Differentiation of isotope-labeled myeloid leukemia cells free in the peritoneal cavity of syngeneic mice

LeuKemia ResearLhVol 6. No 1, pp 117 122, 198-.2 Primed m Great Brilam

0145-2126/S2/010117-0510300/0 © 1982 Pergamon PressLid

BRIEF COMMUNICATION DIFFERENTIATION MYELOID

LEUKEMIA

PERITONEAL

CAVITY

OF

ISOTOPE-LABELED CELLS

OF

FREE

IN THE

SYNGENEIC

MICE

YOSHIO HONMA. MORIAKI HAYASHI, TAKASHI KASUKABE

and MOTOOHozuMI Department of Chemotherapy, Saitama Cancer Center Research Institute, lna-machL Kitaadachi-gun, Saitama-Ken 362, Japan {Received 2 July 1981. Revised 8 September 1981. Accepted 28 September 1981t A ~ t r a c t - - M l cells were labeled in vitro with [3H]thymidine and injected into the peritoneal cavity of syngeneic mice. After several days the peritoneal cells were harvested and [3H]labeled cells were determined by autoradiography. The inoculated isotope-labeled MI cells differentiated in the peritoneal cavity and lipopolysaccharide, an inducer of cell differentiation, significantly stimulated differentiation. These results provide direct evidence that M I cells can be induced to differentiate in vivo under conditions in which leukemia can develop. Key words: Myeloid leukemia cells, differentiation in vivo, lipopolysaccharide, autoradiograph.

INTRODUCTION TREATMENTwith various inducers in vivo causes differentiation of several types of human and mouse myeloid leukemia cells into forms that are functionally and morphologically similar to macrophages and granulocytes [1, 6-9. 12]. Mouse myeloid leukemia MI cells were leukemogenic to syngeneic SL mice, but they could be induced by various inducers to differentiate in vitro into mature granulocytes and macrophages and lose their leukemogenicity in syngenic mice. In a diffusion chamber in syngeneic SL mice, myeloblastic leukemia M1 cells were induced to differentiate into mature granulocytes and macrophages and their proliferation rate decreased [4--6, 10, 11, 13]. Resistant M1 cells, which could not be induced to differentiate into mature cells, were much more leukemogenic than sensitive Ml cells, which could be induced to undergo cell differentiation [4]. These findings, which suggest that leukemogenicity of MI cells in syngeneic mice is related to in citro and in vivo inducibility of differentiation of the cells, may have significant implications for leukemia therapy. Although the diffusion chamber technique is useful for studies on differentiation in cit,o, this method prevents cell-to-cell interaction of inoculated leukemia cells with host immunocompetent cells. In studying the relation between leukemogenicity and in vivo inducibility of differentiation of the cells, it is important to clarify whether leukemia cells are induced to differentiate in trivo under natural conditions without any artificial barrier. MATERIALS AND METHODS AnimaLs Inbred SL mice were maintained as reported previously [4]. Eight-week-old female mice were used for experiments. Abhreciations: PBS. phosphate-buffered saline: LPS, lipopolysaccharide. 117

YOSHIO HONMA et al.

118

Cells and cell culture The M1 cell line of leukemic myeloblasts was established from an SL strain mouse [8]. The cells were cultured in suspension in 6-cm Falcon plastic dishes in Eagle's m i n i m u m essential medium with two-fold concentrations of a m i n o acids and vitamins, supplemented with I0°o heat-inactivated calf serum [4]. The cells were cultured at 37:C in a humid atmosphere of 5°i; CO2 in air and transferred every 3-4 days. Autoradioyraphic examination of cells inoculated into the peritoneal carity The cells were labeled with 1-2,uCi/ml of [3H]thymidine tsp. act, 2 Ci/mmol: New England Nuclearl for 5-7 h. The labeled cells were washed three times with phosphate-buffered saline (PBS: 138 m M NaCI-2.7 m M KCI-8 m M N a z H P O 4 - 1 . 5 m M KH2PO,,, pH 7.4), and then an inoculum of 2 10 × 10~' cells was injected i.p. into a syngeneic mouse. After 3-7 days the cells were harvested by washing the peritoneal cavity with PBS. Autoradiographs of cell smears were prepared as reported previously [3]. Cells with more than 5 grains on the nuclei were counted as labeled cells. Of the peritoneal cells harvested, 7-26°0 were labeled laverage 17.8°.).

RESULTS Two types of cell were used: sensitive M1 cells that could be induced to differentiate in vitro into macrophages and granulocytes by treatment with dexamethasone, a proteinous inducer, or LPS(DS-3 cells), and resistant M1 cells that could not differentiate even with a high concentration of inducer(DR-3 cells) [-5]. In culture, sensitive DS-3 cells were morphologically similar to resistant DR-3 cells with a large round nucleus similar to that of typical myeloblasts. DS-3 cells were prelabeled with [3H]thymidine in vitro, and inoculated into the peritoneal cavity of syngeneic SL mice. After several days the prelabeled DS-3 cells had small irregular-shaped or eccentrically located nuclei, like those frequently seen in mouse granulocytes and macrophages (Table 1 and Fig. 1). The possibility that the peritoneal host macrophages and granulocytes phagocytize or ingest inoculated cells prelabeled by [3H]thymidine and that the label is incorporated into their nuclear DNA is exc|uded by the following findings:(1) in autoradiographs of cell smears, most of the grains formed by [SH]thymidine were located on the nucleus, not the cytoplasm, (2) when the prelabeled cells were treated in vitro with mitomycin C, at 25/~g/ml at 37°C for 60 rain, and then washed with PBS and inoculated into the peritoneal cavity of syngeneic mice, most of the labeled cells were dead and no grains could be observed in mature granulocytes and macrophages in the autoradiographs of the peritoneal cells. Moreover, MI cells prelabeled with [SH]thymidine under these experimental conditions remained undifferentiated in in vitro culture, although excess thymidine or X-irradiation could induce some stages of differentiation in appropriate clones of mouse myeloid leukemia cells [12]. These results show that sensitive MI cells.can be induced to undergo differentiation in the host even under the conditions in which leukemic cells can interact directly with the host cells. The differentiation-stimulating activity of mouse serum on MI cells was enhanced by injection of LPS, and LPS itself induced in vitro differentiation of MI cells [5-7, 12]. We TABLE 1. INDUCTION OF DIFFERENTIATION in vivo OF MI CELLS Morphological changes (%) Days after inoculation

Myeloblastic cells

Cells in intermediate stages

0 3 7

99 59 29

1 23 24

Mature granulocytes Macrophages 0 12 30

0 6 !7

MI cells were labeled in vitro with [3H]thymidine and inoculated into the peritoneal cavity of a syngeneic SL mouse. The peritoneal cells were harvested at the indicated times and the morphology of the labeled cells was examined.

(b) (a

i

J

(c)

(d)

FiG. 1. Autoradiographs of M1 cells inoculated into the peritoneal cavity of syngeneic SL mice. Resistant DR-3 cells (a) and sensitive DS-3 cells (b-d) were prelabeled with [3H'lthymidine in t'itro. The peritoneal cells were harvested three days after intraperitoneat inoculation of prelabeled cells, x 1425.

119

In rivo differentiation of leukemia cells Cell

I21

LPS

DS-3

-I

DR-3

-I ;

V/'A '

%

5'o

'

IO© '

o1' ToTal ceils

FIG. 2. Stimulation by LPS or morphological differentiation of MI cells in the peritoneal cavities of SL mice. Mice were injected intraperitoneally with 5 pg LPS one day after inoculation of M1 cells prelabeled by ['H]thymidine. [], myeloblasts: [], cells in an intermediate stage of differentiation: m, mature granulocytes: I . macrophages.

have found that, in syngeneic SL mice treated with LPS, sensitive MI cells in diffusion chambers are stimulated to differentiate into mature granulocytes and macrophages [5]. The morphology of sensitive MI cells prelabeled by [3H]thymidine in the peritoneal cavity of syngeneic mice change markedly when the mice are treated with LPS (Fig. 2). After three days, only 10~o of the labeled cells in the peritoneal cavity were myeloblastic in LPS-treated mice, whereas 60}~owere myeloblastic in the controls. LPS treatment also slightly increased the numbers of resistant MI cells in intermediate stages of differentiation in the peritoneal cavity. These results suggest that injection of an inducer of differentiation of MI cells can enhance differentiation of the cells under the same condition as those in which the leukemogenicity of the cells is assayed. DISCUSSION A previous report showed that in a pulse labeling experiment in a patient with acute myelomonocytic leukemia only immature cells incorporated [3H]thymidine and that in a continuous labeling experiment the number of labeled cells in some stages of differentiation increased [2]. These results suggest that the morphology of leukemia cells changes in t'ivo. However, there are few reports that myeloid leukemia cells can be induced to differentiate in vivo into mature granulocytes and macrophages under conditions in which leukemia can develop or in the leukemic condition. A group of mice inoculated with 104-106 of sensitive M1 cells survived longer than a group inoculated with a similar number of resistant cells [4, 5]. In the present work we found that M1 cells can differentiate into mature cells under conditions in which leukemia can develop. Our system should be useful for studies on the relation between leukemogenicity and inducibility of differentiation of leukemic cells in vivo. REFERENCES 1. COLLINS S. J.. GALLO R. C. & GALLAGHER R. E. (1977) Continuous growth and differentiation of human myetoid leukaemic cells in suspension culture. Nature, Lond. 270, 347. 2. CLARKSON l., TODO A., OGAWA M., GEE T. & FRIED J. (1971) Consideration of the cell cycle in chemotherapy of acute leukemia. Recent Results Cancer Res. 36, 88. 3. HAYASHI M.. GOTOH O.. OKAaE-KADO J. & HOZUMI M. (1981) Mechanisms controlling the kinetics in proliferation and differentiation of populations of mouse myeloid leukemic cells in vitro. J. cell. Physiol. 108, 123. 4. HONMA Y.. KASUKABET. & HOZUMI M. (1978) Relationship between leukemogenicity and m vivo inducibilit) of normal differentiation in mouse myeloid leukemia cells. J. nam. Cancer Inst. 61,837.

122

YOSHIOHOXVMA et al.

5. HONMAY., KASUKABET.. OKABEJ. & HOZ~MI M. (1979) Prolongation of survival time of mice inoculated with mveloid leukemia cells bv inducers of normal differentiation. Crrncrr Rrs. 39. 3167. 6. HONM~ Y., OKABE J., KAWKA~E T. & HOZUSNM. (1980) Survival of mice inoculated with non-diflerentiating myeloid leukemia cells is prolonged by injection of an inducer of cell differentiation and its sensttizer. Gann 71, 543. 7. HOZUMIM., HONMAY., TOMIDAM., OKABEJ.. KASUKABET.. SVGIYAUAK.. HAVASHIMM.. TA~EUAGAK.. & YAMAMOTO Y. (1979) Induction of differentiation of myeloid leukemia cells with vartous chemicals. .&ttr haemat. Japan. 42, 941.

8. ICHIKAWAY. (1969) Differentiation of a cell line of myeloid leukemia. J. cell PltwYol. 74, 2’73. 9. KOEFFLERH. P. & GOLDED. W. (1980) Human myeloid leukemia cell lines. Blood 56. 344. 10. LOTEMJ. & SACHSL. (1978) In cico induction of normal differentiation in myeloid leukemia cells. PRX~. natn. Acad. Sci. U.S.A. 75, 3781.

11. OKABEJ.. HONMAT., HAYASHIM. & HOZKJMIM. (1979) Actinomycin D restores itI ~YJ sensitivity to differentiation induction of non-differentiating mouse myeloid leukemia cells. Int. J. Cancer 24, 165. 12. SACHS L. (1978) Control of normal cell differentiation and the phenotypic reversion of malignancy in myeloid leukemia. Nature, Lond. 274, 535. Y. & HOZUMIM. (1980) Inhibition of the leukemogenicity of myeloid leukemta 13. TOMIDA,M., YAMAMOTO cells in mice and in uivo induction of normal differentiation of the cells by poly(1). poly(C). Gann 71, 457.