Inhibition of Retrovirus-Induced Disease in Mice by Camptothecin

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1 JOURNAL OF VROLOGY, June 1993, p X/93/ $02.00/0 Copyright 1993, American Society for Microbiology Vol. 67, No. 6 nhibition of Retrovirus-nduced Disease in Mice by Camptothecin ESTHER PREL,1* ESTHER AFLALO,1 GALNA CHECHELNTSKY,l DANEL BENHARROCH,2 MORDECHA ABOUD,' AND SHRAGA SEGAL' Department of mmunology and Microbiology, Faculty of Health Sciences,' and nstitute of Pathology, Soroka Medical Center and Faculty of Health Sciences,2 Ben-Gurion University, Beer-Sheva, srael Received 11 December 1992/Accepted 26 February 1993 We have previously shown that noncytotoxic doses of camptothecin (CPT), a topoisomerase -specific antagonist, inhibit retrovirus replication in acutely and chronically infected cells. To evaluate the efficacy of CPT as an antiretroviral drug in vivo, we injected newborn BALB/c mice with Moloney murine leukemia virus and adult NFS mice with Friend spleen focus-forming virus. The Moloney murine leukemia virus-injected mice developed lymphoma, and the Friend spleen focus-forming virus-injected mice developed erythroleukemia. CPT, administrated together with the virus or 1 or 2 days after virus injection, prevented the onset of the disease in both cases. We showed that repeated CPT treatments increased the effectiveness of the drug when administrated 3 days after virus injection. This ability of CPT to inhibit retrovirus-induced disease in vivo without causing any apparent toxic side effects suggests its application as a legitimate remedy for the treatment of retroviral diseases. The clinical importance of retroviruses which cause disease in humans (i.e., human immunodeficiency virus [HV] and human T-cell leukemia virus) and animals (i.e., equine infectious anemia virus [EAV] and Moloney murine leukemia virus [Mo-MuLV]) has led to intensive attempts to identify specific inhibitors. One basis for developing antiretroviral drugs is a deeper understanding of the viral life cycle and of the proteins and enzymes involved in its replication. We have shown previously that topoisomerase (topo ) activity is associated with retroviral particles (HV and EAV) and that in the case of EAV, this activity is present in the viral core (18). The virus-associated topo activity was inhibited by camptothecin (CPT), a known specific inhibitor of eukaryotic topo (1, 6, 9, 13). The association of topo activity with the viral particles and the possibility that this enzyme is involved in the viral life cycle led us to examine the effect of CPT on retroviral infection. We demonstrated that noncytotoxic doses of CPT inhibited HV replication in acute infection of H9 cells at a high efficacy (>90%) (16). Moreover, we found that CPT inhibited the replication of EAV in chronically infected Cf2Th cells. Continuous exposure of these cells to the drug for 52 days revealed 85 to 92% inhibition of virus production. No effect on the viability or growth rate of the cells was detected (17). CPT is a cytotoxic alkaloid, isolated from Camptotheca acuminata, which has strong antitumor activity against a wide range of experimental tumors (3, 4, 23). n cytotoxic doses, CPT inhibits RNA and DNA synthesis and causes rapid and reversible fragmentation of DNA in mammalian cells (8, 11, 21, 23). These pleiotropic effects were shown to be mediated via a single cellular target, topo (reviewed in reference 13). Our in vitro results indicate that CPT can act as an antiviral drug at noncytotoxic doses. However, an accurate portrayal of human diseases cannot rely exclusively on extrapolations from in vitro data. Therefore, experiments * Corresponding author with animal models are essential for evaluating the efficacy of a specific drug in vivo. We examined the effect of CPT as an antiretroviral drug in mouse models using two different retroviruses Mo-MuLV and Friend spleen focus forming virus (SFFV). Mo-MuLV is a type C retrovirus which cause leukemia in adult mice after a long latency period (reviewed in reference 24). Newborn mice that are given injections of the virus develop lymphoma 2 to 3 months after viral injection (12). Friend murine leukemia retrovirus complex is composed of a replication-competent Friend murine leukemia helper virus and an acutely transforming, replicationdefective spleen focus-forming virus (2, 10, 22). Friend murine leukemia retrovirus complex caused rapid development of erythroleukemia associated with severe immunosuppression when inoculated into newborn or adult mice of susceptible strains. The mice inoculated with SFFV developed the disease 3 to 4 weeks after injection and died after 6 to 7 weeks, thus enabling us to determine the effect of CPT in vivo in a relatively short time. Effect of CPT on Mo-MuLV replication in acutely infected NH 3T3 cells. Before analyzing the effect of CPT on in vivo Mo-MuLV infection, we determined its effect on this virus under in vitro conditions. For this purpose, NH 3T3 cells were infected with MOV3(neo) (a mixture of MOV3, which is a clone of Mo-MuLV [7], and Mo-MuLV bearing the neomycin resistance [Neorj gene) (7). Cells (105 per plate) treated with Polybrene (8,ug/ml) were infected with 5 x 106 PFU of the virus in the presence of various CPT concentrations (0.002 to 2,uM) for 1 h. We found that treatment of the cells with to 0.2,uM CPT was not toxic to the cells (data not shown). Virus-infected cells were selected by growing the cells in the presence of G418 (400,ug/ml), and the number of neomycin-resistant foci was determined. Noncytotoxic CPT doses were found to inhibit Neor focus formation by 40 to 88% (Table 1). To further substantiate the effect of CPT on viral replication, we used the infectious-center technique. Using this method, we could determine the number of virus-producing cells obtained after CPT treatment. For this

2 VOL. 67, 1993 TABLE 1. Effects of various CPT doses on acute infection of NH 3T3 cells by MOV3 (neo) CPT concn No. of Neor % (LM) focia nhibitiona 0 1, ± ± ± ± ± 6.0 a All data presented are means ± standard deviations from three different experiments. purpose, NH 3T3 cells were infected with MOV3 in the presence of various CPT doses. Twenty-four hours after infection, the cells were suspended by trypsinization, and 100,ul of serial dilutions of each suspension was applied onto plates containing subconfluent cultures of FG10 cells (which contain a replication-defective Moloney murine sarcoma virus). The virus produced by the NH 3T3 cells can infect the FG10 cells, and transformed foci will be obtained. The number of the transformed foci obtained is directly correlated with the number of NH 3T3 cells which produce virus. As can be seen in Table 2, noncytotoxic doses of CPT inhibited the formation of infectious centers by 60 to 85%. Effect of CPT on Mo-MuLV-induced disease in newborn mice. To evaluate the in vivo effect of CPT, we analyzed its influence on the development of the disease caused by Mo-MuLV in mice. Newborn BALB/c mice were inoculated with 3 x 105 PFU (19) of Mo-MuLV 24 to 72 h after birth. These mice developed splenomegaly 10 weeks after viral injection. Although it has been claimed that this virus may induce thymic lymphoma (12), in our studies no significant changes in the appearance of the thymus in our BALB/c mice were observed. Figure 1 demonstrates that the average weight of spleens of Mo-MuLV-inoculated mice was mg, while injection of the virus together with CPT (1 mg/kg of body weight) revealed an average spleen weight of 188 ± 60 mg. TABLE 2. Effects of various CPT doses on acute infection of NH 3T3 cells by Mo-MuLV as determined by infectious-center assay Amt of virus CPT concn No. of No. of % (PFU) (GM) transformed focia coloniese nhibition 2 x , x ,330 ± 1, , , ,660 ± ± 1 None 0 2,400 ± ,500 ± ,128 ± ± , ± ± ,550 ± ,600 ± a The number of foci observed was multiplied by the dilution factor. All data are means + standard deviations from three different experiments. " Serial dilutions of uninfected, CPT-treated NH 3T3 cells were made. One hundred microliters of each dilution was applied onto plates, and the number of colonies was determined e00[ NOTES FG. 1. Effect of a single CPT dose on newborn mice inoculated with Mo-MuLV. Newborn BALB/c mice were inoculated with 0.1 ml of medium containing Mo-MuLV (3 x 105 PFU) ( ) or Mo-MuLV plus CPT (1 mg/kg) ( E ). Control uninfected mice were injected with CPT (1 mg/kg) alone ( E ) or 0.1% DMSO (_). Ten weeks post-virus injection, the mice were sacrificed, and their spleens were weighed. Each bar represents the mean spleen weight ± the standard deviation for 10 to 15 mice. The average spleen weight of the uninfected control mice was 100 ± 10 mg. Mice inoculated with 1 mg of CPT alone per kg did not show any effect on the spleen size or any visible side effects. Histopathological analysis clearly showed that the spleens from Mo-MuLV-inoculated mice were heavily populated with lymphoma cells, while the spleens from CPT-treated mice showed the normal spleen cell population (compare Fig. 2A through D). We wanted to see whether repeated CPT treatment would improve the efficacy of the drug. As can be seen in Fig. 3, injection of CPT every 5 days abolished splenomegaly completely. The average spleen weight of mice receiving three drug injections was 150 ± 10 mg. n order to evaluate the efficacy of CPT administered after virus injection, newborn BALB/c mice were given an injection of Mo-MuLV, and CPT was administrated in a single dose 1, 2, or 3 days after virus injection. Figure 4 indicates that CPT administration 1 or 2 days after virus injection prevents or significantly reduces development of splenomegaly, while injection of CPT 3 days after virus injection reduced spleen enlargement by 25%. Since a single CPT dose given 3 days after virus infection was insufficient to reduce splenomegaly, we treated the mice with additional CPT doses 9 and 15 days after virus injection. As can be seen in Fig. 4, the repeated CPT treatment greatly reduced spleen enlargement. Examination of the internal organs revealed that a single CPT injection or repeated CPT injections had no visible toxic effects (no accumulation of peritoneal exudate, hematocrit changes, or necrotic areas in the liver, spleen, or lungs were observed). These results indicate that CPT treatments given a few days after viral infection are effective in preventing or greatly reducing the development of disease caused by Mo-MuLV. Although histological examination of CPT-treated spleens revealed a normal spleen cell population, it is still possible that a few transformed cells are present in the spleen. n order to examine this possibility, spleens from virus-inoculated mice that were either treated with CPT or left untreated were removed, cell suspensions were prepared, and 107 cells were injected into adult BALB/c mice. The spleen weight of mice given injections of spleen cell suspension from virus-

3 3626 NOTES J. VROL. FG. 2. Histopathological examination of spleens from Mo-MuLV-infected mice. Spleens were removed from uninfected mice (A), Mo-MuLV-infected mice (B), and Mo-MuLV- and CPT-treated mice (C and D). Histopathological examination was performed. (A) Photomicrograph of a normal mouse spleen. The lower right portion is occupied by the white pulp. n the upper left portion, megakaryocytes and precursors of the erythrocyte series feature extramedullary hematopoiesis. Magnification, x300. (B) High-power view of a large-cell lymphoma of an Mo-MuLV-treated mouse. The lymphomatous infiltrate almost totally replaces the splenic tissue. Magnification, x300. (C) Photomicrograph of an Mo-MuLV- and CPT-treated mouse spleen. The normal architecture of the spleen, with white pulp and red pulp, is visible. Magnification, x75. (D) High-power view of the white pulp of an Mo-MuLV- and CPT-treated mouse spleen. The population is composed mainly of small round lymphocytes. Magnification, x300. All samples were stained with hematoxylin and eosin. inoculated, non-cpt-treated mice was mg, while mice given injections of cells from CPT-treated mice had an average spleen weight of mg. These results indicate that CPT-treated spleens did not contain transformed cells; this substantiates our hypothesis that CPT inhibits the onset of lymphoma caused by Mo-MuLV and may act as an antiretroviral drug with high efficacy. nhibition of Friend virus-induced disease in mice by CPT. n order to determine the effectiveness of CPT treatment on retrovirus-induced disease in adult mice, the Friend virus- SFFV-infected mouse model was used. A well-characterized stock of SFFV in complex with an amphotropic Friend murine type C virus, which induces only a low incidence of lymphoid and myeloid disease after a long latency (0.1 ml; 105 PFU), was injected intraperitoneally into adult NFS mice. NFS mice were inoculated intraperitoneally in the presence of CPT (5 mg/kg) or 1% dimethyl sulfoxide (DMSO) (CPT was solubilized in DMSO; thus, the same DMSO concentration was injected into the virus-inoculated mice). To determine the efficacy of CPT administrated after

4 VOL. 67, 1993 NOTES 3627 FG. 2-Continued. SFFV inoculation, mice were inoculated intraperitoneally with 5 mg of CPT per kg 3 days after virus infection. Figure 5 demonstrates that CPT administrated concomitantly with the virus prevented the onset of splenomegaly. Moreover, injection of the drug 3 days after virus inoculation was still very effective. The CPT dose used did not produce any visible toxic effect. n conclusion, it is of interest to note that while our previous studies have demonstrated that CPT inhibits the replication of two lentiviruses, HV and EAV (16, 17), the present experiments show a similar effect of this drug with type C retroviruses, indicating that CPT is a general antiretroviral agent. We demonstrated in this study that CPT could efficiently prevent the pathogenicity of two different retroviruses, Mo-MuLV and SFFV, in mice at nontoxic doses. Single doses of the drug given together with the virus or 1 or 2 days after virus infection completely prevented induction of splenomegaly by either of these viruses. t should be noted that we kept virus-inoculated and CPT-treated mice for 15 months and did not observe any splenomegaly, indicating that CPT prevents the onset of the disease rather than just delaying it. However, our results indicated that this drug is very effective during the early stages of virus infection. One of the main problems of in vivo drug administration is the possible toxic side effects caused by the drug. As an anticancer drug, CPT had severe toxic effect when it was given in cytotoxic doses (5, 14, 15, 20). However, the CPT doses which we used in our experiments did not cause any visible toxic effects. On the basis of our in vitro studies, it is reasonable to attribute the inhibitory effect of the drug on the onset of retrovirusinduced diseases to its antiretroviral activity, although we cannot exclude the possibility that the dramatic effect of this drug on the onset of the disease is due to some effect on the cellular targets of the virus in the mice. However, our data

5 3628 NOTES oo S T FG. 3. Effect of repeated CPT treatment on newborn mice infected with Mo-MuLV. Newborn BALB/c mice were inoculated with Mo-MuLV (3 x 105 PFU) or with Mo-MuLV and CPT (1 mg/kg). The Mo-MuLV- and CPT-treated mice were divided into two groups. The first group was given an additional CPT dose 5 days after virus and CPT injection. The second group was given CPT injections 5 and 10 days after virus injection. Each bar represents the mean spleen weight + the standard deviation for 5 to 10 mice. Symbols: _, Mo-MuLV alone; -, 1Z, and X3, Mo-MuLV plus one, two, or three CPT injections, respectively. clearly indicate that these possible cellular effects are not toxic. Should the same results be obtained with lentivirusinduced diseases in animal models, clinical trials may be performed with seropositive HV patients. The mechanism by which this drug inhibits retroviral replication is not clear and is presently under investigation ~600 ~ r FG. 4. Effect of CPT administered to newborn mice at different times after virus injection. Mo-MuLV-infected mice were treated with a single CPT dose (1 mg/kg) 1, 2, or 3 days after virus injection. The mice treated with CPT 3 days after virus injection were divided into two groups. The first group did not receive any further CPT treatment, and the second group was given further CPT doses 9 and 15 days after virus infection. The mice were sacrificed 10 weeks after virus infection, and their spleens were removed and weighed. Each bar represents the mean spleen weight + the standard deviation for 5 to 10 mice. Symbols: _, Mo-MuLV alone; -, Mo-MuLV plus CPT 1 day postinfection; 1, Mo-MuLV plus CPT 2 days postinfection;, Mo-MuLV plus CPT 3 days postinfection; E, Mo-MuLV plus CPT 3, 9, and 15 days postinfection. l 80W 6o FG. 5. Effect of CPT treatment on NFS mice infected with SFFV. NFS mice (6 to 8 weeks old) were inoculated intraperitoneally with 0.1 ml of medium containing 105 PFU of SFFV. Mice were treated with CPT (5 mg/kg) which was injected together with the virus or 3 days after virus infection. Control uninfected mice were given an injection of medium containing 1% DMSO or 5 mg of CPT per kg. Five weeks after virus injection, the mice were sacrificed, and their spleens were weighed. Each bar represents the mean spleen weight + standard deviation for 6 to 10 mice. Symbols: _, DMSO control; E1, CPT control; M, SFFV alone; Ci, SFFV and CPT together; Mi, CPT 3 days after SFFV infection. We thank Donald G. Blair of the National Cancer nstitute, Frederick, Md., for helpful discussions. This work was supported by grants to Esther Priel from the srael Cancer Association and the srael Cancer Research Fund, New York, N.Y. REFERENCES 1. Andoh, T., K. shii, Y. Suzuki, Y. kegami, Y. Kusunoki, Y. Yakemoto, and K. Okado Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase. Proc. Natl. Acad. Sci. USA 84: Friend, C Cell free transmission in adult Swiss mice of a disease having the character of a leukemia. J. Exp. Med. 105: Gallo, R. C., J. Whang-Peng, and R. Adamson Studies on the antitumor activity mechanism of action and cell cycle effects of camptothecin. J. Natl. Cancer nst. 46: Giovanella, B. C., J. S. Steheim, M. E. Wall, M. C. Wani, A. W. Nicholas, L. F. Liu, S. Roberts, and P. Milan DNA topoisomerase -targeted chemotherapy of human colon cancer in xenografts. Science 246: Gottlieb, J. A., and J. K. Luce Treatment of malignant melanoma with camptothecin (NSC ). Cancer Chemother. Rep. 56: Gupta, R. S., R. Gupta, B. Eng, R. B. Lock, W. E. Ross, R. P. Hertzberg, M. J. Carafana, and R. K. Johnson Camptothecin resistant mutant of Chinese hamster ovary cells containing a resistant mutant of DNA topoisomerase. Cancer Res. 48: Heidmann, T., 0. Heidmann, and J. F. Nicolas An indicator gene to demonstrate intracellular transposition of defective retroviruses. Proc. Natl. Acad. Sci. USA 85: Horwitz, M. S., and S. B. Horwitz ntracellular degradation of HeLa and adenovirus type 2 DNA induced by camptothecin. Biochem. Biophys. Res. Commun. 45: Hsiang, Y. H., R. Hertzberg, S. Hecht, and L. F. Liu Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase. J. Biol. Chem. 260: Kabat, D Molecular biology of Friend viral erythroleukemia. Curr. Top. Microbiol. mmunol. 148: Kaufmann, S. H nduction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etopside, camptothecin and other cytotoxic anticancer drugs: a J. VROL.

6 VOL. 67, 1993 cautionary note. Cancer Res. 49: Lee, J. C., and J. N. hle Chronic immunostimulation is required for Moloney leukemia virus induced lymphomas. Nature (London) 288: Liu, L. F DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem. 58: Moertel, C. G., A. J. Schutt, R. J. Reitemeier, and R. G. Hahn Phase study of camptothecin (NSC ) in the treatment of advanced gastrointestinal cancer. Cancer Chemother. Rep. 56: Muggia, F. M., P. J. Creaven, H. H. Hansen, M. H. Cohen, and 0. S. Selawzy Phase clinical trial of weekly and daily treatment with camptothecin (NSC ): correlation with preclinical studies. Cancer Chemother. Rep. 56: Priel, E., S. D. Showalter, and D. G. Blair nhibition of human immunodeficiency virus (HV-1) replication by noncytotoxic doses of camptothecin, a topoisomerase inhibitor. ADS Res. Hum. Retroviruses 7: Priel, E., S. D. Showalter, M. Roberts, S. Oroszlan, and D. G. Blair The topoisomerase inhibitor, camptothecin, inhibits equine infectious anemia virus replication in chronically infected CF2Th cells. J. Virol. 65: Priel, E., S. D. Showalter, M. Roberts, S. Oroszlan, S. Segal, M. NOTES 3629 Aboud, and D. G. Blair Topoisomerase activity associated with human immunodeficiency virus (HV) particles and equine infectious anemia virus core. EMBO J. 9: Row, W. P., W. E. Pugh, and J. W. Hartley Plaque assay techniques for murine leukemia viruses. Virology 42: Schaeppi, U., R. W. Fleischman, and D. A. Cooney Toxicity of camptothecin (NSC ). Cancer Chemother. Rep. 5: Spaturo, A., and D. Kessel Studies on camptothecin induced degradation and apparent reaggregation of DNA from L1210 cells. Biochem. Biophys. Res. Commun. 48: Teich, N., J. Wyke, T. Mak, A. Bernstein, and W. Hardy Pathogenesis of retrovirus-induced disease, p n R. Weiss, N. Teich, H. Varmus, and J. Coffin (ed.), RNA tumor viruses: molecular biology of tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Wall, M. E., M. C. Wan, C. C. Cook, K. H. Polmer, A. T. McPhail, and G. A. Sim The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from camptotheca acuminata. J. Am. Chem. Soc. 88: Wolfe, J. H., and W. D. Hardy, Jr mmunogenetics of murine and feline retroviruses. mmunol. Ser. 43: