant mouse myeloid leukemia cells could not be induced to differentiate in vitro into mature macrophages and granulocytes by incubation with ascitic fluid or dexamethasone as inducer. Neither could endogenous inducers acting on resistant cells maintained in a diffusion chamber in synge- neic SL mice induce differentiation. However, when resistant cells were pretreated in vi tro with low doses of actinomycin D they became sensitive to inducer in vitro or in vivo. The concentration of actinomycin D effective for this effect did not induce differentiation. The effect of actinomycin D was not due to inhibition of cell growth, since sensitivity was not observed when resistant cells were pretreated with the growth inhibitor 5- fluorodeoxyuridine. When resistant cells were kept in a diffusion chamber in mice injected with low doses of actinomycin D they showed significant differentiation, suggesting that the in vivo effect of actinomycin D is partly attributable to sensitiza- tion of the resistant cells to endogenous inducers.

MI cells are strain murine leukemic cells which can be induced to differentiate in vitro into forms that are functionally and morphologically similar to macrophages and granulocytes (Ichikawa, 1969 ; Hozumi et a/., 1974; Honma et al . , 19776; Sachs, 1978). Differentiated characteristics of induced cells are phagocytic and locomotive activity, Fc and C3 receptors, lysosoinal enzyme activity and morpho- logical changes.

Cells which are resistant to the inducer (ascitic fluid or dexamethasone) have been isolated (Honma et al., 1977a; Okabe et al., 19776). However, such resistant cells could be sensitized to the inducer by treatment with dimethyl sulfoxide (Okabe et al., 1977a), some inhibitors of RNA and protein syn- thesis (Okabe et al . , 19776) or some cancer chemo- therapeutic drugs (Hayashi et al . , 1979). Some of these metabolic inhibitors also sensitized a Rauscher virus-induced mouse myeloid leukemia cell line, R453 (Sugiyama et af., 1979). The most effective inhibitor tested was actinomycin D at low concen- trations which did not affect viability of the cells. The resistant cells, however, could not be induced to differentiate by actinomycin D alone.

We previously reported that conditioned medium from resistant M1 cells inhibited induction of differentiation. (Okabe et al., 1978). Moreover, the inhibitory activity in conditioned medium of actinomycin D-treated resistant cells decreased when the cells became sensitive to the inducer. These results suggest that production of the inhibi- tory activity in resistant cells is closely associated with resistance of MI cells to the inducer.

When maintained in diffusion chambers in syngen- eic SL mice, resistant cells remain undifferentiated, but sensitive cells are induced to differentiate into mature macrophages and granulocytes by endogen- ous inducers (Honma et al . , 1978; Lotem and Sachs, 1978). Since it is important, in leukemia therapy, that resistant cells should be made sensitive to endogenous inducers in vivo, we examined in this

0 5 10 0 5 10

(ngh-4 (nglml) Actinomycin D 5-fluodeaxyuridine

FIGURE I - Sensitization in vitra of R-I cells to inducer. R-1 cells ( 5 x 105/ml) were treated with actino- mycin D ( 5 and 10 ng/ml) or 5-fluorodeoxyuridine ( 5 and 10 ng/ml) for 2 days and then washed with PBS. The washed cells ( 5 x 105/ml) were incubated with the inducer (50% ascitic fluid or M dexamethasone) for 2 further days. Phagocytosis was determined as a marker of differentiation. (o), inducer-free; (a), ascitic fluid; (A), dexamethasone.

work the effect of treating resistant cells with actinomycin D in vitro on the resulting sensitivity of these cells to endogenous inducers. We also tested the effect of actinomycin D in vivo on differentiation of resistant leukemia cells.

MATERIAL AND METHODS

Animals All experiments were performed on 2- to 4-month-

old SL strain mice. The animals were maintained by

Received: February 5, 1979.

88 OKABE ET AL.

TABLE 1

MORPHOLOGICAL DIFFERENTIATION OF R-I AND R-18 CELLS TREATED WITH ACTINOMYCIN D '

Morphological changes (%) * Concentration Cell growth

Macrophages (% of control) Intermediate or granulocytes (ng/ml) M yeloblastic

Treatment

R-1 cells Untreated 0 94.7 4.3 1 .o I00 Actinomycin D 2.5 78.7 14.3 6.7 30

5.0 68.3 18.0 13.9 21

Untreated 0 89.0 9.0 2.0 100 Actinomycin D 5.0 60.7 27.0 12.7 25

Untreated 0 78.5 13.8 8.1

R-I 8 cells

S-2 cells

R-l cells ( 5 x I05/ml) were treated with actinomycin D (2.5 and 5.0 ng/ml) for 2 days, washed three times with PBS, suspended in serum- free culture medium (2 x lo5 cellslml) and put into a sterilized diffusion chamber. After 4 days' culture in the diffusion chamber in vivo, the mor- phology of the cells was examined after staining with May-Griinwald-Giemsa solution and the cell number was determined with a Burker-Turk hemocytometer by the trypan blue exclusion test. - Percentages were averages of values in 4-12 diffusion chambers.

sister x brother mating in this laboratory and checked periodically to confirm that they were still specific pathogen-free. The animals were housed in a room at 23" C with a 12 h-12 h light-dark schedule and given commercial stock diet and water ad libitum (Honma et a/., 1978).

Cell line and cell culture The M1 cell line was established from a spon-

taneous myeloid leukemia in a n SL strain mouse (Ichikawa, 1969). In the resistant clones .R-1 and R-18 phagocytic activity, lysozyme activity, loco- motive activity and formation of mature macro- phages and granulocytes could not be induced by the inducers present in ascitic fluid (Okabe et a/., 19776). The sensitive clone, S-2, could be induced to differentiate by ascitic fluid or dexamethasone.

The cells were cultured in Eagle's medium with twice the usual concentration of amino acids and vitamins (Nissui Seiyaku, Tokyo) and 10% inacti- vated (56" C for 30 min) calf serum in a humidified atmosphere of 5 % CO, in air at 37" C. Cells were washed three times with phosphate-buffered isotonic NaCl (PBS) before use. For inoculation into the diffusion chamber, the washed cells were suspended in serum-free culture medium.

Assay of phagocytosis Phagocytic activity was assayed as previously

reported (Okabe et a/., 19776). Cells were inoculated at a concentration of 5 x lo5 cells/ml and incubated with inducer (50% ascitic fluid or M dexa- methasone). After 2 days, thc cells were washed and incubated for 4 h with a suspension of polystyrene

FIGURE 2 - (a) R-l cells cultured for 4 days in an SL mouse. May-Grunwald-Giemsa ( x 1,600). (b-d) R-l cells treated with actino- mycin D (5 ng/ml) for 2 days in vitro. The cells were cultured in diffusion chambers in SL mice. May-Griinwald-Giemsa ( \* 1,600).

DIFFERENTIATION OF LEUKEMIC CELLS

TABLE 11

MORPHOLOGICAL DIFFERENTIATION OF R-l CELLS TREATED WITH 5-FLUORODEOXYURIDINE

89

Momhotoeical changes %) I

Treatment - .

Cell growth . - Concentration

Macrophages ( % of control) Myeloblastic Intermediate or granulocytes

(ngiml)

Untreated 0 96.8 2.8 0.5 100 Actinomycin D 5.0 69.0 19.0 12.0 26 5-fluorodeoxyuridine 5.0 96.8 3.8 0.4 62

10.0 96.5 3.3 0.3 26

' See footnote to Table I

latex particles (2 pl/ml of serum-free culture medium). The cells were vigorously washed three times with PBS and the percentage of phagocytic cells was calculated.

Sensitization of resistant cells by actinomycin D in vitro

Resistant cells (R-1) were treated with actinomycin D (2.5-10 ng/ml) for 2 days, washed twice with PBS and incubated with 50% ascitic fluid or M dexamethasone for 2 further days. Then the phago- cytic activity of the cells was measured as a marker of differentiation.

Diffusion chamber technique The technique of Benestad was used (Benestad,

1970). The diffusion chamber was made by fixing Millipore filters (type GS; pore size, 0.22 pm) to each side of a Millipore Plexiglass ring of 10-mm inner diameter and 2-mni thickness using Millipore M F cement. The chambers were tested for leaks and sterilized with dry heat a t 80" C for 48 h before being filled with 0.1 nil of cell suspension (2 x lo5 cells) and sealed. Two chambers were implanted into the peritoneal cavity of each mouse with no pre- treatment of the animals (Honma et a/., 1978).

Administration of actinomycin D Actinoinycin D (Sigma Chemical Co., St. Louis

Mo., USA) was dissolved in sterilized PBS and 0.2 nil of the solution was injected intraperitoneally 2 and 4 days after implantation of the diffusion chambers. The cells were cultured for 6 days in the diffusion chambers in vivo, then their morphology was examined.

Morphological examination of cells in di//usicn chambers

The chambers were shaken for 1 h at 25" C in 0.5% pronase and 5 % Ficoll in Hanks' balanced salt solution (Benestad, 1970). Cell smears were stained with May-Grunwald-Giemsa solution for examination of ccll morphology.

RESULTS

In vitro differentiation of nctinomycin- D-pretreated resistant cells

Resistant M I cells (R-I) could not be induced to differentiate into mature macrophages and granulo-

cytes by treatment with the inducers tested. However, they became sensitive to inducer when treated with a low concentration ( 5 or 10 ng/ml) of actinomycin D. They did not differentiate after treatment with actinomycin D alone (Fig. la). 5-fluorodeoxyuridine inhibited growth of the R-l cells, but did not sensitize the cells to inducer (Fig. Ib).

In vivo differentiation of actinomycin- D-pretreated resistant cells

Cells which are resistant to the inducer appear spontaneously both in vitro and in vivo. Resistant MI cells did not differentiate into mature macrophages and granulocytes in diffusion chambers in syngeneic SL mice (Honma et a/., 1978). However, the resistant cells could be sensitized to the inducer by treatment with actinomycin D in vitro. Therefore, we examined whether the actinomycin D-treated resistant cells could be induced to differentiate in vivo. For this, R-1 cells ( 5 x 105/ml) were treated in vitro with 2.5 and 5.0 ng/ml of actinomycin D for 2 days, washed three times with PBS and introduced into the diffusion chambers in a volume of 0.1 ml (2 X 10' cells/ml). After culture in viva for 4 days, the untreated R-l cells remained undifferentiated but the cells that had been treated with actinomycin D were induced to differentiate into mature macro- phages and granulocytes (Table I, Fig. 2). Similar results were obtained with another resistant MI clone, R-18 (Table I). After culture in vivo, growth of the R-I cells treated with actinomycin D was significantly inhibited (26% of the growth of control cells). Growth of cells treated in vitro with 5- fluorodeoxyuridine was also inhibited (26% of the growth of control cells) in a diffusion chamber but the cells remained undifferentiated (Table 11). Therefore, resistant cells sensitized in vitro could be induced to differentiate both in vitro and in vivo by the inducers (Fig. la, Table 1).

Induction of dij'jerentiation of resistant cells by administration of actinomycin D in vivo

As shown above, when R-l cells were treated with actinomycin D in vitro, they could be induced to differentiate by inducers both in vitro and in vivo. Therefore, we next examined the effect of systemic administration of actinomycin D on differentiation of R-l cells in diffusion chambers implanted into syngeneic SL mice. In untreated mice most of the R-1 cells in diffusion chambers were still undiffer-

90 OKABE ET AL.

entiated 6 days after implantation. However, in mice treated with low doses of actinomycin D, the cells in the diffusion chambers were induced to differentiate; in treated mice 20% of the cells were mature macrophages and granulocytes, whereas in

I I I I I

30

0 2 10 25 50 Actinomycin D (pg/kg body weight)

FIGURE 3 - Induction by actinomycin D of morpho- logical differentiation of R-1 cells after 6 days’ culture in a diffusion chamber in vivo. Mice were injected with actinomycin D 2 and 4 days after implantation of diffusion chambers. 0, Cells in an intermediate stage of differ- entiation; m, mature macrophages and granulocytes; 0 , percentage of growth of control cells.

untreated mice only 2.0% of the cells differentiated (Fig. 3). Thus treatment with actinomycin D either in vitvo or in vivo sensitizes resistant cells to inducer.

Administration of actinomycin D, at doses of 2.5, 6.25, 12.5, 25.0, 50.0, and 100.0 pg/kg body

weight had no apparent effects on the mice, doses of 200.0 and 400.0 pg/kg body weight appeared to have a toxic effect, doses of over 400.0 ,ug/kg body weight were lethal (data not shown).

DISCUSSION

Resistant MI cells, isolated from sensitive M1 cells, were much more leukemogenic than the sensitive cells (Honma et al., 1978). The sensitive MI cells could be induced to differentiate into mature cells in vitvo by certain inducers (ascitic fluid or dexaniethasone) and in vivo by endogenous inducers, but resistant MI cells could be induced to differ- entiate neither in vitvo nor in vivo. However, resistant cells which had been treated with actinomycin D could be induced to differentiate either in vi t ro (Okabe et a/ . , 19778) or in vivo (Table I). Moreover, the resistant cells could be induced to differentiate in mice injected with low doses of actinomycin D. These results indicate that the resistant cells could be induced to differentiate both in vitro and in vivo by treatment with actinomycin D. Actinomycin D seemed to have a direct effect on the resistant cells, sensitizing them to the inducers (Table I). It may also change the levels of endogenous inducers in the host, since there are reports that it affects normal hematopoiesis in mice (Zuckerman et a/., 1978; Reissman and Ito, 1966). Resistant cells treated with 5-fluorodeoxyuridine in vitvo remained undiffer- entiated both in vitro (Okabe et al., 19776) and in vivo (Table IT), but their growth was markedly inhibited. Therefore, induction of differentiation of resistant cells was not due to inhibition of cell growth.

Our finding that resistant cells become sensitive to inducers either in vitvo or in vivo after treatment with actinomycin D is of clinical significance, because leukemia cells tend spontaneously to become resistant to inducers of differentiation. Many cancer chemotherapeutic drugs currently in clinical use inhibit cell growth and kill cells by various mechanisms. It seems possible that some of these drugs may cause leukemia cells to differentiate into mature cells and, in fact, some cancer chemo- therapeutic drugs do sensitize resistant cells to inducers (Hayashi et al., 1979). Therefore, induction of normal differentiation of leukemia cells with inducers and sensitizers should be a useful new approach in the chemotherapy of leukemia.

L’ACTINOMYCINE D RESTAURE LA SENSlBlLlTE I N VIVO A L‘INDUCTION DE LA DIFFERENCIATION DES CELLULES DE LEUCEMIE MYELOIDE DE SOURIS

QUI NE SE DIFFERENCIAIENT PAS

Les celkules de leuceniie myC1oi;de murine resistantes ne peuvent Eire amenCes A se diffkrencier in vitro en macrophages et granulocytes mCirs par incubation avec du liquide ascitique ou de la dexamethasone. Les inducteurs endogenes agissant sur les cellules resistantes placees dans des chambres A diffusion chez des souris SL synghiques n’induisaient pas non plus la differenciation. Toutefois, lorsque les cellules resistantes etaient pretraitkes in vitro avec de faibles doses d’actinomycine D, elles devenaient sensibles aux inducteurs in vitro ou in vivo. La concentration d’actinomycine D nkessaire pour obtenir cet effet n’induisait pas a elle seule la differenciation. L’effet de I’actinomycine D n’etait pas dCi A une inhibition de la croissance cellulaire, puisque les cellules resistantes ne devenaient pas sensibles lorsqu’elles etaient pretraitees a la 5-fluorodesoxyuridine, qui inhibe la croissance. Lorsque les cellules resistantes etaient maintenues dans une chambre a diffusion chez les souris in- jectees avec de faibles doses d’actinomycine D, elks se differenciaient sensiblement, ce qui donne a penser que I’effet in vivo de I’actinomycine D est partiellement attribuable a une sensibilisation des cellules resistantes aux inducteurs endogenes.

DIFFERENTIATION OF LEUKEMIC CELLS

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