Inhibition of Mwine CFU-C by Vindesine: Restoration of Colony Growth by Colony Stimulating Factor

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1 International Journal of Cell Cloning 1: (1983) Inhibition of Mwine CFU-C by Vindesine: Restoration of Colony Growth by Colony Stimulating Factor Giuseppe Pigoli, Lina Mangoni, Cecilia CaFamatti, Gino Degliantoni, Vittorio Rizzoli Cattedra di Ematologia, Universith di Parma, Italy Key Words. Colony stimulating factor (CSF). Vindesine Antibody to CSF (Anti-CSF) Abstract. Vindesine (VDS) is a new vinca-alkaloid related to vinblastine and vincristine that blocks production of the microtubules in the mitotic phase of the cell cycle. Studies were undertaken to investigate the inhibitory effect of VDS on normal murine bone marrow cell proliferation and the possible interactions between this compound and L-cell derived colony stimulating factor (CSF). One x 107 murine bone marrow cells were exposed to various concentrations of VDS, ranging from 0.1 to 1.5 pg/ml for 1 h at 37 C. Following this period, the cells were plated in agar in the presence of 100 units of CSF. A dose-dependent inhibition of colony formation was noted with increasing doses of the drugs. To determine whether an increased dose of CSF could overcome the inhibitory effect of VDS, further studies compared colony growth in response to 100 and 200 units of CSF. Virtually no inhibition of colony growth was detected in VDS-treated cells exposed to this higher dose of CSF while a dose-dependent reduction in CFU-C was noted with 100 units of CSF. Preincubation of cells with VDS and CSF prevented the inhibition that occurred with VDS alone. The addition of anti-csf serum during the preincubation phase abolished the protective effect of CSF. The studies show that short-term exposure of marrow cells to VDS causes a dosedependent inhibition of in vitro colony formation; this inhibition is prevented by increasing doses of CSF in agar culture or by simultaneous preincubation with CSF. The CSF action appears specific as its protective effect is neutralized by antibody to CSF, suggesting a potential role for CSF in preventing the antimitotic activity of VDS. '"1983 AlphaMed Press, Inc /83/$2.00/0

2 Pigoli/Mangoni/Caramatti/Degliantoni/Rizzoli 143 Introduction Vindesine (VDS) is a semi-synthetic vinca-alkaloid related to vinblastine. VDS has minimal differences in chemical structure from vinblastine and has a similar mechanism of action: it inhibits microtubule formation [ 11 in the mitotic phase of the cell cycle. Furthermore, the antitumor effect of the drug on cancer cells and its reversibility [2] has been described. Because of its wide antitumor activity, VDS is used in the treatment of various types of cancer; namely, leukemias, lymphomas, breast cancer and melanoma. It is generally known that colony stimulating factor (CSF) is a glycoprotein that induces extensive proliferation and differentiation of myeloid progenitor cells in vitro [3]. Incubation of this factor with bone marrow cells in semi-solid gels leads to the development of granulocyte and macrophage colonies. The initial step involved in the action of CSF on responsive cells seems to bind this factor to membrane receptors as observed by incubation and autoradiographic studies [4-91. The present study was undertaken to investigate the inhibitory effect of VDS on in vitro colony growth by normal murine bone marrow cells. Furthermore, we evaluated possible interactions between this compound and L-cell derived CSF by studying the ability of the colony stimulating factor to modify the myelotoxicity of VDS. Materials and Methods Bone marrow cells were obtained from femurs of female CD1 mice (5-7 weeks old). Each femur was removed by aseptic technique and cleansed; cells were flushed forcibly into 2 ml of modified McCoy s 5A medium using a syringe and 23-gauge needle. Cells were dispersed by repeated pipetting. Cell counts were obtained manually to assure complete dispersion of the cell suspension. Murine cell suspensions were overlayed on Ficoll-Hypaque, specific gravity 1.077, and centrifuged at 500 g for 30 min. Cells from the interface (top layer) were washed three times in serum-free medium and counted. In the first set of experiments, marrow cells, at the concentration of 1 X lo7/ ml, were preincubated with increasing doses of VDS (Eldisine Eli-Lilly, Brussels) ranging from 0.1 to 1.5 pg/ml for 60 min at 37 C in 7.5% COZ in a humidified atmosphere. At the end of preincubation, viability was measured by cell count and trypan blue dye exclusion. Cells were washed three times and tested for their proliferative capacity by the granulocyte-monocyte macrophage precursor (CFU-C) agar assay as described by Shadduck and Nugabhushanam [ 101. In brief, 1 X 105 cells were mixed in 1 ml of 0.3% McCoy s agar containing 5% (100 U) or 10% (200

3 Effect of Vindesine on Granulopoiesis 144 U) serum-free L-cell CSF. After 7 days of incubation in a humidified atmosphere with 7.5% CO2, colonies of more than 50 cells were scored using a dissecting microscope. Under these conditions the majority of colonies were composed of macrophages. The CSF used in these studies had a specific activity of 5.7 x 104 units/mg protein. Alternatively, cells were preincubated with the above VDS doses in the presence of 100 units of CSF for 60 min. At the end of the preincubation, cells were washed and plated in agar for CFU-C growth; 100 units of CSF were used to stimulate colony formation. Five plates were prepared for each sample, and all sets of experiments were repeated three times. The rabbit antibody to CSF was kindly provided by Dr. Richard K. Shadduck [ll]. The antiserum used in these studies was prepared using partially purified CSF; the inhibition titer was 1:512. Various dilutions of antibody, ranging from 1 : 1 to 1 :40 were employed. Even the highest dilution of antibody was capable of inhibiting all added CSF. Since the results were similar in six experiments, the effects of all antibody neutralizations were pooled. Results In the initial series of experiments, 1 X 107 bone marrow cells were exposed to increasing doses of vindesine for 60 min prior to plating. A dose-dependent inhibition of colony formation was noted. As shown in Figure 1, a 15% reduction in colony growth was detected when cells were exposed to 0.1 pg/ml of VDS. Increasing doses of the drug led to increasing inhibition of colony growth. At the highest dose of 1.5 pg/ml, 80% inhibition was noted. Higher doses of the drug (5 pg and 10 pg/ml/ 107 cells) yielded no further inhibition of colony formation. To determine whether an increased dose of CSF could overcome the inhibitory effect of VDS, further studies compared colony growth in response to 100 and 200 units of CSF. Marrow cells were preincubated for 60 min in the presence of 3 doses of VDS: 0.1, 0.5 and 1 pg/ml and subsequently plated with 2 dose levels of CSF. The data in Figure 2 show that marrow cells incubated with 100 units or 0.05 ml of CSF revealed a greater reduction of colonies with increasing doses of VDS. In contrast, only a minimal decline in colony formation was observed in the cultures stimulated with 200 units or 0.1 ml of CSF. At the highest level of VDS, a 10% reduction in colony growth was detected in cultures with 200 units of CSF as compared to a 68% reduction in cultures stimulated with 100 units of CSF.

4 Pigoli / Mangoni / Caramat ti / Degliantoni / Rimli 145 Fig. 1. Dose-dependent inhibition of CFU-C by different concentrations of vindesine (pg/ml). Each point represents the mean f SD of three replicate experiments Fig. 2. Colony growth in response to 100 and 200 units of CSF after preincubation with different doses of vindesine. Each point represents the mean f SD of three replicate experiments. H = 0.1 ml CSF; 1-4 = 0.05 ml CSF.

5 Effect of Vindesine on Granulopoiesis Mglml VPS Fig. 3. Protective effect of CSF on marrow toxicity of vindesine during the in vitro preincubation. Cultures contained 100 units of CSF in addition to the stated doses of vindesine. Each point represents the mean f SD of three replicate experiments. H = VDS + CSF; t-] = VDS. In the third set of experiments, cells were preincubated with the above VDS doses and with the addition of 100 units of CSF for 60 min (Fig. 3). After washing, the cells were plated in agar in the presence of 100 units of CSF. As in previous experiments, preincubation with different doses of VDS caused a decrease in colony growth. There was a clear cut difference between cells incubated with VDS and those exposed to VDS and CSF. The L-cell CSF appeared to show a protective effect, as there was no change in the number of colonies at all three levels of VDS. To further investigate the possible role of CSF as a protective agent, cells were preincubated with various doses of the drug, 100 units of CSF or with CSF plus anti-csf serum. As shown in Figure 4, VDS alone caused a marked reduction in colony growth. The addition of CSF during the preincubation phase blocked the inhibitory effect of the drug. The anti-csf serum prevented the protective effect of CSF, thus suggesting that CSF is responsible for counteracting the antimitotic effect of VDS.

6 Pigoli/Mangoni/Caramatti/Degliantoni/Rizzoli 147 Fig. 4. Colony growth after in vitro preincubation of cells with various doses of vindesine in combination with CSF or with CSF plus a specific antibody to CSF. Each point represents the mean f SD of three replicate experiments. - = VDS + CSF; --- = VDS; -*- = VDS + CSF + Anti-CSF. Discussion These studies show that the short-term exposure of marrow cells to VDS causes a dose-dependent inhibition of in vitro colony formation. This inhibition is prevented by increasing doses of CSF in agar cultures or by simultaneous preincubation with CSF. The CSF action appears specific as its protective effect is neutralized by an antibody to CSF. The mechanism of CSF protection is unclear. Similar findings were noted by Yunis and Gross [ 121 when using the drug, chloramphenicol. Inhibition of colony growth was prevented by increasing the concentrations

7 Effect of Vindesine on Granulopoiesis 148 of CSF. Although this drug also inhibited the growth of erythroid colonies (CFU-E), the effect was not reversed by increasing concentrations of erythropoietin [ 131. One possible explanation for the protective effect of CSF in the present studies could be that VDS and CSF compete for the same receptors on the cell membrane. This seems unlikely, since the two compounds have different chemical structures and different mechanisms. However, it has been shown recently that the low molecular weight phorbol ester TPA inhibits the binding of CSF to marrow cell receptors [ 141, Several lines of evidence indicate that CSF is required continuously for colony development. Exposure of marrow cells to CSF for short intervals does not lead to subsequent colony growth [ 151, while the transfer of early cells or clusters that have been exposed to CSF for 2-4 days into new medium devoid of CSF results in a decline in cell number and colony formation [ 161. CSF is also required for the growth and survival of responsive progenitor cells. In addition, CSF is known to induce RNA and protein synthesis in mature granulocytes [ 171 and to induce mature macrophages to become tumoricidal [18]. This suggests that CSF may act on mature cells as well as on progenitor cells, by initiating intracellular programs for both proliferative and differentiated cell functions. The results reported herein suggest that CSF may activate intracellular mechanisms which are blocked by VDS, thus overcoming the antimitotic effect of this drug. Such findings may be useful in further defining the activities of CSF on granulocyte and macrophage progenitor cells. Acknowledgments This work was supported in part by grants from the C.N.R., Nos , and the M.P.I. The authors wish to thank Dr. R.K. Shadduck of the Montefiore Hospital, University of Pittsburgh, for providing L-cell conditioned medium and antibody to CSF. References 1 Paintrand, M.; Pignot, T.; Dantchev, D.; Maral, R.: Comparative effects of vindesine, vincristine and vinblastine on tubular formation in electron microscopy; in Brade, Nagel, Seeber, Proceeding of the International Vinca Alkaloid Symposium-Vindesine, pp. 2-5 (S. Karger, Base1 1980).

8 Pigolil Mangod Caramatti/ Degliantoni/Rizzoli Storme, G.; Mareel, M.; De Bruyne, G.; Van Cauwenberge, R.: Anti-invasive effect of vinca alkaloids; in Brade, Nagel, Seeber, Proceeding of International Vinca Alkaloid Symposium-Vindesine, pp (S. Karger, Base1 1980). Metcalf, D.: Regulation of granulocyte and monocyte-macrophage proliferation by colony stimulating factor (CSF): A review. Exp Hematol 1: (1973). Pigoli, G.; Waheed, A.; Shadduck, R.K.: Observations on the binding and interaction of radioiodinated colony-stimulating factor with murine bone marrow cells in vitro. Blood 59: (1982). Caramatti, C.; Pigoli, G.; Shadduck, R.K.; Waheed, A.: The effect of preincubation of bone marrow cells on the binding of colony stimulating factor. J Lab Clin Med (in press). Shadduck, R.K.; Pigoli, G.; Waheed, A.; Caramatti, C.; Degliantoni, G.; Rizzoli, V.; Porcellini, A.; Schiffer, L.: Identification of hemopoietic cells responsive to colony stimulating factor by autoradiography. Blood (submitted). Guilbert, L.J.; Stanley, E.R.: Specific interaction of murine colony-stimulating factor with mononuclear phagocytic cells, J Cell Biol 85: (1980). Byme, P.V.; Guilbert, L.J.; Stanley, R.: Distribution of cells bearing receptors for a colony-stimulating factor (CSF-1) in murine tissues. J Cell Biol 91: (1981). Chen, D.M.; Hsu, S.; Lin, S.: Interaction of colony stimulating factor (CSF- 1) with murine peritoneal exudate macrophage: binding, internalization and degradation. Exp HematollO: suppl. 11, p. 58 (1982). Shadduck, R.K.; Nagabhushanam, N.G.: Granulocyte colony stimulating factor I. Response to acute granulocytopenia. Blood 38: (1971). Shadduck, R.K.; Metcalf, D.: Preparation and neutralization characteristics of an anti-csf antibody. J Cell Physiol86: (1975). Y unis, A.A.; Gross, M.A.: Drug-induced inhibition of myeloid colony growth: Protective effect of colony-stimulating factor. J Lab Clin Med 86: ( 1975). Yunis, A.A.; Adamson, J.W.: Differential in vitro sensitivity of marrow erythroid and granulocytic colony forming cells to chloramphenicol. Am J Hematol2: (1977). Stuart, R.K.; Sensenbrenner, L.L.; Shadduck, R.K.; Waheed, A.; Caramatti, C.: Phorbol ester stimulated murine myelopoiesis: Role of colony stimulating factors. Blood (submitted). Paran, M.; Sachs, L.: The continued requirement for inducer for the development of macrophage and granulocyte colonies. J Cell Physiol 72: (1968). Metcalf, D.; Foster, R., Jr.: Behavior on transfer of serum stimulated bone marrow colonies. Proc SOC Exp Biol Med 126: (1967). Burgess, A.W.; Metcalf, D.: The effect of colony stimulating factor on the synthesis of ribonucleic acid by mouse bone marrow cells in vitro. J Cell Physiol90: (1977).

9 Effect of Vindesine on Granulopoiesis Wing, E.J.; Waheed, A.; Shadduck, R.K.; Nagle, L.S.; Stephenson, B.A.: Effect of colony stimulating factor on murine macrophages: Induction of antitumor activity. J Clin Invest 69: (1982). Accepted: May 10, 1983 Dr. Vittorio Rizzoli, Cattedra di Ematologia, c/o Clinica Medica Generale, Via Gramsci, 14,43100 Parma (Italy)