Effects of Prostaglandin E on the Proliferation and Differentiation of Leukemic Progenitor Cells in Acute Nodymphocytic Leukemia

Transcription

1 International Journal of Cell Cloning 1 : (1983) Effects of Prostaglandin E on the Proliferation and Differentiation of Leukemic Progenitor Cells in Acute Nodymphocytic Leukemia Keiya Ozawa&.l, Yasusada Miuraa*b, Toshio Sudaa, Kazuo Motoyoshia, Fumimaro TakakubJ adivision of Hemopoiesis, Institute of Hematology and bdepartment of Internal Medicine, Jichi Medical School, Tochigi, Japan Key Words. Prostaglandin E - Leukemic progenitor cells a Differentiation Proliferation Abstract. The effects of prostaglandin El (PGE1) on leukemic colony formation were investigated in seven cases of acute nonlymphocytic leukemia. In contrast to the almost constant results observed for normal myeloid colony formation, in patients there were apparent individual variations. Strong inhibition of colony formation by PGEl was observed in three cases. In one case, enhancement rather than inhibition was observed. These in vitro inhibitory or stimulatory effects of PGEl were not related to the degree of the in vivo proliferation of leukemia cells. Morphologic analysis of colonies revealed that preferential inhibition of monocytic colony formation by PGE1, characteristic of normal bone marrow cultures, was not always observed in leukemia cell cultures. These abnormal responses to PGEl suggest that the proliferation and differentiation of leukemic progenitor cells are regulated in a different way from normal hemopoiesis. Introduction It has been shown that the proliferation and differentiation of normal myeloid progenitors (CFU-GM), which are dependent on the presence of colony-stimulating factor(s) (CSF), are modulated by several compounds 1 Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan O1983 AlphaMed Press, Inc I454/83/$2.00/0

2 ' Ozawa/Miura/Suda/Motoyoshi/Takaku 441 and hormones 11-51, Particularly, prostaglandins of the E series, which are produced by monocytes/macrophages, have been shown to be inhibitory regulators of normal CFU-GM proliferation [6, 71. Moreover, this prostaglandin El, (PGEl)-mediated inhibition was reported to be restricted to monocytoid rather than neutrophilic differentiation [7,8]. Interestingly, abnormal CFU-GM from patients with chronic myelogenous leukemia (CML) was shown to be insensitive to this inhibitory regulation by PGEl [ This phenomenon may be related to the uncontrolled excessive proliferation of CML cells. As for leukemic progenitor cells in acute nonlymphocytic leukemia (ANLL), there is relatively little information regarding their responsiveness to prostaglandins [12, 131. In the present study, we have examined the effects of PGEl on the proliferation and differentiation of leukemic progenitor cells in three cases of acute rnyeloblastic leukemia (AML) and four cases of acute myelomonocytic leukemia (AMMoL). Recently, we have observed that some degrees of differentiation occurred during leukemic colony formation in many cases as determined by dual esterase staining in permanent preparations [ 141. In this study, therefore, morphologic analysis of colonies was performed to compare the effects of PGEl on different types of leukemic colonies. This study wil aid in understanding the nature of leukemic progenitor cells and the regulation of leukemic cell proliferation and differentiation. Materials and Methods Patients Seven cases of ANLL were studied. The patients' peripheral blood or bone marrow specimens contained a sufficient number of leukemic progenitor cells for analysis (Table I). The type of leukemia was classified according to the clinical data and cytochemical findings obtained by dual esterase staining [15]. The cells from AML showed positive naphthol AS-D chloroacetate esterase reaction, and little or no cr-naphthyl butyrate esterase reaction. Both chloroacetate esterasepositive cells and butyrate esterase-positive cells were detected in AMMoL. Leukemic Colony Formation Peripheral blood or bone marrow specimens were obtained from the patients before treatment. Leukemic colonies were formed according to the method of Minden et al. [ 161 with minor modifications. Briefly, mononuclear cells were isolated by Ficoll-Metrizoate (Lymphoprep, Nyegaard, Oslo) density centrifugation at 400 g. In order to avoid the formation of T-lymphocyte colonies, sheep erythrocyte rosette (E-rosette)-forming cells were subsequently removed by the tech-

3 ~~ Effects of PGE on Leukemic Colony Formation 442 Table L Clinical data and colony-forming ability in seven cases of acute nonlymphocytic leukemia Case Diagnosisa Sampleb NCCC Blasts No. of colonies/dishd No. (X 1091~) (%) HPCM PHA-LCM 1 AML PB f 2 61 f 11 2 AML PB f 6 5 f 1 3 AML PB f f 14 4 AMMoL BM f7 5 AMMoL BM f f 17 6 AMMoL BM f f 50 7 AMMoL PB f f 30 a AML, acute myeloblastic leukemia; AMMoL, acute myelomonocytic leukemia. b PB eripheral blood; BM, bone marrow. c N&, nuclear cell count. d E-rosette-negative mononuclear cells were separated from the Sam le. This fraction contained a high ro ortion of blasts. These cr o reserve a cells were cultured in the presence o FH 6 CM or PHA-LCM. 1 X!$cells were plated in cases 1,2, and 3; 2 x 104 cells in cases 4,5, and 7; 4.5 X 104 cells in case 6. Colonies were counted on day 7-9 of culture (mean f SD). nique described by Minden et al. [ 161. About 2% or less of E-rosette-positive cells were left after this rosetting technique. Cells from the E-rosette-negative fraction were stored in 10% dimethyl sulfoxide (DMSO) and 10% fetal calf serum (FCS; Flow Laboratories, Rockville, MD) at -80 C until use. These cells were thawed, washed, resuspended, and cultured at an appropriate concentration in 0.3 7% agar medium containing 20% FCS and 10% CSF. Two different conditioned media were used as the sources of CSF in this study. Phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) was obtained from the supernatant of cultured leukocytes (1 X 106 cells/ml) incubated for 7 days in alpha medium (Flow) with 10% FCS and 1% PHA (Wellcome HA-1 5). Human placenta conditioned medium (HPCM) was prepared according to the method of Burgess et al. [ 171. Culture plates were incubated for 7-9 days at 37 C in a fully humidified atmosphere containing 5% COz in air. Colonies containing 20 or more cells were counted using an inverted microscope. Fresh cells also were cultured in 0.8% methylcellulose (Dow Chemical Co., Midland, MI) instead of agar under the same conditions. Cells within a part of the colonies were pooled and tested for E-rosette formation. T-lymphocyte colony formation was not observed in the present cases.

4 Ozawa/Miura/Suda/Motoyoshi/Takaku 443 Normal Myeloid Colony Formation Bone marrow specimens were obtained from normal human volunteers after receiving written informed consent. E-depleted bone marrow mononuclear cells were cultured at a concentration of 5 x 104 cells/ml in 0.3% agar medium containing 20% FCS and 10% CSF (HPCM or PHA-LCM). After 7-9 days of culture, colonies containing 40 or more cells were counted using an inverted microscope. Efects of PGEl on Colony Formation The effects of PGEl (Sigma Chemical Co., St. Louis, MO) on normal and leukemic myeloid colony formation were tested by the direct addition of PGEl to the culture mixture at concentrations ranging from 10-9 M to 10-5 M. Indomethacin (Sigma), a specific PG synthesis inhibitor, also was added to the cultures at a final concentration of 10-6 M to prevent endogenous PG production. A stock solution of PGEl was prepared in ethanol at 10-2 M and stored at -80 C. Indomethacin was freshly prepared prior to use. Morphologic Analysis of Colonies Permanent preparations of colonies were made [ 181 and stained by dual esterase staining according to the method of Li et al. [ 151. If most of the cells in a colony were chloroacetate esterase-positive or butyrate esterase-positive, the colony was considered a neutrophilic or monocytic (monocyte/macrophage) colony, respectively. If a colony consisted of both chloroacetate esterase-positive cells and butyrate esterase-positive cells, the colony was considered a neutrophil/macrophage (N/M) mixed colony. Esterase reaction of leukemic colonies was often weak, and sometimes deficient. The former were classified as similar to normal myeloid colonies, and the latter, deficient ones seemed to be mostly blastic colonies, on the basis of the results of Wright-Giemsa staining. Furthermore, Scarlet- Bieblich staining [ 191 was performed in normal bone marrow cultures to identify eosinophil colonies. Results Efects of PGEl on Normal Myeloid Colony Formation The addition of PGEl to normal marrow cultures caused a dose-dependent inhibition of colony formation (Fig. 1). In this study two different conditioned media, HPCM and PHA-LCM, were used as the sources of CSF. While HPCM stimulated mainly neutrophilic colony formation, PHA-LCM stimulated the formation of many macrophage and eosinophilic colonies besides neutrophilic ones after 7-9 days culture of normal marrow cells. Compared to a strong inhibitory effect on monocytic colony formation, the effect on neutrophilic lineage was mild, and no apparent inhibition was observed for eosinophilic colony formation. Similar results were obtained in five separate experiments.

5 Effects of PGE on Leukemic Colony Formation 444 I HPCM PHA-LCM Fig. 1. The effects of PGEl on normal myeloid colony formation. E-rosettenegative mononuclear cells were stimulated by HPCM or PHA-LCM in the presence of indomethacin M) and various concentrations of PGE1. The results are expressed as a percentage of control cultures which were stimulated by PHA- LCM without PGEl (mean f SD). (PHA-LCM stimulated more colonies than HPCM). Colony types were determined by dual esterase staining (m = neutrophilic colonies; = neutrophil/macrophage mixed colonies; El = monocyte/ macrophage colonies; 0 = eosinophilic colonies). Efects of PGEl on Leukemic Colony Formation As shown in Figures 2 and 3, the responses of leukemic progenitor cells to PGEl varied from patient to patient, in contrast to the almost constant results observed for normal myeloid progenitors. Strong inhibition of colony formation by PGEl was observed in three cases (cases 3, 4, 5). In case 1 (AML), enhancement rather than inhibition of leukemic colony formation was observed over a wide range of concentrations of PGE1. In cases 6 and 7, in which many esterase-negative blastic colonies were formed, inhibition was mild. These in vitro inhibitory or stimulatory effects of PGEl were not related to the degree of in vivo proliferation of leukemia cells, which was judged by the hematological data when the samples were obtained (data not shown).

6 Ozawa/Miura/Suda/Motoyoshi/Takaku 445 Fig. 2. The effects of PGEl on leukemic colony formation in three cases of AML. E-rosette-negative mononuclear cells were stimulated by HPCM or PHA- LCM in the presence of indomethacin (10-6 M) and various concentrations of PGE1. The results are expressed as a percentage of control cultures which were stimulated by PHA-LCM (cases 1 and 3) or HPCM (case 2) without PGEl (mean f SD). (In cases 1 and 3, PHA-LCM stimulated more colonies than HPCM; and in case 2, HPCM stimulated more colonies than PHA-LCM). Colony types were determined by dual esterase staining = neutrophilic colonies; El = neutrophil/ macrophage mixed colonies; 0 = esterase-negative colonies).

7 Effects of PGE on Leukemic Colony Formation 446 Fig. 3. The effects of PGEl on leukemic colony formation in four cases of AMMoL. E-rosette-negative mononuclear cells were stimulated by HPCM or PHA-LCM in the presence of indomethacin (10-6 M) and various concentrations of PGE1. The resuits are expressed as a percentage of control cultures which were stimulated by HPCM (cases 4, 6, and 7) or PHA-LCM (case 5) without PGEl (mean f SD). (In cases 4, 6, and 7, HPCM stimulated more colonies than PHA- LCM; and in case 5, PHA-LCM stimulated more colonies than HPCM). Colony types were determined by dual esterase staining ( UII = neutrophilic colonies; = neutrophil/macrophage mixed colonies; 0 = monocyte/macrophage colonies; 0 = esterase-negative colonies).

8 Ozawa/Miura/Suda/Motoyoshi/Takaku 447 As reported previously [ 141, the majority of leukemic colonies formed in AML patients were the immature neutrophilic type, and both immature neutrophilic and monocytic colonies were formed in AMMoL patients (Fig. 2, 3). Therefore, the effects of PGEl on different types of leukemic colonies were compared. Various effects, stimulatory or inhibitory, of PGEl were observed on leukemic colony formation of a neutrophilic lineage. The degree of inhibitory effect was often stronger than that observed on normal neutrophilic colony formation. PGEl at concentrations of 10-6 M and lower did not reduce the number of normal neutrophilic colonies. On the other hand, leukemic colony formation of a neutrophilic lineage was inhibited even at a concentration of 10-7 M in case 2 (in HPCM-stimulated cultures), case 3 (in PHA-LCM-stimulated cultures), and case 5. Monocytic colony formation in cases 4 and 5 was strongly inhibited, similar to normal monocytic colony formation. In case 6, monocytic colony formation was not influenced significantly by PGEl addition, and a significant number of monocytic colonies were formed even at a concentration of 10-5 M. The monocytic colonies formed in this case were rather immature, and only a part of the cells in the colonies were butyrate esterasepositive. The effects on esterase-negative blastic colonies in cases 6 and 7 were mild. Patterns of response to PGEl in HPCM-stimulated cultures were similar to those in PHA-LCM-stimulated cultures, except in cases 2 and 4. While neutrophilic colony formation in cases 2 and 4 was inhibited by PGEl in HPCM-stimulated cultures, slight stimulation occurred in PHA- LCM-stimulated cultures. The reasons for this difference are not known. Discussion PGEl has been regarded as one of the modulators of normal myeloid cell proliferation. As reported by several investigators [6-81, normal myeloid colony formation was inhibited dose-dependently by PGE1. This inhibition was more prominent for a monocytic lineage than for a neutrophilic lineage. The effects of PGEl on the proliferation and differentiation of leukemic progenitor cells in ANLL varied from patient to patient, in contrast to the almost constant results observed for normal myeloid progenitors. In this study cryopreserved cells were used to investigate the effects of PGEl on leukemic colony formation. The freezing procedure did not affect the

9 Effects of PGE on Leukemic Colony Formation 448 proportion of colony types, which were influenced mainly by the batches and concentrations of CSF and culture periods (data not shown). Moreover, radiosensitivity and cell-cycle status of leukemic progenitor cells did not change after freezing [20]. Therefore, variations in responsiveness to PGEl seem to be due to the different intrinsic nature of leukemic progenitor cells, but not due to freezing. Enhancement rather than inhibition of colony formation was reported in one case of acute monocytic leukemia (AMoL) [13]. They speculated that AMoL progenitor cells, possessing some properties of monocytic lineage, reacted to PGEl by elaboration of a humoral stimulus to proliferation, because PGEl stimulates CSF release from normal macrophages. In our study, a similar stimulatory effect of PGEl was observed in one case of AML, meaning that such a phenomenon is not specific for AMoL and that the involved mechanisms may be more complicated. The in vitro inhibitory or stimulatory effects of PGEl were not related to the degree of the in vivo proliferation of leukemia cells in the present limited number of cases examined. Morphologic analysis of colonies by dual esterase staining showed that leukemic colonies of a neutrophilic lineage as well as monocytic colonies were markedly inhibited by PGEl in several cases. In other words, unlike normal bone marrow cell cultures, preferential inhibition of monocytic colony formation was not always observed in leukemia cell cultures. It suggests that functional separation between a neutrophilic lineage and a monocytic lineage may be rather incomplete at the progenitor cell level in the leukemic cell differentiation. It is not always easy to demonstrate that colonies formed by the present method of leukemic progenitor assay are derived from leukemic clones but not from a normal clone. However, these colonies have been considered to be leukemic in origin for a number of reasons; e.g., chromosomal markers characteristic of leukemic clones were identified in some colonies The abnormal pattern of colony formation in PGEl-containing cultures shown in this study may be another reason indicating that colonies formed in the present culture system were leukemic in origin. Moreover, the findings that the responses of leukemic progenitor cells to PGEl are abnormal suggest that the proliferation and differentiation of leukemia cells are regulated in a different way from normal hemopoiesis. PGEl has been proposed to modify normal myelopoiesis by altering intracellular cyclic nucleotide concentrations [ 13, 221. Leukemic progenitors from patients with AMoL or CML who showed abnormal re-

10 Ozawa/Miura/Suda/Motoyoshi/Takaku 449 sponses to PGEl were reported to retain their normal patterns of response to cyclic nucleotides [ 131. This finding suggests that abnormal responses of leukemic progenitors may be due, in part, to defects in the PGE1- mediated regulation of intracellular cyclic nucleotide concentrations. Further studies will be necessary to confirm this hypothesis. Acknowledgments The authors would like to thank Misses Sachiko Kurokawa, Michiko Yoshida, and Yasuko Miyazaki for their excellent technical assistance. This study was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and from the Ministry of Education, Science, and Culture of Japan. References Rosenblum, A.L.; Carbone, P.P.: Androgenic hormones and human granulopoiesis in vitro. Blood 43: (1974). Golde, D.W.; Bersch, N.; Quan, S.G.; Cline, M.J.: Inhibition of murine granulopoiesis in vitro by dexamethazone. Am J Hematol (1976). Singer, J.W.; Adamson, J.W.: Steroids and hematopoiesis The response of granulocytic and erythroid colony-forming cells to steroids of different classes. Blood 48: (1976). Golde, D.W.; Cline, M.J.: Hormonal interactions with hematopoietic cells in vitro. Transplant Proc 10: (1978). Kurland, J.I.; Brockman, R.S.; Broxmeyer, H.E.; Moore, M.A.S.: Limitation of excessive myelopoiesis by the intrinsic modulation of macrophage derived prostaglandin E. Science 199: (1978). Kurland, J.I.; Broxmeyer, H.E.; Pelus, L.M.; Brockman, R.S.; Moore, M.A.S.: Role for monocyte-macrophage-derived colony-stimulating factor and prostaglandin E in the positive and negative feedback control of myeloid stem cell proliferation. Blood 52: (1978). Pelus, L.M.; Broxmeyer, H.E.; Kurland, J.I.; Moore, M.A.S.: Regulation of macrophage and granulocyte proliferation. Specificities of prostaglandin E and lactoferrin. J Exp Med 150: (1979). Pelus, L.M.; Broxymeyer, H.E.; Moore, M.A.S.: Regulation of human myelopoiesis by prostaglandin and lactoferrin. Cell Tissue Kinet 14: (1981). Aghetta, M.; Piacibellow, W.; Gavosto, F.: Insensitivity of chronic myeloid leukemia cells to inhibition of growth by prostaglandin E. Cancer Res 40: (1980). Pelus, L.M.; Broxmeyer, H.E.; Clarkson, B.D.; Moore, M.A.S.: Abnormal responsiveness of granulocyte-macrophage committed colony-forming cells from patients with chronic myeloid leukemia to inhibition by prostaglandin E. Cancer Res 40: (1980).

11 Effects of PGE on Leukemic Colony Formation Taetle, R.; Guittard, J.P.; Mendelsohn, J.M.: Abnormal modulation of granulocyte/macrophage progenitor proliferation by prostaglandin E in chronic myeloproliferative disorders. Exp Hematol8: (1980). Koeffler, H.P.; Golde, D.W.: Humoral modulation of human acute myelogenous leukemia cell growth in vitro. Cancer Res 40: (1980). Taetle, R.; Koessler, A.: Effects of cyclic nucleotides and prostaglandins on normal and abnormal human myeloid progenitor proliferation. Cancer Res 40: (1980). Ozawa, K.; Miura, Y.; Suda, T.; Motoyoshi, K.; Takaku, F.: In vitro differentiation of leukemic progenitor cells in various types of acute nonlymphocytic leukemia. Cancer Res 43: (1983). Li, C.Y.; Lam, K.W.; Yan, L.T.: Esterases in human leukocytes. J Histochem Cytochem 21: 1-12 (1973). Minden, M.D.; Buick, R.N.; McCulloch, E.A.: Separation of blast cell and T-lymphocyte progenitors in the blood of patients with acute myeloblastic leukemia. Blood 54: (1979). Burgess, A.W.; Wilson, E.A.M.; Metcalf, D.: Stimulation by human placental conditioned medium of hemopoietic colony formation by human marrow cells. Blood 49: (1977). Kubota, K.; Mizoguchi, H.; Miura, Y.; Suda, T.; Takaku, F.: A new technique for the cytochemical examination of human hemopoietic cells grown in agar gel. Exp Hematol8: (1980). Konwalinka, G.; Glaser, P.; Odavic, R.; Bogusch, E.; Schmalzl, F.; Braunsteiner, H.: A new approach to the morphological and cytochemical evaluation of human bone marrow CFU-c in agar cultures. Exp Hematol 8: (1980). Ozawa, K.; Miura, Y.; Suda, T.; Motoyoshi, K.; Takaku, F.: Radiation sensitivity of leukemic progenitor cells in acute nonlymphocytic leukemia. Cancer Res 43: (1983). Izaguirre, C.A.; McCulloch, E.A.: Cytogenetic analysis of leukemic clones. Blood 52: 287 (1978). Kurland, J.I.; Hadden, J.W.; Moore, M.A.S.: Role of cyclic nucleotides in the proliferation of committed granulocyte-macrophage progenitor cells. Cancer Res 37: (1977). Accepted: July 22,1983 Keiya Ozawa, M.D., The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113 (Japan)