INHIBITION OF THE LEUKEMOGENICITY OF MYELOID LEUKEMIC CELLS IN MICE AND IN VIVO INDUCTION OF NORMAL DIFFERENTIATION OF THE

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1 INHIBITION OF THE LEUKEMOGENICITY OF MYELOID LEUKEMIC CELLS IN MICE AND IN VIVO INDUCTION OF NORMAL DIFFERENTIATION OF THE Mikio TOMIDA, Yuri YAMAMOTO, and Motoo HOZUMI Department of Chemotherapy, Saitama Cancer Center Research Institute* The effects of synthetic RNAs on the leukemogenicity of mouse myeloid leukemic M1 cells were studied in syngeneic SL mice. M1 cells are known to be capable of differentiating into macrophages and granulocytes both in vitro and in vivo. Mice that had been inoculated with three different clones (T-22, R-4 and DR-3) of M1 cells varying in sensitivity to inducers of differentiation died 20 to 40 days after inoculation. However, 10 out of 14 mice inoculated with the slightly prolonged the survival times of mice inoculated with some clones of M1 cells. lating activity, which induced differentiation of M1 cells in vitro, were markedly induced within 3hr in the serum of the mice. cells was examined by implanting diffusion chambers containing the cells into syngeneic SL strain mice. The in vivo inductions of differentiation of three clones (T-22, R-4 and DR-3) of M1 cells in untreated mice varied, but all three clonal cells showed marked differentiation into macrophages and granulocytes in mice These results suggest that stimulation of induction of normal differentiation and interferon to the leukemic cells and immunopotentiating activity of the RNA may also be involved. differentiation-leukemogenicity Mouse myeloid leukmic M1 cells can be induced to differentiate into macrophages and granulocytes both in vitro15,16) and in vivo,8,20,25) and various inducers of in vitro differentiation of M1 cells are known.13,27) A protein inducer(s), designated as D-factor (differentiation-stimulating factor)11,12,22) or MGI (macrophage and granulocyte inducer),27) was found in conditioned media of various cultured cells,14,15,22,29) ascitic serum.4) Macrophages and granuloctyes were suggested to be mainly responsible for the production of D-factor.11,12,15) We previously showed that double-stranded RNAs

2 duction of D-factor by mouse peritoneal macrophages in vitro.29) Furthermore, we found that these RNAs directly affect M1 cells, making them more sensitive to D- factor.32) These RNAs did not themselves induce differentiation of M1 cells, but they markedly stimulated the induction by a low concentration of D-factor. The effect of these RNAs was concluded to be mediated by induced interferon,32) because the RNAs also induced interferon in M1 cells, because partially purified interferon had a stimulatory effect on the differentiation of M1 cells and because the stimulatory effect of the RNAs was abolished by simultaneous treatment of the M1 cells with anti-interferon serum. On the other hand, it was shown that differentiated M1 cells induced in vitro could no longer develop into tumors in syngeneic SL strain mice.5,16) Honma et al.8) also reported a correlation between leukemogenicity and in vivo inducibility of differentiation in several clones of M1 cells. Therefore, we tried to inhibit the development of M1 cell leukemia in syngeneic SL mice by enhancing the induction of normal differentiation of the cells with synthetic RNAs. MATERIALS AND METHODS Animals About 60-day-old, specific pathogenfree SL strain mice maintained in this laboratory were used throughout. Cells Myeloid leukemic M1 cells were originally obtained from a spontaneous myeloid leukemia in an SL strain mouse.15) Clone T-22 cells29) can be induced to differentiate into macrophages and granulocytes by inducers such as D-factor or glucocorticoid hormones. Clone R-424) and DR- 37) cells, which cannot be induced to differentiate in vitro by inducers alone, were also isolated from M1 cells. The cells were cultured in Eagle's minimal essential medium (Nissui Seiyaku Co., Tokyo), supplemented with double the usual concentrations of amino acids and vitamins and M. TOMIDA, ET AL. Culture of Cells in Diffusion Chambers The chambers were made from Millipore mem- scribed previously,2,8) and 0.1ml of cell suspension into each one. Two chambers were implanted into the peritoneal cavity of each mouse that had received no pretreatment. The cells were collected after 4 days by processing the chambers in 0.5% pronase and 5% Ficoll-400 in Hanks' balanced with May-Grunwald-Giemsa stain. More than 200 cells from each chamber were analyzed for the determination of morphological differentiation. Assay of Interferon Interferon in mouse serum was assayed by determining the inhibition of 3H-uridine incorporation into vesicular stomatitis virus RNA in L cells by the method of Kawade et al.17) In this work titers of interferon were measured with NIH reference standard mouse interferon (Cat. No., G ) as a standard and are expressed in international reference units (IU). Assay of D-factor Activity of D-factor in mouse serum was assayed by measuring the induction of phagocytic activity in M1 cells by the serum. M1 (T-22) cells were incubated with various concentrations of the serum for 2 days and then their phagocytic activity was assayed as described previously,29) using polystyrene latex poly(u) and poly(i) were obtained from Yamasa Shoyu, Choshi. RESULTS Effects of Treatment with Synthetic RNAs on the Survival of Mice Inoculated with Various Clones of M1 Cells We inoculated ip 105 cells of three different clones of M1 cells into SL mice. As shown in Fig. 1, all the mice inoculated with T-22 clonal cells, which could be induced to differentiate in vitro by inducers such as D- factor and glucocorticoid, died 29 to 40 days after inoculation. Mean survival times of mice inoculated with R-4 and DR-3 clonal cells, which were less sensitive to inducers, tively. The difference in leukemogenicity between sensitive T-22 cells and resistant R-4 and DR- 3 cells is significant (P<0.001). Honma et al.8) previously showed a clear difference in Gann

3 Fig, 1. Effects of synthetic RNAs on the survival EFFECT OF POLY I:C ON MYELOID LEUKEMIA times of mice inoculated with M1 cells Clone T-22 (a), R-4 (b) or DR-3 (c) cells at 105 cells per 0.2ml of phosphate-buffered saline were inoculated ip into SL mice. Synthetic RNAs in 0.2ml of phosphatebuffered saline were injected ip at the time of cell inoculation and then twice a week until day 93. Groups of leukemogenicity between the resistant and the sensitive cells with a smaller inoculum size (104 cells/mouse). poly(u) and poly(i) were injected ip at the time of cell inoculation and then twice weekly at doses that were not toxic to the mice. Survival of mice inoculated with each clone of M1 cells was markedly prolonged out of 5 mice inoculated with T-22 cells, 3 out of 5 mice inoculated with R-4 cells and 3 out of 4 mice inoculated with DR-3 cells survived for more than 100 days. Three and one of the survivors inoculated with DR-3 cells and R-4 cells, respectively, died of leu- (125 days from the first treatment) with killed at day 165. mouse) for the suppression of leukemia. A similar difference in the effectiveness of the leukemic cells was observed in vitro: a 50- to 100-fold higher concentration of poly quired for the induction of interferon and stimulation of differentiation of T-22 cells.32) We previously showed that T-22 clone cells, but not R-4 or DR-3 clone cells, were induced to differentiate in vitro by treatment days.30) Therefore, we expected an inhibitory effect of poly(i) on the leukemogenicity of the sensitive T-22 cells. The mean survival time of mice injected with T-22 cells Induction of Interferon and Differentiation-stimulating Activity in Mouse feron. We have found that interferon stimulated the induction of differentiation of M1 cells by D-factor (Cancer Res., in press), poly(c) enhanced the production of D- factor in mouse peritoneal macrophages in vitro.29) Hence, we examined the effect of feron and D-factor in vivo. As shown in Fig. 2, the activities of interferon and D-factor were not detectable in the serum of untreated mice (0 time), but both activities were induced within 3hr af- gradually decreased. Interferon disappeared

4 M. TOMIDA, ET AL. induction of differentiation of M1 cells M1 cells were cultured in diffusion chambers in SL of the diffusion chambers. Fig. 2. Activities of interferon and D-factor in Serum was collected from 3 mice at the indicated times and pooled. Activities of interferon and D-factor in the sera were assayed as described in "Materials and Methods." Activity of D-factor was assayed with 1% more rapidly than the differentiation-stimulating activity. Significant activity of D- factor remained for 72hr after treatment Induction of Differentiation of M1 Cells In vivo induction of differentiation of cells was demonstrated by the diffusion chamber technique.8,20,25) In untreated mice, the induction of differentiation of M1 cells was correlated with the in vitro sensitivity of the cells to inducers such as D-factor and dexamethasone. As shown in Fig. 3, T-22 cells, but not R-4 or DR-3 cells, were induced to differentiate into mature macrophages and granulocytes within 4 days after their implan- poly(c) into the mice, even resistant R-4 and DR-3 cells were induced to differentiate (Fig. 4), and differentiation of the sensitive T-22 cells was significantly stimulated. DISCUSSION We clearly demonstrated that injection times of mice inoculated with myeloid leukemic M1 cells and stimulated in vivo induction of differentiation of the cells. The mechanisms of inhibition of leukemogenicity known, but stimulation of the normal differentiation of the leukemic cells seems to be Gann

5 EFFECT OF POLY I:C ON MYELOID LEUKEMIA

6 one of the mechanisms, since it was shown that, on differentiation in vitro, M1 cells lose both proliferating activity and leukemogenicity,5,16) and that the leukemogenicity of each clone of M1 cells is inversely correlated with its in vivo inducibility of differentiation.8) Furthermore, leukemogenicity of M1 cells was recently shown9) to be inhibited by the injection into mice of inducers (lipopolysaccharide31) and dexamethasone) of differentiation of the cells. In the present paper, we showed that M. TOMIDA, ET AL. suited in the induction of D-factor activity and interferon in their sera. Lotem and Sachs21) and we found that an appropriate concentration of interferon stimulated the induction of differentiation of M1 cells by a low concentration of D-factor in vitro (Cancer Res., in press). Therefore, stimulation of in vivo induction of differentiation of M1 cells Lion of D-factor and interferon by the RNA. Although we observed stimulation by activity of macrophages1,28) and natural killer cells.3) Therefore, these various actions ferentiation of M1 cells, including clones which were resistant to inducers in vitro, no definitive correlation was observed between the differentiation of the cells and the survival times of the leukemic mice (Figs. 1 and 3). T-22 cells could differentiate into macrophages and granulocytes in untreated mice to almost the same degree that DR-3 cells most resistant to treatment with the RNA mice. However, mice injected with DR-3 examine myeloid leukemia. To our knowl- better than those that received T-22 cells alone. Therefore, stimulation of the induction poly(c) is not the only mechanism of inhibition of leukemogenicity of the cells by the RNA. One possible mechanism involved may be kemic cells. Treatment of T-22 cells with 5 ug/ml of the RNA in vitro for 2 days reduced DR-3 cells could tolerate even 1mg/ml of the RNA (85 and 95% of the growth of controls, respectively). Therefore, a direct cytotoxic effect of the RNA on T-22 cells may contribute to the prolongation of the survival times of mice inoculated with these cells. Although a difference in the antigenicity of each clone of the M1 cells against syngeneic SL mouse may also affect the leukemogenicity of the cells through immune mechanisms, no such difference in antigenicity was detected among the clones.9) Another possible mechanism of inhibition of the leukemogenicity of the leukemic cells is suppression of growth of the cells by interferon induced by the RNA. Suppression by interferon of in vitro growth of M1 cells was recently observed. Unexpectedly, the resistant clones, R-4 and DR-3, were more sensitive to interferon than the sensitive clones, T-22. recently reported to stimulate the tumoricidal of interferon may also be involved in the (C) of the leukemogenicity of M1 cells. On the other hand, antitumor effects of various tumors. Although Levy et al.19) reported that lymphatic leukemias were the among various tumors of mice, they did not poly(c) markedly inhibits myeloid leukemia. clinically useful26) because of its rapid hydrolysis by nuclease in primate serum.23) Recently, a stabilized material consisting of poly(c) complex may be useful for the therapy of some patients with myeloid leukemia. Gann

7 EFFECT OF POLY I:C ON MYELOID LEUKEMIA This work was supported in part by Grantsin-Aid for Cancer Research from the Ministry of Health and Welfare, and from the Ministry of Education, Science and Culture. (Received January 23, 1980) 14) Hozumi, M., Umezawa, T., Takenaga, K., Ohno, T., Shikita, M., Yamane, I., Cancer 17) Kawade, Y., Matsuzawa, T., Yamamoto, Y., Tsukui, K., Iwakura, Y., Annu. Rep. 18) Levy, H. B., Baer, G., Baron, S., Buckler, C. E., Gibbs, C. J., Iadarola, M. J., London, 10) Hozumi, M., Sugiyama, K., Mura, M., Takizawa, H., Sugimura, T., Matsushima, T., Ichikawa, Y., Proc. 4th Int. Symp. Princess 12) Hozumi, M., Honma, Y., Okabe, J., Tomida, M., Kasukabe, T., Takenaga, K., Sugiyama, K., "Oncogenic Viruses and Host Cell Genes," ed. Y. Ikawa and T. 13) Hozumi, M., Honma, Y., Tomida, M., Okabe, J., Kasukabe, T., Sugiyama, K., Hayashi, M., Takenaga, K., Yamamoto,