A complex of polyinosinic-polycytidylic acid [poly(I)•poly(C)] and cationic liposome (LIC) inhibited the growth of many tumor cell lines at low concentration in vitro, but poly(I)•poly(C) alone had no such antiproliferative effect. The IC50 values of LIC against the tumor cells ranged from 0.1 to 1000 ng/ml. LIC had strong cytotoxic effects on malignant cells of epithelial and fibroblastic origin from various tissues and was also effective against Adriamycin-resistant tumor cells. LIC did not significantly affect the growth of lymphoma cells, leukemia cells, normal diploid fibroblasts, or primary liver cells at concentrations up to 10 μg/ml. The mechanism of the antiproliferative effect of LIC against malignant cells was the induction of apoptosis. LIC induced the fragmentation of nuclear DNA and the degradation of rRNA in tumor cells. The DNA fragmentation occurred within 1–5 h after the addition of LIC, and both the fragmentation and the inhibition of cancer-cell growth were suppressed by a nuclease inhibitor. In contrast, caspase inhibitors did not affect the antiproliferative activity of LIC. These results suggest that LIC induced apoptosis in malignant cells through the direct activation of nucleases and not through the activation of caspases. LIC reduced the incidence and the size of metastatic liver-cancer tumors in two different mouse metastatic liver-cancer models using human colon carcinoma cells. Histochemical analysis revealed that the KM12-HX cells in the tumor nodules were undergoing apoptosis; therefore, LIC also induced the apoptosis of tumor cells in vivo. In these animal models, LIC caused no observed changes in normal hepatocytes.

Previous research on dsRNAs2 has focused almost entirely on their IFN-inducing activity (1, 2, 3, 4, 5, 6, 7). We found that poly(I)•poly(C) and poly(A)•poly(U) had an in vivo antiproliferative effect against many tumor cells in mice but had no detectable in vitro cytotoxic activity against various tumor cell lines when the cells were directly treated with them.3 The antitumor activity of dsRNA in vivo may have been caused by a potentiation of the immune system in the host animals; therefore, the dsRNA may be acting as a biological response modifier (7, 8, 9).

Anionic polymers such as poly(I)·poly(C) do not easily cross the cell membrane. Except for special cells like macrophages, cells cannot incorporate poly(I)·poly(C); therefore, its intracellular effects have not been adequately investigated. We expected that if poly(I)·poly(C) could enter the living cells, drastic and unpredictable biological changes would occur because some enzymes can be activated by poly(I)·poly(C) inside the cells, for example (2′-5′)oligoadenylate synthetase and dsRNA-dependent protein kinase (10, 11, 12).

Cationic liposomes have been extensively used to facilitate the delivery of DNA into living cells (13, 14). The binding of DNA to cationic liposomes is mediated by electrostatic interactions. The mechanism of entry of DNA/cationic liposome complexes into cells was speculated to be cationic-liposome-mediated transfection; this mechanism would involve the fusion of a positively charged complex with the negatively charged plasma membrane, resulting in direct entry of the lipid into the cytosol (13). However, recent evidence suggests that the major mechanism is, in fact, endocytosis of the complex followed by its escape from an endocytotic compartment into the cytosol (15, 16).

In this study, we treated a variety of cell lines with LIC and found that it could directly kill many kinds of tumor cells. As mentioned above, poly(I)·poly(C) alone had no detectable antiproliferative activity in vitro, and the cytotoxic effect of LIC is a previously unreported activity. The evidence presented here suggests that the cytotoxic activity was due to the induction of apoptosis through the activation of cellular nucleases. We also tested LIC in two different mouse models of liver metastasis and found a strong antiproliferative effect. LIC is, therefore, a promising candidate for the treatment of metastatic liver cancer.

Compounds.

Poly(I), poly(C), poly(I)·poly(C), and poly(A)·poly(U) were purchased from Yamasa (Chiba, Japan) and poly(A-U)·poly(A-U), poly(dI)·poly(dC), and poly(G)·poly(C) from Pharmacia Biotech (Uppsala, Sweden). The distribution of chain lengths of the poly(I) and poly(C) used in the preparation of LIC were adjusted by heating to give an apparent maximum at 200–400 bp as determined by gel filtration high-performance liquid chromatography. Positively charged liposomes containing the cationic lipid analogue 2-O-(2-DEAE)-carbamoyl-1,3-O-dioleoylglycerol (synthesized at Nippon Shinyaku Co., Kyoto, Japan) and egg phosphatidylcholine (Nippon Yushi, Osaka, Japan) were prepared as described previously (13). Each nucleic acid polymer/cationic liposome complex was made by mixing a nucleic acid polymer and the cationic liposome freshly for each experiment. The concentration of the complexes was represented as the concentration of the nucleic acid polymer. [3H]poly(C) was synthesized by means of polynucleotide phosphorylase (Amersham, Arlington Heights, IL) using [5-3H]CDP (Amersham) as a substrate. The specific activity of the [3H]poly(I)·poly(C) was 1.3 × 106 dpm/μg. MTT was purchased from Molecular Probes (Eugene, OR), ATA was from Nacalai Tesque (Kyoto, Japan), z-VAD-fmk and DEVD-fmk were from Kamiya Biomedical Company (Seattle, WA), and Hoechst 33342 was from Calbiochem-Novabiochem (San Diego, CA).

Cell Lines and Culture Conditions.

All but two of the cell lines were obtained from American Type Culture Collection (Bethesda, MD) and cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. The culture medium and methods used were as described in the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (8th edition, 1994). Human primary hepatocytes and CS-C serum-free medium were purchased from Applied Cell Biology Research Institute (Street Kirkland, WA). HCC-T cells were a gift from Dr. Hideaki Saito of Keio University (Tokyo, Japan). BALB/c AMuLVA.6R.1 is derived from the normal BALB/c embryonic fibroblast line BALB/c CL.7 by transformation with Abelson murine leukemia virus, and 293 is derived from primary human embryonic kidney cells by transformation with sheared human adenovirus type 5 DNA.

Assessment of Cell Growth.

Cells from monolayer cultures were placed in 96-well microplates (Corning Costar, Corning, NY) at a density of 104 cells/well (100 μl/well). After 5 h, compounds were added to each well at concentrations of up to 10 μg/ml, and the incubation was continued at 37°C. Cells from suspension cultures were seeded into 96-well microplates at a density of 104 cells/well (100 μl/well) and treated with up to 10 μg/ml of each compound at 37°C for 3 days. The number of cells surviving after 3 days was determined by MTT assay (17). IC50 values were determined in triplicate in each experiment, and each experiment was done twice under identical conditions. IC50 was defined as the drug concentration that induced 50% cell death in comparison with untreated controls and was calculated by nonlinear regression analysis.

DNA Fragmentation.

Cells were treated with LIC (1 μg/ml) for 5 h (A431 cells) or 7.5 h (KM12-HX cells). The cells were harvested after trypsinization and lysed with 5 mm Tris-HCl (pH 8), containing 10 mm EDTA and 0.5% (v/v) Triton X-100. The lysate was centrifuged at 13,000 × g for 20 min (Himac CR15D refrigerated centrifuge; Hitachi, Tokyo, Japan) to separate the fragmented DNA (supernatant) from intact chromatin (pellet). The soluble DNA was treated with RNase A (100 μg/ml) at 37°C for 1 h and then with proteinase K (200 μg/ml) in 1% (w/v) SDS at 50°C for 2 h. The DNA was extracted sequentially with phenol and phenol/chloroform and then precipitated with ethanol. The DNA was analyzed on 1.8% (w/v) agarose gels containing 0.5 μg/ml ethidium bromide, and the DNA was visualized under UV light. To measure the amount of fragmented DNA, the cells were plated at a density of 2.8 × 105 cells/well (6-well plates) and prelabeled with [3H] thymidine (2 μCi/well) for 8 h before the addition of LIC (1 μ g/ml). The fragmented DNA was extracted as described previously. The radioactivities of the fragmented DNA and the total DNA were measured with a liquid scintillation counter (TRI-CARB4640; Packard, Meriden, CT).

Analysis of rRNA.

Cells were plated at a density of 1.8 × 106 cells per 10-cm plate and treated with LIC (1 μg/ml) for 4 h. Cells were harvested after trypsinization, washed with PBS, and lysed with 5 mm Tris-HCl (pH 7.6), containing 20 mm KCl, 1.25 mm magnesium acetate, and 1.25% (v/v) glycerol. The lysate was centrifuged at 16,000 × g for 15 min at 4°C (Himac CR15D). Then the supernatant was laid on a cushion of 1 m sucrose in 5 mm Tris-HCl (pH 7.6), containing 1 mm DTT and 0.1 mm EDTA, and centrifuged at 150,000 × g for 5 h at 4°C (Optima TLX ultracentrifuge; Beckman, Palo Alto, CA) to obtain the ribosomal fraction (pellet). rRNA was prepared by extraction with acid/guanidinium thiocyanate/phenol-chloroform (18) and analyzed by electrophoresis on a 1.8% (w/v) agarose gel containing 2.2 m formaldehyde. The gel was stained with 0.1 m ammonium acetate containing 0.5 μg/ml ethidium bromide, and the RNA was visualized under UV light.

Effects of a Nuclease Inhibitor and Caspase Inhibitors.

A431 cells were plated at a density of 104 cells/well (96-well plates). After 1 day, cells were treated with 30 μm ATA (a nuclease inhibitor; Ref. 19), 100 μm z-VAD-fmk (an ICE inhibitor), or 100 μm DEVD-fmk (a CPP32 inhibitor). LIC was added to the cells 2 h after treatment with inhibitors. The incubation was continued for 3 days, and cell growth was measured by MTT assay.

Incorporation of LIC into Cells.

Each cell line was seeded at a density of 5 × 104 cells/well (24-well plates). One day later, the culture medium was changed to fetal bovine serum-deficient medium. [3H]LIC (1 μg/ml) was added to the medium, and the incubation was continued at 37°C. Then the cells were washed three times with PBS, detached by trypsinization, and collected by centrifugation. The radioactivity incorporated into the cells was measured with a liquid scintillation counter (LSC-3500; Aloka, Tokyo, Japan).

Microinjection of Poly(I)·Poly(C).

The cells were plated at a density of 5 × 103 cells/well in 4-well plates with microgrid coverslips (Eppendorf, Hamburg, Germany). One day later, poly(I)·poly(C) (1 mg/ml, dissolved in 0.9% NaCl solution) was microinjected into the cells with an Eppendorf Transinjector 5246 and Eppendorf Micromanipulator 5171 under an Olympus IMT-2 microscope (Olympus, Tokyo, Japan). After microinjection, the cells were washed with PBS and photographed in culture medium. For nuclear-staining analysis, cells were fixed with 4% (v/v) formaldehyde-PBS solution and stained with 100 μm Hoechst 33342.

Hepatic Metastasis Models.

Male BALB/c nu/nu mice were purchased from Clea Japan (Tokyo, Japan). In one model, BALB/c nu/nu mice were anesthetized with pentobarbital, and the spleen was exposed to allow the direct injection of 106 viable KM12-HX cells in 0.05 ml of PBS. The lienal artery and vein were clamped 10 min after the inoculation of the tumor cells, the spleen was removed, and the abdomen and skin were closed with glue. The mice were allowed to recover and were randomized before the first treatment. In a second model, BALB/c nu/nu mice were intrasplenically injected with 3 × 106 Ls174T cells and thereafter treated as described above.

Antitumor Activity of LIC in Mice with Hepatic Metastasis of KM12-HX Cells.

BALB/c nu/nu mice (9-week-old, male) were injected with KM12-HX cells by intrasplenic injection on day 0. Mice were divided at random into five groups of six. LIC was administered i.v. two times per week (every 3 or 4 days) from day 7 under schedule A or from day 28 under schedule B. The total number of administrations of LIC was nine or three, respectively. Control mice were injected i.v. two times per week from day 7 with vehicle only [10% (w/v) maltose solution]. In the nontreated group, mice were not injected with tumor cells but their spleens were removed. Mice were killed on day 37, and the liver was removed for weighing.

Antitumor Activity of LIC in Mice with Hepatic Metastasis of Ls174T Cells.

Ls174T cells were injected intrasplenically into BALB/c nude mice (5-week-old, male). Six mice per group were used. Under schedule A, LIC was administered i.v. two times a week (every 3 or 4 days) for 3 weeks starting on day 6. Under schedule B, LIC was administered i.v. once a day from day 20 to day 25. The total number of administrations was six under both schedules. The weights of the livers were measured on day 28.

Inhibition of Cell Growth by Nucleic Acid Polymer/Cationic Liposome Complexes in A431 Cells.

The antiproliferative activity of LIC and several other nucleic acid/cationic liposome complexes in vitro were compared by measuring growth inhibition in A431 cells by means of the MTT assay. LIC inhibited the growth of A431 cells in a dose-dependent manner from 3 ng/ml to 1 μg/ml (Fig. 1,A). Poly(I)·poly(C) alone and cationic liposome alone had no effect up to 100 μg/ml and 10 μg/ml, respectively (data for high concentrations of both compounds are not shown). Neither poly(C)/cationic liposome complex nor poly(I)/cationic liposome complex inhibited cell growth (Fig. 1,B). The cationic liposome complex of poly(A-U)·poly(A-U), an alternating copolymer, had about the same effect as LIC, but the effect of the cationic liposome complex of poly(A)·poly(U) was almost 100 times weaker. Poly(G)· poly(C)/cationic liposome complex had no effect (Fig. 1,C), and poly(dI)·poly(dC), a cationic liposome complex of double-stranded DNA, also had no effect (Fig. 1 C).

Inhibition of Cell Growth by LIC in Various Cell Lines in Vitro.

LIC was tested on more than 60 cell lines. It had a potent antiproliferative effect on tumor cells of epithelial or fibroblastic origin, with IC50 values ranging from 0.1 to 1000 ng/ml (Table 1). LIC acted on a wide variety of cell types, including breast, colon, bladder, ovary, prostate, cervix, lung, bone, liver, pancreas, skin, and muscle. Among these cell lines, four that were resistant to Adriamycin (OVCAR3, PC-3, KBC.1, and MDA-MB-231) were sensitive to LIC, which was also effective against two virus-transformed cell lines (BALB/c AMuLVA.6R.1 and 293). LIC did not significantly affect the growth of lymphoma cells or leukemia cells (Table 1), nor did it affect the growth of normal diploid fibroblasts or embryonic or normal liver cells (Table 2). We also examined the cyotoxicity of LIC toward human primary hepatocytes (Table 2). Both Adriamycin and mitomycin (data not shown) were cytotoxic toward human primary hepatocytes, with respective IC50 values of 0.19 μg/ml and 0.15 μg/ml, but LIC was not cytotoxic at concentrations up to 10 μg/ml.

LIC-induced DNA Fragmentation in Tumor Cells.

We investigated whether the growth-inhibitory activity of LIC against tumor cells was a result of the induction of DNA fragmentation. We treated A431 and KM12-HX cells with LIC and subjected them to a procedure for extracting fragmented DNA. In untreated cells, no fragmented DNA was observed, but in treated cells, fragmentation was clearly observed (Fig. 2,A). We determined the amount of fragmented DNA prelabeled by [3H]thymidine in A431 cells (Fig. 2 B). Only 1 h after the addition of LIC, the fragmentation had already begun, and after 9 h, it had reached a maximum of 60% of the total DNA.

LIC-induced Fragmentation of rRNA in Tumor Cells.

rRNA was extracted from A431, MDA-MB-468, KB, HeLaS3, and MCF7 cells. 28S and 18S rRNAs were clearly separated by agarose gel electrophoresis, and no degraded RNA was seen in untreated cells. Four h after the addition of LIC, however, both 28S and 18S rRNAs were degraded in all five of the cell lines (Fig. 3). Only 1 h after the addition of LIC, both types of rRNAs had begun to degrade, and after 16 h, both types were almost completely degraded (data not shown).

Effect of a Nuclease Inhibitor or Caspase Inhibitors on the Antiproliferative Effect of LIC.

We have found that ATA inhibited the DNA fragmentation caused by LIC (data not shown). We also tested its effect on the inhibition of tumor-cell growth brought about by LIC. The exposure of A431 cells to 30 μ m ATA almost completely suppressed the antiproliferative effect of LIC (Fig. 4). We tested whether caspase inhibitors such as DEVD-fmk [a caspase-3 (CPP32) inhibitor] or z-VAD-fmk [a caspase-1 (ICE) inhibitor] influence the antiproliferative activity of LIC, but neither inhibitor had any effect (Fig. 4).

Cellular Uptake of Poly(I)·Poly(C).

The amounts of poly(I)·poly(C) incorporated by six different cell lines were measured by means of radioactive LIC containing [3H]poly(I)·poly(C) (Fig. 5). In SK-HEP1, HCC-T, and A431 cells, which died about a day after the addition of LIC, the uptake of poly(I)·poly(C) into cells was still increasing 3 h after the addition. However, there was no relationship between the sensitivity of the cells to LIC and the amount of poly(I)·poly(C) incorporated. In insensitive cell lines, such as human primary hepatocytes, CHO-K1 cells, and U937 cells, the levels of poly(I)·poly(C) incorporated were very low compared with those of the sensitive cell lines. Even in LIC-sensitive cells, when poly(I)· poly(C) was added alone, no incorporation was observed; and when LIC was added at 4°C, only very low incorporation was observed (data not shown).

Microinjection of Poly(I)·Poly(C).

%When poly(I)·poly(C) was injected into HeLaS3 cells, cell death was seen 1 day after microinjection (Fig. 6). When LIC-treated HeLaS3 cells were stained with Hoechst 33342, which stains DNA, the fragmented and condensed nuclei indicative of apoptosis were observed (Fig. 6,D). Therefore, the biological activity of LIC resided in its poly(I)·poly(C) component. CHO-K1 (Fig. 6 F) or BHK21 cells (data not shown) were not killed by the microinjection of poly(I)· poly(C).

Inhibition of Tumor Growth by LIC in Mouse Models of Metastatic Liver Cancer.

After i.v. administration to mice, over 50% of the injected LIC accumulated in the liver (data not shown). This observation led us to speculate that LIC would be effective in the treatment of animal models of liver cancer. We evaluated the antitumor activity of LIC in two different mouse models of metastatic liver cancer. KM12-HX cells (human colon carcinoma cells) were injected intrasplenically into nude mice on day 0, and metastatic liver cancer was observed on day 37 (Fig. 7). The carcinoma cells grew in control mice to give an average liver weight of 2.5 g (the average liver weight in sham-operated mice was 1.5 g). Pathological analysis revealed that the tumor cells in the nodules were poorly differentiated epidermal adenoma cells that produced calcium and formed calcified areas beside the nodules. At this stage, tumor necrosis or apoptosis, which can be brought about by a lack of nutrition, was not seen in the nodules of control mice. LIC was administered i.v. according to two different schedules. Under schedule A (twice weekly for 5 weeks, starting on day 7), LIC inhibited metastatic liver cancer growth in a dose-dependent manner. The inhibition of growth was 98% in the 100 μg/kg group and 48% in the 10 μg/kg group. In mice treated with 100 μg/kg of LIC, few liver-cancer nodules were seen, but the calcified areas remained. Under schedule B (twice weekly for 2 weeks, starting on day 28), the growth inhibition was 84% in the 100 μ g/kg group. A small number of nodules was observed, and the apoptotic bodies of the KM12-HX cells were seen in these nodules. This data suggested that LIC induced the apoptosis of KM12-HX cells and strongly inhibited tumor-cell growth in the liver. We also examined the antiproliferative effect of LIC in another metastatic liver-cancer model using Ls174T cells (human colon carcinoma cells; Fig. 8). In this model, the average liver weight of control mice had reached 2.1 g by day 28. Under schedule A (twice weekly for 3 weeks, starting on day 6), LIC inhibited the growth of Ls174T cells in the liver in a dose-dependent manner. The growth inhibition was 92% in the 100 μ g/kg group and 54% in the 30 μg/kg group. Under schedule B (once daily from day 20 to day 25), the growth inhibition was 54% in the 100 μg/kg group.

DISCUSSION

We used cationic liposomes to facilitate the entry of poly(I)· poly(C) into tumor cells. The presence of a receptor to poly(I)· poly(C) has been postulated and a Mr 60,000 candidate protein was identified (20), but it is not yet clear whether this protein is the actual receptor. In any event, using cationic liposomes, we found that poly(I)·poly(C) strongly inhibited the growth of many kinds of tumor cells in vitro, whereas poly(I)·poly(C) without the cationic liposome did not (Fig. 1). The microinjection of poly(I)·poly(C) into HeLaS3 cells showed a similar cytotoxic effect (Fig. 6). This evidence suggests that intracellular poly(I)·poly(C) induced tumor cell death, and this cytotoxicity is a new biological activity of poly(I)·poly(C). Poly(A-U)·poly(A-U) had almost the same cytotoxic activity as poly(I)·poly(C), whereas poly(A)·poly(U) had a weaker effect. Although poly(A-U)·poly(A-U) and poly(A)·poly(U) both form double-stranded structures, their antiproliferative activities with cationic liposomes were very different. The melting temperature of poly(A-U)·poly(A-U) is higher than that of poly(A)·poly(U) (21); therefore, the stability of the complexes would be expected to be different. Poly(G)· poly(C)/cationic liposome complex, poly(dI)·poly(dC)/cationic liposome complex, and single-stranded RNA/cationic liposome complexes had no cytotoxic activity. Thus, this activity may depend on the three-dimensional structure of dsRNA.

The antiproliferative activity of LIC, in contrast to that of Adriamycin, is characteristic of the cell type. Epithelial and fibroblastic tumor cell lines (adherent cell type) tend to have high sensitivities, whereas leukemia and lymphoma (suspension cell type) do not respond (Table 1). Fibroblasts and normal liver cells were insensitive to LIC (Table 2). Gene-transfection experiments with cationic liposomes reveal a large difference in the transfection efficiency between adherent and suspension cell types (22); therefore, we speculated that sufficient poly(I)·poly(C) could not enter suspension cell types or nonmalignant cell types to inhibit cell growth. To test this idea, we measured the uptake of radiolabeled poly(I)·poly(C) by several cell lines after the addition of LIC (Fig. 5). In three tumor cell lines sensitive to LIC, the incorporation of poly(I)·poly(C) was still increasing 3 h after LIC addition. In contrast, three insensitive cell lines showed very low incorporation of poly(I)·poly(C). This result indicated that differences in the amount of LIC taken up is a possible reason for the differences in cellular sensitivity to LIC. We considered the possibility that poly(I)·poly(C) may merely bind tightly to receptors on the surface of sensitive cells without being internalized. To test this possibility, we repeated the uptake experiment and exposed the cells to RNase A in addition to washing them with PBS, but essentially the same results were obtained.

The microinjection experiment suggested another possible explanation for differences in sensitivity to LIC. HeLaS3 cells (Fig. 6) and A431 cells (data not shown) underwent apoptosis after the microinjection of poly(I)·poly(C). In LIC-sensitive tumor cells, apoptosis occurred if sufficient poly(I)· poly(C) could get inside the cells either by the addition of LIC or by the microinjection of poly(I)·poly(C). LIC-insensitive CHO-K1 cells and BHK-21 cells were not killed by the microinjection of poly(I)·poly(C). In these cells, poly(I)·poly(C) inside the cells did not induce apoptosis. These microinjection experiments suggest that the signal transduction mechanisms of poly(I)·poly(C) in these two cell types is different.

Poly(I)·poly(C) is an IFN inducer in vivo(23) and, under special conditions, in vitro(24), and IFN is reported to inhibit cell growth (27). But in our in vitro experiments, neither IFN-α nor IFN-β inhibited the growth of tumor cell lines as LIC did, even at concentrations up to 1000 IU/ml. Moreover, anti-IFN-α and anti-IFN-β antibodies, which can neutralize the activity of these IFNs, did not reduce the antiproliferative effect of LIC, nor did 1000 IU/ml IFN-β induce DNA fragmentation in HeLaS3 cells. These data indicated that the antiproliferative effect of LIC is not primarily related to IFN induction.

We found that DNA was fragmented and RNA was degraded in tumor cells after the addition of LIC (Figs. 2 and 3). In addition, the nuclease inhibitor ATA suppressed both the antiproliferative activity of LIC (Fig. 4) and the DNA fragmentation it caused (data not shown). The nuclease inhibitors ATA at a concentration of 30 μm and ZnCl2 (at 0.4 mm) inhibited the DNA fragmentation caused by LIC, but cycloheximide (20 μm) did not (data not shown). This observation suggested that poly(I)·poly(C) activated one or more nucleases whose activity was inhibited by ATA or ZnCl2, and that LIC did not induce the synthesis of nucleases or other proteins that activated the nucleases. Therefore, the ability of poly(I)·poly(C) to activate nucleases, both DNases and RNases, seems to be significant. We are now working to identify the nucleases involved. It has already been reported that there are several DNases responsible for apoptosis. Two DNases, nuc-58 and nuc-40, were detected in CTLL2 cells (25), Ca2+/Mg2+-dependent endonuclease was detected in human spleen (26), and DNase II was detected in CHO cells (27). NUC-18 has been purified from rat thymocytes (28, 29) and was found to be homologous with cyclophilin (30). DNase was also identified in rat thymocytes (31), and its DNA sequence has been published (32). Additional studies are needed to determine which nucleases may be the target proteins of poly(I)·poly(C) and what is the mechanism of activation by poly(I)·poly(C).

The apoptosis signaling pathway is mediated by a family of cysteine proteases known as caspases, which cleave other caspases sequentially in the apoptosis cascade (37, 38). In our study, caspase inhibitors did not affect the antiproliferative activity of LIC (Fig. 4); therefore, LIC-induced apoptosis was not related to the activation of caspases.

Apoptosis is reported to be involved in various diseases, including AIDS, hepatitis, neuronal cell death in CAG repeat diseases, and brain or heart injury by ischemia (39, 40, 41). However the induction of apoptosis in specific cell types is potentially a very useful therapy against cancer, viral infection, and autoimmune diseases. Therefore, the antiproliferative activity of LIC against tumor cells is very significant. LIC strongly inhibited the tumor cell growth in two mouse models of metastatic liver cancer, and our histochemical analysis suggests that the effect was through the induction of apoptosis. LIC may, therefore, be a promising candidate for a therapeutic agent to treat metastatic liver cancer.

Fig. 1.

Inhibition of A431 cell growth by nucleic acid polymer/cationic liposome complexes. A431 cells were plated in 96-well microplates at a density of 104 cells/well. Compounds were added to each well and the incubation was continued for 3 days. The numbers of viable cells were determined by MTT assay. A, effect of components of LIC; ○, LIC; ▴, poly(I)-poly(C) alone; □, cationic liposome alone. B, effect of single-stranded RNA/cationic liposome complexes; ○, LIC; ▵, poly(C)/cationic liposome; ▪, poly(I)/cationic liposome. C, effect of dsRNA/cationic liposome complexes; ○, LIC; •, poly(A-U)·poly(A-U)/cationic liposome; □, poly(A)·poly(U)/cationic liposome; ▵, poly(G)· poly(C)/cationic liposome; ▪, poly(dl)·(dC)/cationic liposome.

Fig. 1.

Inhibition of A431 cell growth by nucleic acid polymer/cationic liposome complexes. A431 cells were plated in 96-well microplates at a density of 104 cells/well. Compounds were added to each well and the incubation was continued for 3 days. The numbers of viable cells were determined by MTT assay. A, effect of components of LIC; ○, LIC; ▴, poly(I)-poly(C) alone; □, cationic liposome alone. B, effect of single-stranded RNA/cationic liposome complexes; ○, LIC; ▵, poly(C)/cationic liposome; ▪, poly(I)/cationic liposome. C, effect of dsRNA/cationic liposome complexes; ○, LIC; •, poly(A-U)·poly(A-U)/cationic liposome; □, poly(A)·poly(U)/cationic liposome; ▵, poly(G)· poly(C)/cationic liposome; ▪, poly(dl)·(dC)/cationic liposome.

Close modal
Fig. 2.

DNA fragmentation by LIC in A431 cells and KM12-HX cells (A) and the extent of DNA fragmentation in A431 cells (B). In A, each cell line was treated with LIC. The cells were harvested after 5 h (A431) or 7.5 h (KM12-HX), lysed with Triton X-100, and centrifuged to separate fragmented DNA from intact chromatin. The fragmented DNA was then extracted with phenol-chloroform and electrophoresed on 1.8% agarose gels. In B, the fragmented DNA was extracted as described for A at the time periods indicated. The extent of DNA fragmentation was defined as the radioactivity of the fragmented DNA as a percentage of that of the total DNA.

Fig. 2.

DNA fragmentation by LIC in A431 cells and KM12-HX cells (A) and the extent of DNA fragmentation in A431 cells (B). In A, each cell line was treated with LIC. The cells were harvested after 5 h (A431) or 7.5 h (KM12-HX), lysed with Triton X-100, and centrifuged to separate fragmented DNA from intact chromatin. The fragmented DNA was then extracted with phenol-chloroform and electrophoresed on 1.8% agarose gels. In B, the fragmented DNA was extracted as described for A at the time periods indicated. The extent of DNA fragmentation was defined as the radioactivity of the fragmented DNA as a percentage of that of the total DNA.

Close modal
Fig. 3.

Fragmentation of rRNA by LIC in five different tumor cell lines. Five tumor cell lines were treated with LIC for 4 h. Cells were harvested and ribosomal fractions prepared; then RNA was extracted with acid/guanidinium/phenol/chloroform. The rRNA was electrophoresed on a 1.8% agarose gel containing formaldehyde and stained with ethidium bromide. Arrows, the bands of fragmented rRNA.

Fig. 3.

Fragmentation of rRNA by LIC in five different tumor cell lines. Five tumor cell lines were treated with LIC for 4 h. Cells were harvested and ribosomal fractions prepared; then RNA was extracted with acid/guanidinium/phenol/chloroform. The rRNA was electrophoresed on a 1.8% agarose gel containing formaldehyde and stained with ethidium bromide. Arrows, the bands of fragmented rRNA.

Close modal
Fig. 4.

Effect of a nuclease inhibitor or caspase inhibitors on the antiproliferative activity of LIC against A431 cells. A431 cells were plated in 96-well microplates at a density of 104 cells/well. Inhibitors were added to each well 2 h before the treatment with LIC, and the incubation was continued for 3 days. The numbers of viable cells were determined by MTT assay. •, without inhibitor (LIC only); , LIC plus 30μm ATA (a nuclease inhibitor); , LIC plus 100 μm DEVD-fmk (a caspase 3 inhibitor); LIC plus 100 μ m z-VAD-fmk (a caspase inhibitor).

Fig. 4.

Effect of a nuclease inhibitor or caspase inhibitors on the antiproliferative activity of LIC against A431 cells. A431 cells were plated in 96-well microplates at a density of 104 cells/well. Inhibitors were added to each well 2 h before the treatment with LIC, and the incubation was continued for 3 days. The numbers of viable cells were determined by MTT assay. •, without inhibitor (LIC only); , LIC plus 30μm ATA (a nuclease inhibitor); , LIC plus 100 μm DEVD-fmk (a caspase 3 inhibitor); LIC plus 100 μ m z-VAD-fmk (a caspase inhibitor).

Close modal
Fig. 5.

Uptake of LIC by various cell lines. Cells were exposed to 1 μg/ml [3H]-labeled LIC for the time periods indicated. The radioactivity associated with the cells was determined as described in “Materials and Methods.”, HCC-T; SK-HEP-1; , A431; •, human primary hepatocyte; ▪, CHO-K1; ▴, U937.

Fig. 5.

Uptake of LIC by various cell lines. Cells were exposed to 1 μg/ml [3H]-labeled LIC for the time periods indicated. The radioactivity associated with the cells was determined as described in “Materials and Methods.”, HCC-T; SK-HEP-1; , A431; •, human primary hepatocyte; ▪, CHO-K1; ▴, U937.

Close modal
Fig. 6.

Microinjection of poly(l)·poly(C) into HelaS3 cells and CHO-K1 cells. Cells were microinjected with poly(l)·poly(C) by a transinjector under a microscope. The nucleus was stained with Hoechst 33342. HelaS3 cells are shown 30 min (A and C) and 24 h (B and D) after microinjection. HeLaS3 cells were stained with Hoechst 33342 (C and D). CHO-K1 cells are shown 30 min (E) and 24 h (F) after microinjection.

Fig. 6.

Microinjection of poly(l)·poly(C) into HelaS3 cells and CHO-K1 cells. Cells were microinjected with poly(l)·poly(C) by a transinjector under a microscope. The nucleus was stained with Hoechst 33342. HelaS3 cells are shown 30 min (A and C) and 24 h (B and D) after microinjection. HeLaS3 cells were stained with Hoechst 33342 (C and D). CHO-K1 cells are shown 30 min (E) and 24 h (F) after microinjection.

Close modal
Fig. 7.

Inhibition of tumor growth (KM12-HX) by LIC in a mouse model of metastatic liver cancer (A) and the appearance of their livers on day 37 (B). KM12-HX cells (106 cells/mouse) were intrasplenically inoculated into nude mice on day 0. LIC was administered under two different schedules as described in “Materials and Methods.” The liver weights were measured on day 37. Columns (A), mean ± SE (n = 6); **, significantly different from control by Dunnett’s test; P < 0.01.

Fig. 7.

Inhibition of tumor growth (KM12-HX) by LIC in a mouse model of metastatic liver cancer (A) and the appearance of their livers on day 37 (B). KM12-HX cells (106 cells/mouse) were intrasplenically inoculated into nude mice on day 0. LIC was administered under two different schedules as described in “Materials and Methods.” The liver weights were measured on day 37. Columns (A), mean ± SE (n = 6); **, significantly different from control by Dunnett’s test; P < 0.01.

Close modal
Fig. 8.

Inhibition of tumor growth (Ls174T) by LIC in a mouse model of metastatic liver cancer (A) and the appearance of their livers on day 28 (B). Ls174T cells (3 × 106 cells/mouse) were intrasplenically inoculated into nude mice on day 0. LIC was administered under two different schedules as described in “Materials and Methods.” The liver weights were measured on day 28. Columns (A), mean ± SE (n = 6); **, significantly different from control by Dunnett’s test; P < 0.01; *, significantly different from control by Dunnett’s test; P < 0.05.

Fig. 8.

Inhibition of tumor growth (Ls174T) by LIC in a mouse model of metastatic liver cancer (A) and the appearance of their livers on day 28 (B). Ls174T cells (3 × 106 cells/mouse) were intrasplenically inoculated into nude mice on day 0. LIC was administered under two different schedules as described in “Materials and Methods.” The liver weights were measured on day 28. Columns (A), mean ± SE (n = 6); **, significantly different from control by Dunnett’s test; P < 0.01; *, significantly different from control by Dunnett’s test; P < 0.05.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

The abbreviations used are: dsRNA, double-stranded RNA; poly(I), polyinosinic acid; poly(C), polycytidylic acid; poly(A), polyadenylic acid; poly(U), polyuridylic acid; poly(G), polyguanylic acid; poly(dI), deoxyinosinic acid; poly(dC), deoxycytidylic acid; LIC, poly(I)•poly(C)/cationic liposome complex; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ATA, aurintricarboxylic acid; DEVD-fmk, Asp-Glu-Val-Asp-fluoromethyl ketone; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone; ICE, interleukin-1β converting enzyme.

3

Unpublished data.

Table 1

Inhibition of the growth of tumor cell lines by LIC or Adriamycin

Each cell line was seeded at a density of 104 cells/well (96-well plates). After 5 h, drugs were added to each well, and the incubation was continued. The number of cells surviving after 3 days was determined by MTT assay. The percentage inhibition was calculated, and the activity of the compound was tabulated as the IC50.
Cell lineCell typeIC50 (ng/ml)
LICADRa
MCF7 Breast adenocarcinoma 0.09 73 
A-204 Rhabdomyosarcoma 0.71 76 
Malme 3M Malignant melanoma 0.80 114 
MDA-MB-468 Breast adenocarcinoma 0.89 57 
LoVo Colon adenocarcinoma 1.3 65 
RT4 Bladder papilloma 1.6 58 
T47D Breast ductal carcinoma 2.1 114 
OVCAR3 Ovary adenocarcinoma 2.4 >1000 
HT1376 Bladder carcinoma 4.3 217 
MDA-MB-453 Breast carcinoma 5.4 143 
A431 Epidermoid carcinoma 5.7 52 
PC-3 Prostate adenocarcinoma 6.4 >1000 
RD Rhabdomyosarcoma 6.9 208 
SK-BR-3 Breast adenocarcinoma 8.1 78 
HeLaS3 Cervix epitheloid carcinoma 8.6 49 
Ls174T Colon adenocarcinoma 8.8 96 
MDA-MB-134VI Breast ductal carcinoma 14 81 
WM-266-4 Melanoma 15 206 
CALU6 Lung anaplastic carcinoma 21 282 
TSU-PR-1 Prostate cancer 26 572 
KM12-HX Colon cancer 33 130 
SAOS2 Osteogenic sarcoma 34 75 
SK-HEP-1 Liver adenocarcinoma 38 302 
HT144 Malignant melanoma 38 219 
DU145 Prostate carcinoma 39 236 
COLO320DM Colon adenocarcinoma 40 838 
SK-OV-3 Ovary adenocarcinoma 40 252 
G-361 Malignant melanoma 52 146 
SW480 Colon adenocarcinoma 53 97 
KB Oral epidermoid carcinoma 71 25 
NC65 Pancreatic cancer 137 73 
293 AD 5 transformed embryonic kidney cell 145 39 
HCC-T Liver cancer 153 76 
BALB/c AMuLVA.6R.1 Virus-transformed BALB/c CL.7 199 37 
PLC/PRF/5 Hepatoma 207 42 
KBC.1 Drug-resistant oral epidermoid carcinoma 209 >1000 
A546 Lung carcinoma 263 831 
AsPC-1 Pancreatic adenocarcinoma 268 800 
Hs0578T Breast ductal carcinoma 282 760 
A673 Rhabdomyosarcoma 407 104 
HepG2 Hepatocellular carcinoma 581 465 
MDA-MB-231 Breast adenocarcinoma 735 >1000 
WiDr Colon adenocarcinoma 1000 157 
U-937 Histiocytic lymphoma >1000 39 
K-562 Chronic myelogenous leukemia >1000 200 
MOLT-4 Acute lymphoblastic leukemia >1000 24 
CCRF-CEM Acute lymphoblastic leukemia >1000 110 
HL-60 Promyelocytic leukemia >1000 52 
MOLT-3 Acute lymphoblastic leukemia >1000 13 
Each cell line was seeded at a density of 104 cells/well (96-well plates). After 5 h, drugs were added to each well, and the incubation was continued. The number of cells surviving after 3 days was determined by MTT assay. The percentage inhibition was calculated, and the activity of the compound was tabulated as the IC50.
Cell lineCell typeIC50 (ng/ml)
LICADRa
MCF7 Breast adenocarcinoma 0.09 73 
A-204 Rhabdomyosarcoma 0.71 76 
Malme 3M Malignant melanoma 0.80 114 
MDA-MB-468 Breast adenocarcinoma 0.89 57 
LoVo Colon adenocarcinoma 1.3 65 
RT4 Bladder papilloma 1.6 58 
T47D Breast ductal carcinoma 2.1 114 
OVCAR3 Ovary adenocarcinoma 2.4 >1000 
HT1376 Bladder carcinoma 4.3 217 
MDA-MB-453 Breast carcinoma 5.4 143 
A431 Epidermoid carcinoma 5.7 52 
PC-3 Prostate adenocarcinoma 6.4 >1000 
RD Rhabdomyosarcoma 6.9 208 
SK-BR-3 Breast adenocarcinoma 8.1 78 
HeLaS3 Cervix epitheloid carcinoma 8.6 49 
Ls174T Colon adenocarcinoma 8.8 96 
MDA-MB-134VI Breast ductal carcinoma 14 81 
WM-266-4 Melanoma 15 206 
CALU6 Lung anaplastic carcinoma 21 282 
TSU-PR-1 Prostate cancer 26 572 
KM12-HX Colon cancer 33 130 
SAOS2 Osteogenic sarcoma 34 75 
SK-HEP-1 Liver adenocarcinoma 38 302 
HT144 Malignant melanoma 38 219 
DU145 Prostate carcinoma 39 236 
COLO320DM Colon adenocarcinoma 40 838 
SK-OV-3 Ovary adenocarcinoma 40 252 
G-361 Malignant melanoma 52 146 
SW480 Colon adenocarcinoma 53 97 
KB Oral epidermoid carcinoma 71 25 
NC65 Pancreatic cancer 137 73 
293 AD 5 transformed embryonic kidney cell 145 39 
HCC-T Liver cancer 153 76 
BALB/c AMuLVA.6R.1 Virus-transformed BALB/c CL.7 199 37 
PLC/PRF/5 Hepatoma 207 42 
KBC.1 Drug-resistant oral epidermoid carcinoma 209 >1000 
A546 Lung carcinoma 263 831 
AsPC-1 Pancreatic adenocarcinoma 268 800 
Hs0578T Breast ductal carcinoma 282 760 
A673 Rhabdomyosarcoma 407 104 
HepG2 Hepatocellular carcinoma 581 465 
MDA-MB-231 Breast adenocarcinoma 735 >1000 
WiDr Colon adenocarcinoma 1000 157 
U-937 Histiocytic lymphoma >1000 39 
K-562 Chronic myelogenous leukemia >1000 200 
MOLT-4 Acute lymphoblastic leukemia >1000 24 
CCRF-CEM Acute lymphoblastic leukemia >1000 110 
HL-60 Promyelocytic leukemia >1000 52 
MOLT-3 Acute lymphoblastic leukemia >1000 13 
a

ADR, Adriamycin.

Table 2

The effect of LIC or Adriamycin on the growth of nontumor cell lines

Inhibition of growth was measured as described in Table 1.
Cell lineCell typeIC50 (ng/ml)
LICADRa
System-Hc cells Human primary hepatocytes >1000 190 
WRL68 Human embryonic liver cell >1000 283 
BNL CL.2 Mouse embryonic liver cell >1000 398 
Clone 9 Rat normal liver cell >1000 228 
Swiss/3T3 Mouse embryonic cell >1000 903 
NIH/3T3 Mouse embryonic cell >1000 102 
BALB/c CL.7 Mouse normal fibroblast >1000 117 
BALB/c3T3 cloneA31 Mouse embryonic cell >1000 427 
NRK-49F Rat normal kidney fibroblast >1000 >1000 
CHO-K1 Chinese hamster ovary cell >1000 20 
BHK-21 Hamster kidney cell >1000 47 
Inhibition of growth was measured as described in Table 1.
Cell lineCell typeIC50 (ng/ml)
LICADRa
System-Hc cells Human primary hepatocytes >1000 190 
WRL68 Human embryonic liver cell >1000 283 
BNL CL.2 Mouse embryonic liver cell >1000 398 
Clone 9 Rat normal liver cell >1000 228 
Swiss/3T3 Mouse embryonic cell >1000 903 
NIH/3T3 Mouse embryonic cell >1000 102 
BALB/c CL.7 Mouse normal fibroblast >1000 117 
BALB/c3T3 cloneA31 Mouse embryonic cell >1000 427 
NRK-49F Rat normal kidney fibroblast >1000 >1000 
CHO-K1 Chinese hamster ovary cell >1000 20 
BHK-21 Hamster kidney cell >1000 47 
a

ADR, Adriamycin.

We thank Dr. Mitsuo Oshimura of Tottori University for valuable discussions and Dr. Hideaki Saito of Keio University for providing HCC-T cells.

1
Ball J. K., McCarter J. A. Effect of polyinosinic-polycytidylic acid on induction of primary or transplanted tumors by chemical carcinogen or irradiation.
J. Natl. Cancer Inst.
,
46
:
1009
-1014,  
1971
.
2
Bloksma N., Kuper C. F., Hofhuis F. M. A., Benaissa-Trouw B., Willers J. M. N. Antitumor activity of endotoxin, concanavalin A and poly I:C and their ability to elicit tumor necrosis factor, cytostatic factors and interferon in vivo.
Cancer Immunol. Immunother.
,
16
:
35
-39,  
1983
.
3
Davies M. E., Field A. K Effect of poly I:C/poly-l-lysine (poly ICL) on the development of murine osteogenic sarcoma.
J. Interferon Res.
,
3
:
89
-95,  
1983
.
4
DeClercq E., Zhang Z-X., Huygen K., Leyten R. Inhibitory effect of interferon on the growth of spontaneous mammary tumors in mice.
J. Natl. Cancer Inst.
,
69
:
653
-657,  
1982
.
5
Levy H. B., Asofsky R., Riley F., Garapin A., Cantor H., Adamson R. The mechanism of the antitumor action of poly(I)· poly(C).
Ann. NY Acad. Sci.
,
173
:
640
-648,  
1970
.
6
Youn J. K., Kim B. S., Min J. S., Lee K. S., Choi H. J., Lee Y. B., Lee D. W., Koh E. H., Kim K. W., Lee K. B., Michelson A. M. Adjuvant treatment of operable stomach cancer with polyadenylic.polyuridylic acid in addition to chemotherapeutic agents. Differential effect on natural killer cell and antibody-dependent cellular cytotoxicity.
Int. J. Immunopharmacol.
,
9
:
313
-324,  
1987
.
7
Greene J. J., Ts’o P. O. P., Strayer D. R., Carter W. A. Therapeutic applications of double-stranded RNAs Strayer D. R. eds. .
Interferons and Their Applications
,
:
535
-555, Springer-Verlag Inc. New York  
1984
.
8
Tramelli D., Varesio L. Activation of murine macrophages. 1. Different pattern of activation by poly-I:C than by lymphokine or LPS.
J. Immunol.
,
127
:
58
-63,  
1981
.
9
Weiss R. A., Meunier P. C., Kelley A., Klinkner A., Badger A. M., Bugelski P. J. Activation of rat pulmonary lavage cells by intratracheal administration of polyinosinic-polycytidylic acid.
J. Biol. Response Modif.
,
9
:
411
-419,  
1990
.
10
Kerr I., Brown R. ppA2′p5′AZp5′A: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon treated cells.
Proc. Natl. Acad. Sci. USA
,
75
:
256
-260,  
1978
.
11
Kerr I., Brown R., Hovanessian A. Nature of inhibitor of cell free protein synthesis formed in response to interferon double stranded RNA.
Nature (Lond.)
,
268
:
540
-542,  
1977
.
12
Revel M. Interferon induced translation regulation.
Tex. Rep. Biol. Med.
,
35
:
212
-220,  
1977
.
13
Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofectin: a highly efficient, lipid-mediated DNA-transfection procedure.
Proc. Natl. Acad. Sci. USA
,
84
:
7413
-7417,  
1987
.
14
Gao X., Huang L. A novel cationic liposome reagent for efficient transfection of mammalian cells.
Biochem. Biophys. Res. Commun.
,
179
:
280
-285,  
1991
.
15
Zabner J., Fasbender A. J., Moninger T., Poellinger K. A., Welsh M. J. Cellular and molecular barriers to gene transfer by a cationic lipid.
J. Biol. Chem.
,
270
:
18997
-19007,  
1995
.
16
Pinnaduage P., Schmitt L., Huang L. Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse l-cells.
Biochim. Biophys. Acta
,
985
:
33
-37,  
1989
.
17
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
J. Immunol. Methods
,
65
:
55
-63,  
1983
.
18
Tsuji T., Nakamura T. .
Jikken Igaku
,
9
:
99
-101,  
1991
.
19
Blumenthal T., Landers T. A. The inhibition of nucleic acid-binding proteins by aurintricarboxylic acid.
Biochem. Biophys. Res. Commun.
,
55
:
680
-688,  
1973
.
20
Yoshida I., Azuma M., Kawai H., Fisher H. W., Suzutani T. Identification of a cell membrane receptor for interferon induction by poly rI:rC.
Acta Virol.
,
36
:
347
-358,  
1992
.
21
Arnott S., Selsing E. Structures for the polynucleotide complexes poly(dA)·poly(dT) and poly(dT)poly(dA)·poly(dT).
J. Mol. Biol.
,
88
:
509
-533,  
1974
.
22
Farhood H., Bottega R., Epand R. M., Huang L. Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity.
Biochim. Biophys. Acta
,
1111
:
239
-246,  
1992
.
23
DeClercq E., Merigan T. C. Local and systemic protection by synthetic polyanionic interferon inducers in mice against vescular stomatitis virus.
J. Gen. Virol.
,
5
:
359
-367,  
1969
.
24
Van Damme J., Billiau A. Large-scale production of human fibroblast interferon.
Methods Enzymol.
,
78
:
101
-119,  
1981
.
25
Deng G., Podack E. R. Deoxyribonuclease induction in apoptotic cytotoxic T lymphocytes.
FASEB J.
,
9
:
665
-669,  
1995
.
26
Meireeles J., Carson D. A. Ca2+/Mg2+-dependent endonuclease from human spleen: purification, properties, and role in apoptosis.
Biochemistry
,
32
:
9129
-9136,  
1993
.
27
Barry M. A., Eastman A. Identification of deoxyribonuclease II as an endonuclease involved in apoptosis.
Arch. Biochem. Biophys.
,
300
:
440
-450,  
1993
.
28
Compton M. M., Cidlowski J. A. Identification of a glucocorticoid induced nuclease in thymocytes.
J. Biol. Chem.
,
262
:
8288
-8292,  
1987
.
29
Gaido M. L., Cidlowski J. A. Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes.
J. Biol. Chem.
,
266
:
18580
-18585,  
1991
.
30
Montague J. W., Gaido M. L., Frye C., Cidlowski J. A. A calcium-dependent nuclease from apoptotic rat thymocytes is homologous with cyclophilin.
J. Biol. Chem.
,
269
:
18877
-18880,  
1994
.
31
Shiokawa D., Ohyama H., Yamada T., Takahashi K., Tanuma S. Identification of endonuclease responsible for apoptosis in rat thymocytes.
Eur. J. Biochem.
,
226
:
23
-30,  
1994
.
32
Shiokawa D., Tanuma S. Molecular cloning and expression of a cDNA encoding an apoptotic endonuclease DNase γ.
Biochem. J.
,
332
:
713
-720,  
1998
.
33
Enari M., Sakahira H., Yokoyama H., Okawa K., Iwamatsu A., Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.
Nature (Lond.)
,
391
:
43
-50,  
1998
.
34
Sakahira H., Enari M., Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis.
Nature (Lond.)
,
391
:
96
-99,  
1998
.
35
Enari M., Talanian R. V., Wong W. W., Nagata S. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis.
Nature (Lond.)
,
380
:
723
-726,  
1996
.
36
Enari M., Hase A., Nagata S. Apoptosis by a cytosolic extract from Fas-activated cells.
EMBO J.
,
14
:
5201
-5208,  
1995
.
37
Nagata S. Apoptosis by death factor.
Cell
,
88
:
355
-365,  
1997
.
38
Fraser A., Evan G. A license to kill.
Cell
,
85
:
781
-784,  
1996
.
39
Webb S. J., Harrison D. J., Wyllie A. H. Apoptosis. an overview of the process and its relevance in disease.
Adv. Pharmacol.
,
41
:
1
-34,  
1997
.
40
Solary E., Dubrez L., Eymin B. The role of apoptosis in the pathogenesis and treatment of diseases.
Eur. Respir. J.
,
9
:
1293
-1305,  
1996
.
41
McConkey D. J., Zhivotovsky B., Orrenius S. Apoptosis-molecular mechanisms and biomedical implications.
Mol. Aspects Med.
,
17
:
1
-110,  
1996
.