- Email: [email protected]
Leukemia cell lines require self-secreted stem cell growth factor (SCGF) for their proliferation
Available online at www.sciencedirect.com
Leukemia Research 32 (2008) 1623–1640
Letters to the Editor
Leukemia cell lines require self-secreted stem cell growth factor (SCGF) for their proliferation Stem cell growth factor (SCGF) is a cytokine of the Ctype lectin family, and acts on hematopoietic stem/progenitor cells to support their proliferation [1–3]. Within the bone marrow, they, but not other mature cells, produce SCGF [4–6]. The skeletal tissues, particularly proliferating osteoblasts and chondroblasts, themselves express scgf gene [2], thus the osseous microenvironment is speculated to foster hematopoietic stem/progenitor cells by virtue of SCGF. Serum SCGF level increases along a hematopoietic recovery after bone marrow transplantation, but does not in the case of delayed engraftment [4]. These findings implicate not only a property of SCGF as one of the stem cell markers but also an autocrine or paracrine SCGF regulation of hematopoietic stem/progenitor cells. Most of leukemia cell lines also produce SCGF [1,7], in fact SCGF cDNA has been cloned from the cDNA library prepared from KPB-M15 cells, a human myeloid cell line established from a patient with chronic myeloid leukemia in blast crisis [1]. Here, we address the question of whether self-secreted SCGF triggers proliferation of leukemia cell lines by the neutralization experiment using anti-SCGF antibody. Five SCGF-producing cell lines [1,7] representative of each hematopoietic lineage, KPB-M15 (myeloid), HL-60 (promyelocyte), U937 (monocyte), Raji (B cell) and MOLT-4 (T cell) (the latter four were obtained from Health Science Research Resources Bank, Sennan, Japan), were weekly 1:5- (all but 1:3.5- for HL-60) split cultured with 10% fetal calf serum (FCS)-containing Iscove’s modified Dulbecco’s medium (IMDM) with or without 10 g/ml goat anti-human SCGF antibody (Abcam, Cambridge, MA) or 10 g/ml goat non-immunized isotype IgG (Abcam). Viable cells in the culture of each cell line with anti-SCGF antibody remained unchanged at week 1, but dramatically declined at week 2, and totally disappeared by week 3 or 4, while cells vigorously proliferated in the culture with or without isotype IgG (Fig. 1A). An anti-SCGF antibody-induced decrease in the viable cell counts paralleled a decrease in the cellular viability (data not shown). A long-term culture without the medium exchange was avoided because of a difficulty in discriminat-
0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.
ing the cytoreductive effect of antibody neutralization from a harmful influence of nutritional exhaustion and metabolite accumulation in the medium. The damaged cells in the culture with anti-SCGF antibody appeared apoptotic, e.g. pyknotic nuclei, karyorrhexis, loss of nucleoli and shrunk cytoplasm (Fig. 1B). The present study demonstrates that all leukemia cell lines tested require self-secreted SCGF for their proliferation, indicating that a putative autocrine SCGF mechanism is working and the loop blockade with neutralizing antibody deprives extracellular SCGF to invite apoptosis. An SCGF concentration in the medium with 10% FCS is estimated 1 ng/ml or so [4,8], which contributes little to the biologic activity. Alternatively, less possible, anti-SCGF antibody abrogates an inhibitory activity of SCGF on some unknown apoptotic factor to enhance apoptosis. Uncontrolled autonomous proliferation of leukemia cell lines could be partially explained by the SCGF-mediated stimulation, irrespective of viral, chemical and radiation leukemogenesis. Analysis on SCGF receptor unidentified yet will provide more information for detailed proliferative mechanisms. SCGF is predicted to bind to SCGF receptor through a protein-protein interaction as does tetranectin molecularly akin to SCGF, although SCGF has a conserved carbohydrate-recognition domain of C-type lectin motif sequence at the COOH-terminal. Autocrine vascular endothelial growth factor (VEGF) [9] or insulin-like growth factor-I (IGF-I) [10] loop promotes growth of myeloid cell lines. However, anti-VEGF antibody (bevacizumab; Avastin® ) is ineffective for acute myelogenous leukemia cells since it is inert to the “private” VEGF loop [9], and it is unclear if anti-IGF-I antibody blocks the “public” IGF-I loop. No leukemia cell line has been reportedly inhibited to grow by anti-cytokine antibody except the factor-dependent cell lines. Relatively late-onset suppression by anti-SCGF antibody was seen around the culture period of week 2, indicating a possible action of SCGF on a certain subset of leukemia cell lines, for example, equivalent to the leukemic stem cells. They occupy a part of CD34+ CD38− population of acute myelogenous leukemia cells, reside within the osteoblast-rich endosteal region of the bone marrow when transplanted into the irradiated immunodeficient mice, and survive chemotherapy to originate a relapse of leukemia [11]. Anti-SCGF
1624
Letters to the Editor / Leukemia Research 32 (2008) 1623–1640
Fig. 1. Inhibition of leukemia cell line proliferation by anti-SCGF antibody. (A) A culture of each KPB-M15, HL-60, U937, Raji and MOLT-4 cell line was weekly fed with 10% FCS-containing IMDM with or without 10 g/ml anti-SCGF antibody or 10 g/ml isotype IgG, when viable cells (mean ± S.D. of three separate experiments) were counted immediately before the medium exchange. “No antibody” (blue) indicates the culture with medium alone. (B) Morphology of U937 cells (400× magnification). Cytospin preparations were May-Gr¨unwald-Giemsa stained at the culture period of week 3.
antibody is expected to eradicate even leukemic stem cells if it can arrest their interaction with the microenvironment harboring them. An SCGF concentration in the serum of leukemia patients is 10-100 times higher than the disease-free controls [8] and leukemia will be hopefully cured by normalizing the elevated SCGF level. Consequently a stem cell-specific SCGF-targeting medicine would enable a new potential master key therapy for any type of leukemia.
Acknowledgements This work is not supported by grants. This work is completely original and has not been published previously.
References [1] Hiraoka A, Sugimura A, Seki T, Nagasawa T, Ohta N, Shimonishi M, et al. Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells. Proc Natl Acad Sci USA 1997;94:7577–82.
[2] Hiraoka A, Yano K, Kagami N, Takeshige K, Mio H, Anazawa H, et al. Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells. Hematol J 2001;2:307–15. [3] Gehling UM, Ergun S, Schumacher U, Wagener C, Pantel K, Otte M, et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood 2000;95:3106–12. [4] Ito C, Sato H, Ando K, Watanabe S, Yoshiba F, Kishi K, et al. Serum stem cell growth factor for monitoring hematopoietic recovery following stem cell transplantation. Bone Marrow Transpl 2003;32:391–8. [5] Ma X, Husain T, Peng H, Lin S, Mironenko O, Maun N, et al. Development of a murine hematopoietic progenitor complementary DNA microarray using a subtracted complementary DNA library. Blood 2002;100:833–44. [6] Ng YY, van Kessel B, Lokhorst HM, Baert MRM, van den Burg CM, Bloem AC, et al. Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stemcell activity. J Leuk Biol 2004;75:314–23. [7] Bannwarth S, Giordanengo V, Lesimple J, Lefebvre J-C. Molecular cloning of a new secreted sulfated mucin-like protein with a C-type lectin domain that is expressed in lymphoblastic cells. J Biol Chem 1998;273:1911–6. [8] Ando K, Hotta T, Ito C, Sato H, Furuya A, Shitara K, et al. Method of judging leukemia, pre-leukemia or aleukemic malignant blood disease and diagnostic therefor. PCT/JP2003/004531.
CONTROL
80 60
ATRA
40 ATO
CONTROL
80 60
ATRA
40 20
ATRA+ATO
0
24 H
doi: 10.1016/j.leukres.2008.01.003
100
72 H
Available online 4 March 2008
Fig. 2. The effect of ATRA, ATRA + ATO on CD11b expression of NB4 cells.
1 M/l DNR for 24 h, 48 h, 72 h and 168 h. Control group were untreated NB4 cells. Then CD11b expression on NB4 cells was assessed by direct immunofluorescence of flow cytometric analysis. We found that ATRA significantly upregulated CD11b expression in a dependent-time manner and the change of CD11b expression was most evident with an approximately sixfold increase in the median of the percentage of positive cells after 168 h exposure (Fig. 1). 1 M ATO alone did not cause the significant change of CD11b expression at any tested times (Fig. 1). When we added ATO to ATRA, CD11b expression did not change after 24 h and 48 h exposure, but increased after 72 h and 168 h exposure (Fig. 2). When we added ATO altogether with daunorubicin to ATRA, ATRA-induced a particularly high increase in CD11b expression was reversed (Fig. 3). The increase of CD11b expression induced with ATRA is a key event in the development of the hyperleukocytosis 100 CONTROL
80 60
ATRA
40 ATRA+ATO+
20
DNR
16 8H
H
48
H
0
24 H
percentage of positive cells
The clinical effectiveness of all-trans retinoic acid (ATRA) and trioxide arsenic (ATO) in the treatment of acute promyelocytic leukemia (APL) have been widely confirmed. Unfortunately, in the course of induction-APL with ATRA or ATO, 70% of patients suffer from hyperleukocytosis and 5–25% of patients develop leukostasis, which may lead to life-threatening APL differentiation syndrome. Because of its poor prognosis and high mortality, the early diagnosis and effective treatment of this syndrome need to be seriously considered. According to recent studies [1], the hyperleukocytosis and APL differentiation syndrome are associated with the adhesion change of APL cells during ATRA-induced differentiation. The CD11b is the adhesion molecular of the mark of APL cells-differentiation. Recent studies have showed that increase of CD11b expression induced with ATRA is an important cause of the hyperleukocytosis and acute promyelocytic leukemia differentiation syndrome [2]. At present, ATRA combined with ATO and anthracycline has been used to treat APL patients, which not only notably improves curative effect, but also reduces the incidence rate of the hyperleukocytosis and APL differentiation syndrome. To explore the role of CD11b expression and the effects of these drugs in hyperleukocytosis and APL differentiation syndrome, we studied the effects of ATRA, ATO and daunorubicin (DNR) on CD11b expression of NB4 cells that is the only true human promyelocytic leukemia cell line with the characteristic chromosomal translocation t (15; 17) (p21; q23). NB4 cells were cultured respectively with 1 M/l ATRA, 1 M/l ATO, 1 M/l ATRA combined with 1 M/l ATO, 1 M/l ATRA combined with both 1 M/l ATO and
168H
Fig. 1. The effect of ATRA, ATO on CD11b expression of NB4 cells.
7 January 2008
Effect of the combination of ATRA, ATO and DNR on CD11b expression in NB4 cells
72H
24H
0
48H
20
72
Atsunobu Hiraoka ∗ Department of Internal Medicine, Osaka Dental University, 1-5-17 Ohtemae, Chuo-ku, Osaka 540-0008, Japan ∗ Tel.: +81 669101111; fax: +81 669438051. E-mail address: [email protected]
1625
100
percentage of positive cells
[9] Santos SCR, Dias S. Internal and external autocrine VEGF/KDR loops regulate survival of subsets of acute leukemia through distinct signaling pathways. Blood 2004;103:3883–9. [10] Doepfner KT, Spertini O, Arcaro A. Autocrine insulin-like growth factor-I signaling promotes growth and survival of human acute myeloid leukemia cells via the phosphoinositide 3-kinase/Akt pathway. Leukemia 2007;21:1921–30. [11] Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S, et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nature Biotechnol 2007;25:1315–21.
percentage of positive cells
Letters to the Editor / Leukemia Research 32 (2008) 1623–1640
Fig. 3. The effect of ATRA, ATRA + ATO + DNR on CD11b expression of NB4 cells.
Leukemia Research 32 (2008) 1623–1640
Letters to the Editor
Leukemia cell lines require self-secreted stem cell growth factor (SCGF) for their proliferation Stem cell growth factor (SCGF) is a cytokine of the Ctype lectin family, and acts on hematopoietic stem/progenitor cells to support their proliferation [1–3]. Within the bone marrow, they, but not other mature cells, produce SCGF [4–6]. The skeletal tissues, particularly proliferating osteoblasts and chondroblasts, themselves express scgf gene [2], thus the osseous microenvironment is speculated to foster hematopoietic stem/progenitor cells by virtue of SCGF. Serum SCGF level increases along a hematopoietic recovery after bone marrow transplantation, but does not in the case of delayed engraftment [4]. These findings implicate not only a property of SCGF as one of the stem cell markers but also an autocrine or paracrine SCGF regulation of hematopoietic stem/progenitor cells. Most of leukemia cell lines also produce SCGF [1,7], in fact SCGF cDNA has been cloned from the cDNA library prepared from KPB-M15 cells, a human myeloid cell line established from a patient with chronic myeloid leukemia in blast crisis [1]. Here, we address the question of whether self-secreted SCGF triggers proliferation of leukemia cell lines by the neutralization experiment using anti-SCGF antibody. Five SCGF-producing cell lines [1,7] representative of each hematopoietic lineage, KPB-M15 (myeloid), HL-60 (promyelocyte), U937 (monocyte), Raji (B cell) and MOLT-4 (T cell) (the latter four were obtained from Health Science Research Resources Bank, Sennan, Japan), were weekly 1:5- (all but 1:3.5- for HL-60) split cultured with 10% fetal calf serum (FCS)-containing Iscove’s modified Dulbecco’s medium (IMDM) with or without 10 g/ml goat anti-human SCGF antibody (Abcam, Cambridge, MA) or 10 g/ml goat non-immunized isotype IgG (Abcam). Viable cells in the culture of each cell line with anti-SCGF antibody remained unchanged at week 1, but dramatically declined at week 2, and totally disappeared by week 3 or 4, while cells vigorously proliferated in the culture with or without isotype IgG (Fig. 1A). An anti-SCGF antibody-induced decrease in the viable cell counts paralleled a decrease in the cellular viability (data not shown). A long-term culture without the medium exchange was avoided because of a difficulty in discriminat-
0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.
ing the cytoreductive effect of antibody neutralization from a harmful influence of nutritional exhaustion and metabolite accumulation in the medium. The damaged cells in the culture with anti-SCGF antibody appeared apoptotic, e.g. pyknotic nuclei, karyorrhexis, loss of nucleoli and shrunk cytoplasm (Fig. 1B). The present study demonstrates that all leukemia cell lines tested require self-secreted SCGF for their proliferation, indicating that a putative autocrine SCGF mechanism is working and the loop blockade with neutralizing antibody deprives extracellular SCGF to invite apoptosis. An SCGF concentration in the medium with 10% FCS is estimated 1 ng/ml or so [4,8], which contributes little to the biologic activity. Alternatively, less possible, anti-SCGF antibody abrogates an inhibitory activity of SCGF on some unknown apoptotic factor to enhance apoptosis. Uncontrolled autonomous proliferation of leukemia cell lines could be partially explained by the SCGF-mediated stimulation, irrespective of viral, chemical and radiation leukemogenesis. Analysis on SCGF receptor unidentified yet will provide more information for detailed proliferative mechanisms. SCGF is predicted to bind to SCGF receptor through a protein-protein interaction as does tetranectin molecularly akin to SCGF, although SCGF has a conserved carbohydrate-recognition domain of C-type lectin motif sequence at the COOH-terminal. Autocrine vascular endothelial growth factor (VEGF) [9] or insulin-like growth factor-I (IGF-I) [10] loop promotes growth of myeloid cell lines. However, anti-VEGF antibody (bevacizumab; Avastin® ) is ineffective for acute myelogenous leukemia cells since it is inert to the “private” VEGF loop [9], and it is unclear if anti-IGF-I antibody blocks the “public” IGF-I loop. No leukemia cell line has been reportedly inhibited to grow by anti-cytokine antibody except the factor-dependent cell lines. Relatively late-onset suppression by anti-SCGF antibody was seen around the culture period of week 2, indicating a possible action of SCGF on a certain subset of leukemia cell lines, for example, equivalent to the leukemic stem cells. They occupy a part of CD34+ CD38− population of acute myelogenous leukemia cells, reside within the osteoblast-rich endosteal region of the bone marrow when transplanted into the irradiated immunodeficient mice, and survive chemotherapy to originate a relapse of leukemia [11]. Anti-SCGF
1624
Letters to the Editor / Leukemia Research 32 (2008) 1623–1640
Fig. 1. Inhibition of leukemia cell line proliferation by anti-SCGF antibody. (A) A culture of each KPB-M15, HL-60, U937, Raji and MOLT-4 cell line was weekly fed with 10% FCS-containing IMDM with or without 10 g/ml anti-SCGF antibody or 10 g/ml isotype IgG, when viable cells (mean ± S.D. of three separate experiments) were counted immediately before the medium exchange. “No antibody” (blue) indicates the culture with medium alone. (B) Morphology of U937 cells (400× magnification). Cytospin preparations were May-Gr¨unwald-Giemsa stained at the culture period of week 3.
antibody is expected to eradicate even leukemic stem cells if it can arrest their interaction with the microenvironment harboring them. An SCGF concentration in the serum of leukemia patients is 10-100 times higher than the disease-free controls [8] and leukemia will be hopefully cured by normalizing the elevated SCGF level. Consequently a stem cell-specific SCGF-targeting medicine would enable a new potential master key therapy for any type of leukemia.
Acknowledgements This work is not supported by grants. This work is completely original and has not been published previously.
References [1] Hiraoka A, Sugimura A, Seki T, Nagasawa T, Ohta N, Shimonishi M, et al. Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells. Proc Natl Acad Sci USA 1997;94:7577–82.
[2] Hiraoka A, Yano K, Kagami N, Takeshige K, Mio H, Anazawa H, et al. Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells. Hematol J 2001;2:307–15. [3] Gehling UM, Ergun S, Schumacher U, Wagener C, Pantel K, Otte M, et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood 2000;95:3106–12. [4] Ito C, Sato H, Ando K, Watanabe S, Yoshiba F, Kishi K, et al. Serum stem cell growth factor for monitoring hematopoietic recovery following stem cell transplantation. Bone Marrow Transpl 2003;32:391–8. [5] Ma X, Husain T, Peng H, Lin S, Mironenko O, Maun N, et al. Development of a murine hematopoietic progenitor complementary DNA microarray using a subtracted complementary DNA library. Blood 2002;100:833–44. [6] Ng YY, van Kessel B, Lokhorst HM, Baert MRM, van den Burg CM, Bloem AC, et al. Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stemcell activity. J Leuk Biol 2004;75:314–23. [7] Bannwarth S, Giordanengo V, Lesimple J, Lefebvre J-C. Molecular cloning of a new secreted sulfated mucin-like protein with a C-type lectin domain that is expressed in lymphoblastic cells. J Biol Chem 1998;273:1911–6. [8] Ando K, Hotta T, Ito C, Sato H, Furuya A, Shitara K, et al. Method of judging leukemia, pre-leukemia or aleukemic malignant blood disease and diagnostic therefor. PCT/JP2003/004531.
CONTROL
80 60
ATRA
40 ATO
CONTROL
80 60
ATRA
40 20
ATRA+ATO
0
24 H
doi: 10.1016/j.leukres.2008.01.003
100
72 H
Available online 4 March 2008
Fig. 2. The effect of ATRA, ATRA + ATO on CD11b expression of NB4 cells.
1 M/l DNR for 24 h, 48 h, 72 h and 168 h. Control group were untreated NB4 cells. Then CD11b expression on NB4 cells was assessed by direct immunofluorescence of flow cytometric analysis. We found that ATRA significantly upregulated CD11b expression in a dependent-time manner and the change of CD11b expression was most evident with an approximately sixfold increase in the median of the percentage of positive cells after 168 h exposure (Fig. 1). 1 M ATO alone did not cause the significant change of CD11b expression at any tested times (Fig. 1). When we added ATO to ATRA, CD11b expression did not change after 24 h and 48 h exposure, but increased after 72 h and 168 h exposure (Fig. 2). When we added ATO altogether with daunorubicin to ATRA, ATRA-induced a particularly high increase in CD11b expression was reversed (Fig. 3). The increase of CD11b expression induced with ATRA is a key event in the development of the hyperleukocytosis 100 CONTROL
80 60
ATRA
40 ATRA+ATO+
20
DNR
16 8H
H
48
H
0
24 H
percentage of positive cells
The clinical effectiveness of all-trans retinoic acid (ATRA) and trioxide arsenic (ATO) in the treatment of acute promyelocytic leukemia (APL) have been widely confirmed. Unfortunately, in the course of induction-APL with ATRA or ATO, 70% of patients suffer from hyperleukocytosis and 5–25% of patients develop leukostasis, which may lead to life-threatening APL differentiation syndrome. Because of its poor prognosis and high mortality, the early diagnosis and effective treatment of this syndrome need to be seriously considered. According to recent studies [1], the hyperleukocytosis and APL differentiation syndrome are associated with the adhesion change of APL cells during ATRA-induced differentiation. The CD11b is the adhesion molecular of the mark of APL cells-differentiation. Recent studies have showed that increase of CD11b expression induced with ATRA is an important cause of the hyperleukocytosis and acute promyelocytic leukemia differentiation syndrome [2]. At present, ATRA combined with ATO and anthracycline has been used to treat APL patients, which not only notably improves curative effect, but also reduces the incidence rate of the hyperleukocytosis and APL differentiation syndrome. To explore the role of CD11b expression and the effects of these drugs in hyperleukocytosis and APL differentiation syndrome, we studied the effects of ATRA, ATO and daunorubicin (DNR) on CD11b expression of NB4 cells that is the only true human promyelocytic leukemia cell line with the characteristic chromosomal translocation t (15; 17) (p21; q23). NB4 cells were cultured respectively with 1 M/l ATRA, 1 M/l ATO, 1 M/l ATRA combined with 1 M/l ATO, 1 M/l ATRA combined with both 1 M/l ATO and
168H
Fig. 1. The effect of ATRA, ATO on CD11b expression of NB4 cells.
7 January 2008
Effect of the combination of ATRA, ATO and DNR on CD11b expression in NB4 cells
72H
24H
0
48H
20
72
Atsunobu Hiraoka ∗ Department of Internal Medicine, Osaka Dental University, 1-5-17 Ohtemae, Chuo-ku, Osaka 540-0008, Japan ∗ Tel.: +81 669101111; fax: +81 669438051. E-mail address: [email protected]
1625
100
percentage of positive cells
[9] Santos SCR, Dias S. Internal and external autocrine VEGF/KDR loops regulate survival of subsets of acute leukemia through distinct signaling pathways. Blood 2004;103:3883–9. [10] Doepfner KT, Spertini O, Arcaro A. Autocrine insulin-like growth factor-I signaling promotes growth and survival of human acute myeloid leukemia cells via the phosphoinositide 3-kinase/Akt pathway. Leukemia 2007;21:1921–30. [11] Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S, et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nature Biotechnol 2007;25:1315–21.
percentage of positive cells
Letters to the Editor / Leukemia Research 32 (2008) 1623–1640
Fig. 3. The effect of ATRA, ATRA + ATO + DNR on CD11b expression of NB4 cells.