Int. J. Cancer: 71, 231–236 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer rIFN-g-ACTIVATED RAT NEUTROPHILS INDUCE TUMOR CELL APOPTOSIS BY NITRIC OXIDE Tetsuro UCHIDA1, Takao YAMASHITA1*, Akemi ARAKI1, Hiroshi WATANABE2 and Fujiro SENDO1 of Immunology and Parasitology, Yamagata University School of Medicine, Yamagata, Japan 2Department of Nursing, Yamagata University School of Medicine, Yamagata, Japan 1Department We have previously shown that 1) neutrophils activated with various cytokines, including rat recombinant interferon g (rIFN-g), inhibit tumor cell growth and that 2) nitric oxide (NO) is the effector molecule in tumor inhibition by rIFN-gstimulated rat peritoneal exudate neutrophils. In this study, we examined the nature of tumor cell death induced by rat peritoneal neutrophils activated by rIFN-g in order to clarify the mechanism of apoptosis in neoplastic tumor cell death. DNA of 3 syngeneic rat tumor cell lines was significantly fragmented within 3 hr of incubation in the presence of rIFNg-activated neutrophils, and this effect was dependent on both the concentration of rIFN- g and the number of neutrophils. This DNA fragmentation was inhibited by L-N-(1iminoethyl)-ornithine (L-NIO), a NO synthase inhibitor, but not by superoxide dismutase (SOD). Tumor cells treated with the activated neutrophils were shown by electron microscopy to be apoptotic, exhibiting necrotic features with a longer incubation. On the other hand, cytolysis of tumor cells, as determined by a [3H]-uridine release assay, was first observed only at 24 hr of incubation with the rIFN- g-activated neutrophils. Taken together, our results suggest that tumor cell apoptosis by activated neutrophils is NO-dependent and that apoptotic tumor cells undergo necrosis as a secondary process. We suggest that tumor cell apoptosis induced by activated neutrophils plays an important role in regulation of neoplastic tumor cell growth and death in vivo. Int. J. Cancer 71:231–236, 1997. r 1997 Wiley-Liss, Inc. We have previously shown that neutrophils activated by various immunomodulators (Kimura et al., 1987; Fukase et al., 1987) and cytokines (Inoue and Sendo, 1983; Kuzu, 1988; Hayashi et al., 1988; Miyake et al., 1988) exhibit cytotoxic activity against tumor cells. Human neutrophils treated with interferon gamma (IFN-g) were particularly prone to inhibit the growth of various tumor cells (Miyake et al., 1988). We have also reported that recombinant rat interferon gamma (rIFN-g)-activated rat neutrophils inhibit the growth of tumor cells in vitro and that nitric oxide (NO) is the effector molecule for cytostasis of tumor cell (Yamashita et al., this issue). There are 2 forms of cell death, apoptosis and necrosis (Wyllie et al., 1980). Apoptosis plays a pivotal role in the mechanisms of various types of cell death and is involved in regulation of cell number and elimination of damaged cells in tissues and organs. Apoptosis is characterized by specific morphological features, such as reduced cell volume, nuclear and cytoplasmic condensation and formation of apoptotic bodies. DNA of apoptotic cells is fragmented at the molecular level by a specific endonuclease (Wyllie et al., 1980; Gerschenson and Rotello, 1992). The balance between tumor cell proliferation and spontaneous cell death via apoptosis plays an important role in the regulation of neoplastic tumor cell growth. Apoptosis is involved in tumor cell death induced by various types of chemotherapeutic agents in vitro (Barry et al., 1990; Walker et al., 1991; Ling et al., 1993). We have focused our attention on the nature of tumor cell death induced by rIFN-g-activated rat neutrophils. We show that tumor cell apoptosis elicited by rIFN-g-activated neutrophils is NOdependent, and that apoptotic tumor cells undergo necrosis as a secondary process. MATERIAL AND METHODS Culture media and cytokines rIFN-g and monoclonal antibodies (MAbs) to rIFN-g were kindly donated by Mrs. M. de Labie (TNO Health Research, Rijswijk, The Netherlands). N-iminoethyl-L-ornithine (L-NIO) (McCall et al., 1991) was purchased from Alexis (Laufelfingen, Switzerland). Superoxide anion dismutase (SOD) was purchased from Wako (Tokyo, Japan). RPMI-1640 medium (GIBCO, Grand Island, NY) was supplemented with 10% fetal calf serum (FCS) (Whittaker, Walkersville, MD), 100 U/ml penicillin (Banyu, Tokyo, Japan), 100 µg/ml streptomycin (Meiji Seika, Tokyo, Japan), 2 mg/ml NaHCO3 (Wako) and HEPES (Dojin, Kumamoto, Japan). The hypotonic detergent buffer for tumor cell lysis consisted of 10 mM Tris-HCl, 1 mM EDTA (Wako) and 0.2% Triton X-100 (Wako). Animals Specific pathogen-free (SPF) WKA/Hkm rats obtained from Funabashi (Shizuoka, Japan) were kept in an environment free of specific pathogens. Ten to 12-week-old female rats were used in all experiments. Tumor cells We used KMT-17 (a 20-methylcholanthrene-induced sarcoma of WKA/Hok rats), WRT-7 (a myelomonocytic leukemia cell line of WKA/Hok rats) and KDH-8 (a dimethyl-azobenzanthraceneinduced liver cell cancer in WKA/Hok rats) as target cells. These tumor cells were maintained in 25-cm2 culture flasks (Nunc, Roskilde, Denmark) in RPMI-1640 medium. Rat neutrophils To obtain peritoneal exudated cells including neutrophils, 15 ml of sterilized 3% proteose peptone (Difco, Detroit, MI) were injected into the peritoneal cavity. Twelve hours later, the same volume of proteose peptone was injected again, and 3 hr later, exudate cells were collected in Eagle’s minimum essential medium (MEM) (Nissui, Tokyo, Japan). Neutrophils were purified from exudate cells by density gradient centrifugation at 400g on colloidal silica (Percoll; Pharmacia, Uppsala, Sweden) for 60 min at room temperature, according to the method of Inoue and Sendo (1983). The bottom layer containing neutrophils was collected, and after lysis of red blood cells using 0.15 M Tris-0.75% NH4Cl, cells were washed and resuspended in culture medium. The purity of the neutrophil suspensions was .95%, as determined by May-Giemsa Contract grant sponsor: Ministry of Education, Science and Culture of Japan, contract grant number 06281117. *Correspondence to: Dr. Takao Yamashita, Department of Immunology and Parasitology, Yamagata University School of Medicine, 2-2-2 Iida Nishi, Yamagata 990-23, Japan. Fax: 81-236-28-5267. E-mail: firstname.lastname@example.org Received 5 June 1996; revised 16 December 1996 232 UCHIDA ET AL. FIGURE 1 – DNA fragmentation of 3 tumor cell lines induced by rat neutrophils activated with rIFN-g. Tumor cells (a, KMT-17; b, WRT-7; c, KDH-8) (4 3 104 cells/well) were incubated with rIFN-g-activated neutrophils (4 3 105 or 1 3 106 cells/well) in 48-well plates for the indicated periods of time. DNA fragmentation of tumor cells was determined by the DNA fragmentation assay described in Material and Methods. Results of 4 different experiments are expressed as means 6 SD. *p , 0.001; **p , 0.05. staining. The viability of neutrophils was shown to be .99% using the Trypan blue dye exclusion test. DNA fragmentation assay The method described by Greenblatt and Elias (1992) was used for quantitation of DNA fragmentation. Tumor cells (3.0 3 106 cells/flask) were labeled with [3H]-thymidine (5 µCi/ml) in a 25-cm2 culture flask (Nunc) overnight, and then tumor cells were washed free of [3H]-thymidine with 2 washings in RPMI-1640 medium and resuspended in culture medium. Rat neutrophils (4.0 3 105 cells/well–2.0 3 106 cells/well) were incubated in the presence or absence of rIFN-g (0.1–100 U/ml) in 48-well plates (Sumitomo Bakelite, Tokyo, Japan) at 37°C for 1 hr in a humidified atmosphere of 5% CO2 and 95% air. After addition of labeled tumor cells (4.0 3 104 cells/well), the reaction mixtures were incubated for varying periods of time. In some experiments, in order to determine effector molecules, tumor cells were incubated for 1 hr with rIFN-g-treated neutrophils in the presence of L-NIO or SOD in 48-well plates at 37°C in a humidified atmosphere of 5% CO2 and 95% air. An anti-rIFN-g MAb was also added to the mixture of neutrophil preparations and rIFN-g. At various intervals, cells exposed to each experimental condition were collected and washed twice with phosphate-buffered saline (PBS), and then lysed in a hypotonic detergent buffer for 30 min at room temperature. Lysates were centrifuged at 12,000g for 30 min at room temperature. The radioactivity of [3H]-thymidine in supernatants and pellets was quantitated using a liquid scintillation analyzer (Packard, Tri-Carb 1500; Meriden, CT). The percentage of DNA fragmentation was calculated from the ratio of fragmented DNA to total (fragmented 1 intact) DNA. The following formula was used for the calculation. % DNA fragmentation 5 radioactivity in supernatants (fragmented DNA) radioactivity in supernatants radioactivity in pellets 1 (intact chromatin DNA) (fragmented DNA) 3 100 Cytolysis assay Tumor cell cytostasis was determined by a previously reported method (Inoue and Sendo, 1983). Briefly, tumor cells (3.0 3 106 cells/flask) were labelled for 4 hr with [3H]-uridine (5 µCi/ml) in a 25-cm2 culture flask, then washed free of [3H]-uridine with the culture medium. To quantitate cytolysis of tumor cells, neutrophils TUMOR CELL APOPTOSIS INDUCED BY rIFN-g-ACTIVATED NEUTROPHILS 233 (2.0 3 cells/ml and 5.0 3 cells/ml) were cultured with tumor cells (2.0 3 105 cells/ml) in the presence or absence of rIFN-g (10 U/ml) in 96-well plates (Sumitomo Bakelite) at 37°C for various intervals in a humidified atmosphere of 5% CO2 and 95% air. Plates were then centrifuged at 400g for 5 min, and the radioactivity of supernatants was determined by means of a liquid scintillation analyzer. The percentage of cytolysis was calculated using the following formula: 106 % lysis 5 106 experimental release 2 spontaneous release maximal release 2 spontaneous release 3 100 (%) Electron microscopy For electron microscopy, we used a Hitachi H-700H electron microscope (Tokyo, Japan) following a method described elsewhere (Koike et al., 1993). Briefly, the specimens were immersed in sodium cacodylate buffer (pH 7.4, 0.1 M) containing 2.5% glutaraldehyde. Samples were postfixed with 1% osmium tetroxide in the same buffer and stained en block with 2% uranyl acetate. Next, dehydration of samples was carried out using 60, 70, 80, 90, 95, 97 and 99.5% graded ethanol and propylene monoxide, and samples were then embedded in Epon 812 resin. Serial sections of each specimen were cut with a diamond knife, mounted on formvar film-coated single-slot grids and then stained with uranyl acetate and lead citrate aqueous solutions. FIGURE 2 – Inhibitory effect of MAbs to rIFN-g on DNA fragmentation of tumor cells (KMT-17) induced by rIFN-g-activated neutrophils. Tumor cells (4 3 104 cells/well) were incubated with rIFN-g-activated neutrophils (1 3 106 cells/well) in the presence of anti-rIFN-g MAbs (12.5 and 50 µg/ml) in 48-well plates for 24 hr. Results of 4 different experiments are expressed as means 6 SD. *p , 0.05. Statistical analysis All data are expressed as means 6 SD. When comparing different tumor cell conditions, we used an unpaired, two-sided t-test to assess statistical significance. Significance is a calculated p value of ,0.05. RESULTS Induction of tumor cell apoptosis by rIFN-g-activated rat neutrophils We have previously shown that rIFN-g-activated rat neutrophils inhibit growth of tumor cells in vitro (Yamashita et al., this issue). In order to clarify the biological characteristics of this inhibition, we investigated whether these neutrophils induce DNA fragmentation of 3 types of tumor cells, KMT-17, WRT-7 and KDH-8. Figure 1a shows the time course of DNA fragmentation of [3H]-thymidinelabeled KMT-17 cells incubated with rIFN-g (10 U/ml)-treated neutrophils. DNA fragmentation was detected as early as 3 hr from the start of incubation at an effector-to-target ratio (E/T ratio) of 25 in the presence of rIFN-g-treated neutrophils compared with target cells alone. The extent of fragmentation was directly related to the incubation time and the E/T ratio. On the other hand, DNA fragmentation of tumor cells incubated in the presence of rIFN-g alone or medium-treated neutrophils remained low during the entire test period. After 24 hr of incubation, the amount of DNA fragmentation at an E/T ratio of 25 in the presence of rIFN-g was approximately 60% of the cellular DNA and was about 4 times the control level. Figure 1b and c show DNA fragmentation of the 2 other tumor cell types (WRT-7 and KDH-8), which were incubated with rIFN-g (10 U/ml)-treated neutrophils. Fragmentation patterns of both types were extremely similar to that of KMT-17, as shown in Figure 1a. These findings indicate that tumor cell apoptosis was induced by these activated neutrophils. We then examined the inhibitory effect of anti-rIFN-g MAb on DNA fragmentation of tumor cells (KMT-17) induced by these activated neutrophils. Although DNA fragmentation was 60% at an E/T ratio of 25 in the presence of rIFN-g (10 U/ml), it was reduced to ,30% in the presence of anti-rIFN-g MAb, indicating that tumor cell DNA fragmentation was really induced by rIFN-g (Fig. 2). We next examined what effect the concentration of rIFN-g used for neutrophil activation had on the DNA fragmentation of KMT-17 tumor cells. As shown in Figure 3, when the E/T ratio was FIGURE 3 – rIFN-g concentration dependence of DNA fragmentation of tumor cells (KMT-17) induced by rIFN-g-activated neutrophils. Tumor cells (4 3 104 cells/well) were incubated with neutrophils (4 3 105 cells/well) activated with various concentrations of rIFN-g (0.1, 1, 10 and 100 U/ml) in 48-well plates for the indicated periods of time. DNA fragmentation of tumor cells was determined by the DNA fragmentation assay described in Material and Methods. Results of 4 different experiments are expressed as means 6 SD. *p , 0.001. 10 at 24 hr of incubation, dependence on the rIFN-g concentration was observed. DNA fragmentation induced by neutrophils activated with various rIFN-g concentrations reached a plateau at 10 U/ml. Moreover, almost the same patterns of dependence were observed with the other 2 cell lines (data not shown). In the following experiments, 10 U/ml rIFN-g were used for neutrophil activation. Effect of a NO synthase inhibitor, L-NIO and SOD on induction of DNA fragmentation in tumor cells We then tried to determine which effector molecules induced DNA fragmentation in these cells. We focused on nitric oxide 234 UCHIDA ET AL. (NO), which had exhibited antitumor activity in our previous work (Yamashita et al., this issue). Figure 4 shows the effect of L-NIO and SOD on DNA fragmentation of KMT-17 tumor cells induced by the activated neutrophils. L-NIO is a novel, potent, rapid-in-onset and irreversible inhibitor of NO synthase (NOS) in phagocytic cells, and SOD is a scavenger of superoxide anion (O22 ). Tumor cells were cultured with rIFN-g-activated neutrophils at an E/T ratio of 25 in the presence of L-NIO (1 and 10 µM) or SOD (5 and 50 U/ml). DNA fragmentation of these cells was not inhibited by SOD at either 12 or 24 hr. However, in the presence of L-NIO in cocultures, marked inhibition was noted at 12 hr from the start of incubation, and this effect continued until 24 hr. There were similar inhibition patterns using L-NIO with the other 2 types of tumor cells (data not shown). No cytotoxicity was demonstrable using L-NIO or SOD on tumor cell cultures alone at the concentrations used. Electron microscopic features of tumor cells treated with rIFN-g-activated neutrophils In order to confirm that tumor cell apoptosis had indeed occurred, we used electron microscopy to examine the morphological features of tumor cells treated in various ways. Untreated KMT-17 cells, as shown in Figure 5a, were characterized by a large nucleus and numerous well-developed mitochondria. Figure 5b shows typical morphological features of tumor cells incubated for 24 hr with rIFN-g-treated rat neutrophils at an E/T ratio of 25. Cells with an apoptotic nucleus appeared to have a cell membrane which remained intact. The cell shape had become rounded, and the nucleus was divided into several apoptotic nuclear fragments. Mitochondria were markedly reduced in number and their shape was atrophic. Cytoplasmic vacuolization was also seen. These features are characteristic of apoptotic changes, but the reduced number of mitochondria and the existence of cytoplasmic vacuolization suggest the start of secondary necrotic changes. Figure 5c shows a representative feature of tumor cells treated with rIFN-gactivated rat neutrophils for a period longer than 24 hr, at which time tumor cells shown in Figure 5b were obtained. The membrane was disrupted with the collapse of cellular organelles and showed typical characteristics of necrosis. Thus, the electron microscopy findings suggest that apoptosis of tumor cells was followed by secondary necrosis. Induction of secondary tumor cell necrosis by rIFN-g-activated rat neutrophils As described above, rIFN-g-activated rat neutrophils induced tumor cell apoptosis. However, the electron microscopic results described above suggest that tumor cells which had undergone apoptosis further underwent necrosis (Fig. 5). To evaluate necrosis of tumor cells treated with rIFN-g-activated rat neutrophils, we performed a [3H]-uridine release assay, as described in Material and Methods. [3H]-uridine-labeled tumor cells were cultured with rIFN-g-activated rat neutrophils. At 12 hr from the start of incubation, at which time DNA fragmentation had already started to appear (Fig. 1), cytolysis of KMT-17 tumor cells remained low (Fig. 6). After 24 hr of incubation, cytolysis of tumor cells at an E/T ratio of 25 in the presence of rIFN-g had increased to approximately 20%. This result indicates that tumor cell necrosis is initiated later than apoptosis, suggesting that the former is a secondary process. DISCUSSION We have shown that rat peritoneal exudate neutrophils stimulated with rIFN-g induce NO-dependent tumor cell apoptosis. There have been many reports demonstrating that neutrophils exert a cytotoxic effect on various tumors (Kimura et al., 1987; Fukase et al., 1987; Inoue and Sendo, 1983; Kuzu, 1988; Hayashi et al., 1988; Miyake et al., 1988; Yamashita et al., this issue). As for the effector molecules responsible, superoxide, hydrogen peroxide (Morikawa et al., 1985) and cationic proteins (Hayashi et al., 1988; Clark et al., 1976) have all been reported as possible candidates. However, little is known about the precise mechanisms involved. Our present results clearly indicate that NO can also be considered an effector molecule, especially when cytolysis occurs after long incubation periods (Inoue and Sendo, 1983), and when the form of tumor cell death is apoptosis induced by activated neutrophils. Our results are in accordance with those of studies using macrophages, showing that these cells induce apoptosis of a target cell, P815, through NO-dependent mechanisms (Cui et al., 1994). Although in that study, apoptosis of another type of target cell, L929, was induced via NO-independent mechanisms (Cui et al., 1994), in our present experiments, neutrophil-mediated apoptosis of all 3 target cell types was NO-dependent. Further experiments using other FIGURE 4 – Effect of L-NIO and SOD on DNA fragmentation of tumor cells (KMT-17) induced by rat neutrophils activated with rIFN-g. Tumor cells (4 3 105 cells/well) were incubated with neutrophils (1 3 106 cells/well) in the presence of rIFN-g (10 U/ml) together with L-NIO (1 and 10 µM) or SOD (5 and 50 U/ml) in 48-well plates for the indicated periods of time. DNA fragmentation of tumor cells was determined by the DNA fragmentation assay described in Material and Methods. Results of 4 different experiments are expressed as means 6 SD. *p , 0.001. TUMOR CELL APOPTOSIS INDUCED BY rIFN-g-ACTIVATED NEUTROPHILS 235 FIGURE 5 – Electron micrograph of tumor cells (KMT-17) incubated for 24 hr with rIFN-g-activated neutrophils. (a) Representative structure of untreated KMT-17, characterized by a large nucleus and numerous well-developed mitochondria. (b) Typical morphological features of KMT-17 cell apoptosis. Cells with an apoptotic nucleus retained their cell membrane intact. (c) Typical of necrotic KMT-17 cells, the cell membrane was disrupted with the collapse of cellular organelles. FIGURE 6 – Cytolysis of tumor cells (KMT-17) induced by neutrophils activated with rIFN-g. Tumor cells (2 3 104 cells/well) were incubated with neutrophils (2 3 105 or 5 3 105 cells/well) in the presence of rIFN-g (10 U/ml) in 96-well plates for the indicated periods of time. Cytolysis of tumor cells was determined by the [3H]-uridine release assay described in Material and Methods. Results of 4 different experiments are expressed as means 6 SD. *p , 0.05. target cells may be required to ascertain whether rIFN-g-activated neutrophil-mediated tumor cytotoxicity is always governed by NO-dependent mechanisms or whether other effector molecules are also involved, as in the case of macrophage-mediated tumor cytotoxicity. Concerning the relationship between apoptosis and necrosis of tumor cells induced by rIFN-g-activated neutrophils, assays of DNA fragmentation and cytolysis as well as electron microscopy clearly show that apoptosis occurs as a primary process and is followed by necrosis (Figs. 1, 5). As already shown in many reports, neutrophils produce various reactive oxygen intermediates. With respect to the possible involvement of active oxygen forms other than NO, our result that SOD did not inhibit the reaction suggests that O22 was not responsible for induction of tumor cell apoptosis by the rIFN-g-activated neutrophils. Furthermore, this also suggests that another powerful oxidant, peroxynitrite anion (ONOO2 ), which is produced by reaction of NO with O22 (Beckman et al., 1990; Koppenol et al., 1992), and which plays a pivotal role in various types of tissue injuries (Beckman et al., 1990; Beckman, 1991; Matheis et al., 1992), is not involved in the tumor cell apoptosis observed in our present experiments. In our previous report, we demonstrated that SOD not only failed to inhibit tumor cytostasis but actually enhanced it (Yamashita et al., this issue). However, in these experiments, this was not observed. This discrepancy may be explained as follows. In the 236 UCHIDA ET AL. previous study, enhancement of cytostasis by SOD was observed only when E/T ratios were ,10, suggesting that at high effector-totarget ratios, the amount of NO produced by activated neutrophils was too large to be significantly influenced by inhibition of the reaction (NO 1 O22 = ONOO2 ) with SOD. On the other hand, tumor cell DNA fragmentation, as an indicator of apoptosis induced by rIFN-g-activated neutrophils, could be observed at an E/T ratio of 25, at which point the effect of SOD on the total amount of NO released is negligible. Alternatively, the mechanisms of cytostasis and apoptosis may differ slightly from each other. These factors may account for the lack of enhancement of apoptosis of tumor cells by addition of SOD, as well as for the difference between our present results and those of the previous report. The result on DNA fragmentation in one tumor cell line (KMT-17) which was induced after a short incubation time (3 hr) needs to be discussed further. Indeed, it was previously shown that in vitro production of NO requires more than 3 hr of incubation (Miles et al., 1995). In a preliminary experiment, the amount of NO released by peritoneal neutrophils increased slightly at 3 hr of incubation in the presence of IFN-g (data not shown). This result may explain the above-mentioned result that DNA fragmentation of tumor cells started after 3 hr of incubation. Experiments are underway to further explore the possible mechanisms of tumor cell apoptosis elicited by activated neutrophils. REFERENCES BARRY, M.A., BEHNKE, C.A. and EASTMAN, A., Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharmacol., 40, 2353–2362 (1990). BECKMAN, J.S., The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J. Develop. Physiol., 15, 53–59 (1991). BECKMAN, J.S., BECKMAN, T.W., CHEN, J., MARSHALL, P.A. and FREEMAN, B.A., Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. nat. Acad. Sci. (Wash.), 87, 1620–1624 (1990). CLARK, R.A., OLSSON, I. and KLEBANOFF, S.J., Cytotoxicity for tumor cells of cationic proteins from human neutrophil granules. J. Cell Biol., 70, 719–725 (1976). CUI, S., REICHNER, J.S., MATEO, R.B. and ALBINA, J.E., Activated murine macrophages induce apoptosis in tumor cells through nitric oxidedependent or -independent mechanisms. Cancer Res., 54, 2462–2467 (1994). FUKASE, S., INOUE, T., ARAI, S. and SENDO, F., Tumor cytotoxicity of polymorphonuclear leukocytes in Beige mice: linkage of high responsiveness to linear b-1, 3-D-glucan with the beige gene. Cancer Res., 47, 4842–4847 (1987). GERSCHENSON, L.E. and ROTELLO, R.J., Apoptosis: a different type of cell death. FASEB J., 6, 2450–2455 (1992). GREENBALT, M.S. and ELIAS, L., The type B receptor for tumor necrosis factor-a mediates DNA fragmentation in HL-60 and U937 cells and differentiation in HL-60 cells. Blood, 80, 1339–1346 (1992). HAYASHI, T., ARAI, S. and SENDO, F., The mechanisms of cytotoxicity to tumor cells by polymorphonuclear leukocytes stimulated with cytokines. Jpn. J. Cancer Res., 79, 375–383 (1988). INOUE, T. and SENDO, F., In vitro induction of cytotoxic polymorphonuclear leukocytes by supernatant from a concanavalin A-stimulated spleen cell culture. J. Immunol., 131, 2508–2514 (1983). KIMURA, S., INOUE, T., YAMASHITA, T., MIDORIKAWA, Y., ARAI, S. and SENDO, F., Production of factor(s) that render polymorphonuclear leukocytes cytostatic from spleen cells stimulated with a streptococcal preparation, OK-432. Cancer Res., 47, 6204–6209 (1987). KOIKE, K., WATANABE, H., HIROI, M. and TONOSAKI, A., Gap junction of stratum granulosum cells of mouse follicles: immunohistochemistry and electron microscopy. J. Electron Microsc., 42, 94–106 (1993). KOPPENOL, W.H., MORENO, J.J., PRYOR, W.A., ISCHIROPOULOS, H. and BECKMAN, J.S., Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem. Res. Toxicol., 5, 834–842 (1992). KUZU, H., Augmentation of neutrophil tumor cytotoxicity by human recombinant interleukin 1. Yamagata med. J., 6, 71–83 (1988). LING, Y.-H., PRIEBE, W. and PEREZ-SOLER, R., Apoptosis induced by anthracycline antibiotics in P388 parent and multidrug-resistant cells. Cancer Res., 53, 1845–1852 (1993). MATHEIS, G., SHERMAN, M.P., BUCKBERG, G.D., HAYBRON, D.M., YOUNG, H.H. and IGNARRO, L.J., Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Amer. J. Physiol., 262, H616–H620 (1992). MCCALL, T.B., FEELISCH, M., PALMER, R.M.J. and MONCADA, S., Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. Brit. J. Pharmacol., 102, 234–238 (1991). MILES, A.M., OWENS, M.W., MILLIGAN, S., JOHNSON, G.G., FIELDS, J.Z., ING, T.S., KOTTAPALLI, V., KESHAVARZIAN, A. and GRISHAM, M.B., Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes. J. Leukocyte Biol., 58, 616–622 (1995). MIYAKE, Y., AJITSU, S., YAMASHITA, T. and SENDO, F., Enhancement by recombinant interferon-g of spontaneous tumor cytostasis by human neutrophils. Mol. Biother., 1, 37–42 (1988). MORIKAWA, K., KAMEGAYA, S., YAMAZAKI, M. and MIZUNO, D., Hydrogen peroxide as a tumoricidal mediator of murine polymorphonuclear leukocytes induced by a linear b-1, 3-D-glucan and some other immunomodulators. Cancer Res., 45, 3482–3486 (1985). WALKER, P.R., SMITH, C., YOUDALE, T., LEBLANCE, J., WHITFIELD, J.F. and SIKORSKA, M., Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes. Cancer Res., 51, 1078–1085 (1991). WYLLIE, A.H., KERR, J.F.R. and CURRIE, A.R., Cell death: the significance of apoptosis. Int. Rev. Cytol., 68, 251–306 (1980). YAMASHITA, T., UCHIDA, T., ARAKI, A. and SENDO, F., Nitric oxide is an effector molecule in inhibition of tumor cell growth by IFN-g activated rat neutrophils. Int. J. Cancer, this issue.