5-FU high-proliferative-potential. megakaryocyte mixed HPP-Meg-Mix cell fms-like tyrosine kinase-3 Flt-3 ligand. high-proliferative-potential

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1 Vol. 30, pp , 2002 high-proliferative-potential megakaryocyte mixed HPP-Meg-Mix cell fms-like tyrosine kinase-3 Flt-3 ligand FU high-proliferative-potential megakaryocyte mixed colony forming cell HPP-Meg-Mix CFC HPP-CFC CFU-GM CFU-Meg recombinant mouse fms-like tyrosine kinase-3 ligand Flt-3 rhg-csf rhepo rhtpo rmscf rmil-3 rmil-6 rmil BALB/c 5-FU 150 mg/kg 2 Flt HPP-Meg-Mix Flt-3 + SCF G-CSF + Epo + Tpo + SCF + Flt-3 5-FU 14 HPP-Meg-Mix Flt-3 HPP-Meg-Mix SCF Flt-3 HPP-Meg-Mix fms-like tyrosine kinase-3 ligand Flt-3 ligand interleukin-1α IL-1α IL-3 IL-4 IL-6 IL-11 stem cell factor SCF colony stimulating factor CSF -1 granulocyte-macrophage-csf GM-CSF granulocyte-csf G-CSF erythropoietin Epo basic fibroblast growth factor bfgf leukemia inhibitory factor LIF thrombopoietin Tpo Fms-like tyrosine kinase-3 ligand Flt-3 III Flt-3 B 1~3 in vivo G-CSF 4 high-proliferative-potential colony forming 105

2 cell HPP-CFC Lowry et al. 6 5-fluorouracil 5-FU interleukin-1α IL-1α IL- 3 IL-4 GM-CSF G-CSF HPP-CFC mm HPP-CFC HPP-Meg-Mix HPP-CFC FU G-CSF Epo SCF IL-3 Tpo HPP-Meg-Mix HPP-Meg- Mix SCF 5-FU Flt-3 HPP-Meg-Mix cell 5-FU Flt-3 G-CSF IL-3 SCF IL- 11 IL-6 Epo Tpo HPP- Meg-Mix 8 10 BALB/c, CE- 2, rhg-csf, rhepo epoetin alpha, rhtpo rmil-3 rmscf rmflt-3 ligand R&D Systems, USA rmil-6 Genzyme Techne, USA rmil-11 8 G-CSF SCF Tpo Epo 4 Flt-3 IL-3 IL-6 IL-11 8 Flt-3 G-CSF SCF Tpo Epo IL-3 IL-6 IL rhg-csf; 10,000 U (100 ng)/ml rmscf; 100 ng/ml rhtpo; 10 ng/ml rhepo; 2 U/ml rmflt-3 ligand; 2 ~ 1,000 ng/ml rmil-3; 50 U (29 ng)/ml rmil-6; 100 ng/ml rmil-11; 100 ng/ml 5-FU 150 mg/kg kg 3 mg/body 5-FU, FU 2 5-FU D2 BM5-FU D2 SP 5-FU D2 BM 23 1 ml Iscove's modified Dullbecco's medium IMDM; Sigma Chemical, USA 15 ml 5-FU D2 BM 5-FU D2 SP IMDM 5-FU D2 SP 5-FU D2 BM SP IMDM 90% viability /ml 0.5% 0.3% 9 10 agar noble, DIFCO Laboratories, USA 0.5 g 0.3 g 25 ml 43 C 2 IMDM IMDM 2.5 ml 10% fetal calf serum; FCS, HyClone 106

3 HPP-Meg-Mix Flt-3 ligand Laboratories, USA 1.0 ml bovine serum albumin; BSA, Cohn fraction V, Sigma Chemical 0.5 ml 5.0 ml IMDM 35 mm FALCON 1008, Becton Dickinson, USA ml IMDM 2.5 ml FCS 1.0 ml BSA 0.5 ml 5-FU D2 BM 5-FU D2 SP 0.5 ml 5.0 ml IMDM 1.0 ml CO2 air 95%, CO2 5%, 37 C 14 BSA mixed bed resin, AG 501X8 (D), 20 ~ 50 mesh, Bio-Rad Laboratories, USA 7 acetyl- cholinesterase; AChE 10 mg 0.1 mol/l ph ml 0.1 mol/l 1.0 ml 30 mmol/l 2.0 ml 5 mmol/l 2.0 ml AChE 65 mm 85 mm Whatman No.1, Whatman International, England 6% Ladd Research, USA AChE 1 1 ml mol/l, ph AChE HE AChE HE Harris' Hematoxylin,, 3 HPP-Meg-Mix AChE AChE 4 CFU- Meg 40 AChE CFU-GM mm 5.0 mm HPP- CFC 20 Lowry et al. 6 HPP-Meg-Mix SD Mann-Whitney U p 0.05 Flt-3 G-CSF Epo Tpo SCF 4 Flt-3 Fig. 1 5-FU D2 BM HPP-Meg-Mix Flt ng/ml 6 Flt ng/ml HPP-Meg-Mix 5- FU D2 SP Flt ng/ml HPP- Meg-Mix 200 ng/ml Flt-3 Flt-3 direct effects of Flt-3 Flt-3 Flt-3 + G-CSF Flt-3 + SCF Flt-3 + Tpo Flt-3 + Epo Flt-3 + IL-3 Flt-3 + IL-6 Flt-3 + IL- 11 Table 1 107

4 Fig. 1 Flt-3 dose response curve. Varying concentration of Flt-3 used to stimulate HPP-Meg-Mix colonies, derived from 5-FU D2 BM are shown with mean ± SD. *Greater-than-additive effect of concentration of Flt-3 compared with the other concentrations; p<0.05. Flt-3 Flt-3 + Epo Flt-3 + IL-11 5-FU D2 BM 5-FU D2 SP Flt-3 + SCF 5-FU D2 BM HPP-Meg-Mix 4.4 ± 1.9 mean ± SD Flt-3 + SCF HPP-Meg-Mix HPP-CFC 5-FU D2 BM Flt-3 G-CSF SCF Tpo IL-3 IL-6 SCF p<0.05 CFU-Meg Flt-3 SCF Tpo IL-3 5-FU D2BM Flt-3 Tpo p<0.05 CFU-GM Flt-3 G-CSF SCF Tpo IL-3 IL-6 5-FU D2 BM SCF Tpo CFU-GM p<0.05 Flt-3 linage expression of Flt-3 Flt-3 G-CSF Epo SCF Tpo 4 GEST Flt-3 IL-3 IL-6 IL-11 5-FU D2 BM Fig. 2 5-FU D2 SP Fig. 3 5-FU D2 BM HPP-Meg-Mix GEST GEST + Flt-3 3 ± ± 4.0 Flt-3 p<0.05 GEST IL-3 IL-6 IL-11 Flt-3 HPP-Meg-Mix HPP-CFC CFU-Meg GEST Flt-3 Table 1 Effect on 5-FU D2 BM and 5-FU D2 SP of Flt-3 alone or in combination with rhg-csf, rmscf, rhtpo, rhepo, rmil-3, rmil-6, and rmil

5 HPP-Meg-Mix Flt-3 ligand Fig. 2 Mean number of HPP-Meg-Mix colonies, HPP-CFC colonies, CFU-Meg colonies, and total colonies, derived from the 5-FU D2 BM are shown with standard deviation (SD). Cells were maximally stimulated with rhg-csf, rhepo, rmscf, rhtpo, rmil-3, rmil-6, and rmil-11, without and with rmflt-3 ligand at plateau concentrations. * Greater-than-additive effect of Flt-3 ligand compared with the same groups not supplemented with Flt-3 ligand; p<0.05. ** Greater-than-additive effect of Flt-3 ligand plus other cytokines compared to the other cytokines with Flt-3 ligand; p<0.05. Fig. 3 Mean number of HPP-Meg-Mix colonies, HPP-CFC colonies, CFU-Meg colonies, and total colonies, derived from the 5-FU D2 SP are shown with standard deviation (SD). Cells were maximally stimulated with rhg-csf, rhepo, rmscf, rhtpo, rmil-3, rmil-6, and rmil-11, without and with rmflt-3 ligand at plateau concentrations. * Greater-than-additive effect of Flt-3 ligand compared with the same groups not supplemented with Flt-3 ligand; p<0.05. ** Greater-than-additive effect of Flt-3 ligand plus other cytokines compared to the other cytokines with Flt-3 ligand; p< FU D2 SP HPP-Meg-Mix GEST GEST + Flt-3 3 ± ± 0.5 Flt-3 p<0.05 GEST IL-3 IL-6 IL-11 Flt-3 HPP-Meg-Mix HPP-CFC Flt-3 CFU-Meg GEST GEST + Flt-3 0 ± ± 2.8 Flt-3 p<0.05 Table 2 HPP-Meg-Mix 5-FU Day 0 Day 2 Day 21 Day 35 Day 14 Day 2 Day

6 Table 2 Colony counts on the days post 5-FU injection Fig. 4 Spleen weight of mice after the injection of 5-FU. p<0.05 HPP-CFC CFU-Meg CFU-GM Day 21 Day 14 Day 21 5-FU Day 2 HPP-Meg-Mix Day 2 p< FU ± g Day ± g Day ± g Day 60 Fig. 4 HPP-Meg-Mix Day ± FU Day 14 HPP-CFC 5-FU Day ± ± 10.4 Day ± 7.3 Day 21 Day 35 5-FU Day 2 CFU-Meg Day 2 Day 21 CFU-GM Day 2 Day 21 CFU-Meg CFU- GM 5-FU Day 2 Day 2 p< FU Day 35 5-FU 2 5-FU D2 BM HPP-CFC high-proliferative-potential colony forming cell-1 HPP-CFC-1 11~13 Flt-3 ligand III receptor tyrosine kinase; RTK Flt-3 fms-like tyrosine kinase-3; FLK2; CD135 G-CSF Flt-3 CD34 pro/pre-b 17 5-FU D2 BM SP 8 Flt-3 G-CSF IL-3 SCF IL-11 IL-6 Epo Tpo 110

7 HPP-Meg-Mix Flt-3 ligand HPP-Meg-Mix 5-FU 60 Flt-3 20 ng/ml HPP- Meg-Mix 200 ng/ml Flt-3 HPP-Meg-Mix Zeigler et al. 18 Flt-3 G0 Flt-3 5-FU D2 BM HPP-Meg-Mix Flt-3 + SCF Flt-3 Flt-3 + SCF SCF Flt-3 CFU-Meg Flt-3 + SCF Flt-3 + Tpo Flt-3 + Tpo Flt-3 Tpo Flt-3 SCF Tpo CFU-GM Flt-3 4 G-CSF Epo SCF Tpo Flt-3 HPP-Meg-Mix Kobayashi et al. 19 SCF Flt-3 ligand Tpo IL-3 G0 IL-3 IL-3 HPP-Meg-Mix HPP-CFC HPP-Meg-Mix Flt-3 Flt-3 IL-6 IL-11 HPP-Meg-Mix IL-6 IL-11 HPP-Meg-Mix HPP-Meg-Mix Flt-3 IL-6 IL-11 5-FU FU HPP-Meg-Mix Day 14 HPP-CFC CFU-Meg CFU-GM Day 2 Day 14 Day 14 Day 21 Day 14 1 Hannum C, Culpepper J, Campbell D, McClanahan T, Zurawski S, Bazan JF, Kastelein R, Hudak S, Wagner J, Mattson J, Luh J, Duda G, Martina N, Peterson D, Menon S, Shanafelt A, Muench M, Kelner G, Namikawa R, Rennich D, Roncarolo M-G, Zlotnik A, Rosnet O, Dubreuil P, Bimbaum D and Lee F. Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs. Nature 1994; 368: Matthews W, Jordan CT, Gavin M, Jenkins NA, Copeland NG and Lemischka IR. A receptor tyrosine kinase cdna isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit. Proc Natl Acad Sci USA 1991; 88 (20): Hudak S, Hunte B, Culpepper J, Menon S, Hannum C, Thompson-Snipes L and Rennick D. FLT3/FLK2 111

8 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units. Blood 1995; 85 (10): Pless M, Wodnar-Filipowicz A, John L, Baldomero H, Lyman SD, Nissen C and Gratwohl A. Synergy of growth factors during mobilization of peripheral blood precursor cells with recombinant human Flt3- ligand and granulocyte colony-stimulating factor in rabbits. Exp Hematol 1999; 27 (1): McNiece IK, Bertoncello I, Kriegler AB and Quesenberry PJ. Colony-forming cells with high proliferative potential (HPP-CFC). Int J Cell Cloning 1990; 8(3): Lowry PA, Deacon DM, Whitefield P, Rao S, Quesenberry M, Quesenberry PJ. The high-proliferative-potential megakaryocyte mixed (HPP-Meg-Mix) cell: a trilineage murine hematopoietic progenitor with multiple growth factor responsiveness. Exp Hematol 1995; 23 (10): High-Proliferative-Potential Megakaryocyte Mixed (HPP-Meg-Mix) colony, Thrombopoietin (Tpo), Granulocyte-Colony Stimulating Factor (G-CSF), Erythropoietin (Epo), Stem Cell Factor (SCF) Interleukin-3 (IL-3) : 5-Fluorouracil (5-FU). 1998; 26: Ku H, Hirayama F, Kato T, Miyazaki H, Aritomi M, Ota Y, D'Andrea AD, Lyman SD and Ogawa M. Soluble thrombopoietin receptor (Mpl) and granulocyte colony-stimulating factor receptor directly stimulate proliferation of primitive hematopoietic progenitors of mice in synergy with steel factor or the ligand for Flt3/Flk2. Blood 1996; 88 (11): McNiece IK, Stewart FM and Deacon DM and Quesenberry PJ. Synergistic interactions between hematopoietic growth factors as detected by in vitro mouse bone marrow colony formation. Exp Hematol 1988; 16 (5): McNiece IK, Robinson BE and Quesenberry PJ. Stimulation of murine colony-forming cells with high proliferative potential by the combination of GM-CSF and CSF-1. Blood 1988; 72 (1): McNiece IK, Bertoncello I, Keller JR, Ruscetti FW, Hartley CA and Zsebo KM. Transforming growth factor beta inhibits the action of stem cell factor on mouse and human hematopoietic progenitors. Int J Cell Cloning 1992; 10 (2): Kovacs CJ, Evans MJ, Roberts C, Harrell J, Abernathy R, Gooya J and Johnke RM. Temporal recovery of short-term repopulating HSC subpopulations in marrow following schedule-dependent administrations of IL-1 alpha and M-CSF. Exp Hematol 1995; 23 (9): Kriegler AB, Verschoor SM, Bernardo D and Bertoncello I. The relationship between different high proliferative potential colony-forming cells in mouse bone marrow. Exp Hematol 1994; 22 (5): Rosnet O, Buhring HJ, delapeyriere O, Beslu N, Lavagna C, Marchetto S, Rappold I, Drexler HG, Birg F, Rottapel R, Hannum C, Dubreuil P and Birnbaum D. Expression and signal transduction of the FLT3 tyrosine kinase receptor. Acta Haematol 1996; 95 (3-4): Yokota S, Kiyoi H, Nakao M, Iwai T, Misawa S, Okuda T, Sonoda Y, Abe T, Kashima K, Matsuo Y and Naoe T. Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines. Leukemia 1997; 11 (10): Kiyoi H, Naoe T, Nakano Y, Yokota S, Minami S, Miyawaki S, Asou N, Kuriyama K, Jinnai I, Shimazaki C, Akiyama H, Saito K, Oh H, Motoji T, Omoto E, Saito H, Ohno R and Ueda R. Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood 1999; 93 (9): Small D, Levenstein M, Kim E, Carow C, Amin S, Rockwell P, Witte L, Burrow C, Ratajczak MZ, Gewirtz AM and Civin CI. STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34 + human bone marrow cells and is involved in the proliferation of early progenitor/stem cells. Proc Natl Acad Sci USA 1994; 91 (2): Zeigler FC, Bennett BD, Jordan CT, Spencer SD, Baumhueter S, Carroll KJ, Hooley J, Bauer K and Matthews W. Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells. Blood 1994; 84 (8): Kobayashi M, Laver JH, Lyman SD, Kato T, 112

9 HPP-Meg-Mix Flt-3 ligand Miyazaki H and Ogawa M. Thrombopoietin, steel factor and the ligand for flt3/flk2 interact to stimulate the proliferation of human hematopoietic progenitors in culture. Int J Hematol 1997; 66 (4): Nilsson SK and Bertoncello I. Age-related changes in extramedullary hematopoiesis in the spleen of normal and perturbed osteopetrotic (op/op) mice. Exp Hematol 1994; 22 (4): Abstract The Flt-3 Ligand Supports the Generation of High-Proliferative-Potential Megakaryocyte Mixed Colony Forming Cells (HPP-Meg-Mix CFC) with Multiple Growth Factor Responsiveness on 5-Fluorouracil (5-FU) Treated Murine Bone Marrow and Spleen Shinji Yoshioka Object: The effects of recombinant mouse fms-like tyrosine kinase-3 ligand (rmflt-3 ligand), recombinant human granulocyte-colony stimulating factor (rhg-csf), rh erythropoietin (rh Epo), rh thrombopoietin (rhtpo), rm stem cell factor (rmscf), rm interleukin-3 (rmil-3), rm interleukin-6 (rmil-6) and rm interleukin-11 (rmil-11) on high-proliferative-potential megakaryocyte mixed colony forming cells (HPP-Meg-Mix CFC) were investigated in murine bone marrow and spleen. Materials and Methods: Bone marrow and spleen cells from female BALB/c mice treated with 150 mg/kg 5-FU were collected after 2 to 60 days. These cells were cultured in combination with multiple growth factors including an Flt-3 ligand, in double-layer semi-solid agar for 14 days. Results: HPP-Meg-Mix colony formation was not seen with Flt-3 alone or in the absence of SCF. The quantity of HPP-Meg-Mix colonies increased in the combined presence of rmflt-3 ligand + rmscf in day 2 bone marrow, and G-CSF + Epo + SCF + Tpo + IL-3 in day 14 spleen. The quantity of more mature colonies such as CFU-Meg and CFU-GM increased until day 21 in the spleen, but HPP-Meg-Mix colony formation was observed only on day 2 in bone marrow with the combination of SCF and other cytokines. Conclusion: These findings suggest that the rmflt-3 ligand might stimulate the proliferation of HPP-Meg-Mix CFC, and rmscf might stimulate more primitive stem cells than with the rmflt-3 ligand. (St. Marianna Med. J., 30: , 2002) Division of Hematology and Oncology, Department of Internal Medicine St. Marianna University School of Medicine, Sugao, Miyamae-ku, Kawasaki , Japan 113

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