Forming Cells from Patients with Chronic Myeloid Leukemia to Inhibition by Prostaglandin E1
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[CANCER RESEARCH 40. 251 2-2515, July 1980] 0008-5472/80/0040-OOOOS02.00 Abnormal Responsiveness of Granulocyte-Macrophage Committed Colony- forming Cells from Patients with Chronic Myeloid Leukemia to Inhibition by Prostaglandin E1 Louis M. Pelus,2 Hal E. Broxmeyer,3 Bayard D. Clarkson, and Malcolm A. S. Moore Departments of Developmental Hematopoiesis [L. M. P., H. E. B., M. A. S. M.] and Hematopoietic Cell Kinetics [B. D. C.J. Sloan-Kettering Institute for Cancer Research. New York, New York 10021 ABSTRACT the negative feedback regulation of leukemic cell proliferation have been reported. Mature granulocytes from patients with The formation of myeloid colonies in soft-agar cultures of chronic myeloid leukemia are quantitatively deficient in a col normal human marrow was markedly inhibited by prostaglandin ony-inhibitory activity (3), subsequently identified as lactoferrin E. Morphological characterization of colonies in the presence (5), which inhibits monocyte CSF production. Moreover, CSF or absence of prostaglandin EI showed that inhibition was production by monocytes from these patients was less sensitive restricted to monocytoid rather than neutrophil differentiation. than normal to inhibition by colony-inhibitory activity (lactofer Myeloid colony formation by granulocyte-macrophage-commit- rin) derived from normal mature granulocytes. In acute leuke ted colony-forming cells from patients with chronic myeloid mia, a leukemic-inhibitory activity can be obtained from low- leukemia was not inhibited even by high concentrations of density nonadherent blood and bone marrow cells which di prostaglandin E and was independent of colony morphology. rectly inhibits the proliferation of normal but not leukemic The altered sensitivity of leukemic colony-forming cells to pros colony-forming cells (4). taglandin E was observed at all stages of the disease and This communication extends the concept of abnormal regu persisted following chemotherapy-induced reversion to a par lation in leukemia and demonstrates that CFU-GM from patients tial or complete Philadelphia chromosome-negative bone mar with Ph'-positive chronic myeloid leukemia are insensitive to row status. This evidence suggests that altered myeloid stem normal feedback regulation by prostaglandin E. Furthermore, cell sensitivity to a normal regulatory factor may play a role in insensitivity to prostaglandin E-mediated inhibition persists in the pathophysiology of chronic myeloid leukemia. patients whose bone marrow has temporarily reverted to Ph1- negative status following intensive chemotherapy (1, 8). INTRODUCTION Marrow culture studies have documented that committed MATERIALS AND METHODS myeloid stem cells, CFU-GM,4 proliferate and differentiate in Patients. Bone marrow aspirates for the assessment of gran- vitro, forming granulocyte and/or macrophage colonies, under ulocyte-macrophage colony formation were obtained from nor the influence of obligatory humoral stimulatory glycoproteins termed CSF (6, 12, 20). Under normal circumstances, myelo- mal volunteers following informed consent or from patients with poiesis is limited by at least 2 naturally occurring negative chronic myeloid leukemia undergoing routine bone marrow biopsy at the time of diagnosis or for the assessment of clinical feedback controls. Monocytes and macrophages synthesize status and response to therapy. Eight Ph'-positive patients with and release prostaglandin E (10,19), which preferentially limits the proliferation of colony-forming cells committed to mono chronic myeloid leukemia (2 newly diagnosed and 6 patients with chronic-phase disease on maintenance therapy) and 3 cytoid differentiation (19), whereas normal polymorphonuclear patients shown to be completely or partially Ph1 negative fol neutrophils elaborate lactoferrin, which decreases the produc tion and/or release of CSF by monocytes and macrophages lowing intensive chemotherapy were studied. Maintenance therapy for patients with chronic-phase disease (5, 19). Investigation of leukemic cell regulation has demonstrated consisted of daily p. o. administration of hydroxyurea and/or that, while the proliferation of CFU-GM from patients with 6-mercaptopurine, or busulfan. Assay of colony formation was leukemia, like normal CFU-GM, is dependent upon the pres performed a minimum of 1 week following cessation of therapy. ence of CSF (16), karyotypic analysis of proliferating cells (7, No difference in the morphological types of proliferating mye 14) and abnormal in vitro growth patterns (13,15,16) indicate loid colonies and clusters was observed between untreated a derivation from leukemic clones. Moreover, abnormalities in newly diagnosed patients and those on maintenance therapy. Aggressive chemotherapy for patients with chronic myeloid 1 Supported by Grants CA-08748, CA-23528, and CA-20194, and the Gar leukemia consisted of daunorubin, 1-yS-D-arabinofuranosylcy- Reichman Foundation. 2 Recipient of National Service Award F32-CA-05904 from the National Can tosine, and 6-thioguanine (1, 8). Assay of colony formation was cer Institute, Department of Health, Education, and Welfare. Special fellow of performed 3 weeks following therapy. The Leukemia Society of America. To whom requests for reprints should be Physical Separation of Colony-forming Cells. Marrow col addressed, at Department of Developmental Hematopoiesis, Sloan-Kettering ony-forming cells (CFU-GM) were obtained by neutral-density Institute for Cancer Research, 1250 First Avenue. New York, N. Y. 10021. 3 Scholar of the Leukemia Society of America. (density cut) centrifugation in bovine serum albumin (density, ' The abbreviations used are: CFU-GM, granulocyte-macrophage-committed colony-forming cell; CSF, colony-stimulating factorts); Ph', Philadelphia chro 1.070 g/ml; 270 mOsm). Buoyant mononuclear cells were mosome marker. subsequently subjected to an adherence procedure to remove Received December 26, 1979; accepted March 12. 1980. endogenous CSF-producing cells (2). 2512 CANCER RESEARCH VOL. 40 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. Defective Regulation of Chronic Myeloid Leukemia Stem Cells Assay for Colony-forming Capacity. The ability of normal mation (Chart 1). The effects of prostaglandin E were dose dependent over a concentration range of 10~5 to 10~12 M. and patient bone marrow cells to form colonies of granulocytes and macrophages was assayed in soft-agar culture. One Significantly decreased cell growth was observed in 8 of 10 normal donors (p < 0.005) at 10~9 M and in 5 of 9 donors (p hundred thousand light-density nonadherent mononuclear cells <0.01)at 10~10M prostaglandinEi concentrations. Incontrasi, were plated in a 1.0-ml layer of 0.3% agar (Difco Laboratories, Inc., Detroit, Mich.) in McCoy's 5A modified medium containing the addition of prostaglandin E to marrow cultures from patients 10% fetal calf serum (Microbiological Associates, Inc., Be- with Ph1-positive chronic myeloid leukemia had no significant thesda, Md.). CFU-GM proliferation was stimulated by the effect on CFU-GM proliferation at all concentrations tested inclusion of 10% (v/v) placenta! cell-derived CSF prepared as (Chart 2). described by Nicola ef al. (17). No colony or cluster formation Colony and Cluster Morphology. Morphological and cyto- by light-density nonadherent bone marrow cells from normal chemical examination of proliferating colonies and clusters individuals or patients with chronic myeloid leukemia was ob from normal bone marrow cultures indicated that the effects of served in the absence of exogenously added CSF. Prostaglan- prostaglandin E on CFU-GM proliferation were manifested pref dins were added directly to the culture dish just prior to the erentially on monocyte-macrophage and to a lesser extent on addition of the bone marrow suspension. Indomethacin, at a mixed monocytoid-neutrophilic colonies and clusters, with no final concentration of 10~6 M, was routinely incorporated into significant inhibition of neutrophil colony or cluster formation observed with concentrations of prostaglandin E, below 10~5 all agar cultures to prevent endogenous prostaglandin produc tion. At this concentration, indomethacin inhibits greater than M (Table 1). Morphological analysis of proliferating clones from 90% of the prostaglandin production by normal mouse mac 3 patients with chronic-phase disease demonstrated that mon rophages and human monocytes, neoplastic macrophages, ocyte-macrophage and mixed monocytoid-neutrophilic colo and murine and human leukemic cell lines (9,10,18). Colonies nies and clusters were insensitive to inhibition even at concen (>50 cells) and clusters (3 to 50 cells) in quadruplicate cultures trations of prostaglandin E, which produce a 70% inhibition of were scored on Day 7 of culture. monocyte-macrophage and 60% inhibition of mixed monocy- Morphology and Cytochemistry. Morphological examina toid and neutrophilic colony and cluster formation by normal bone marrow cells (Table 1). Prostaglandin F2nat a concentra tion of proliferating clones was performed in situ following tion of 10~5 M had no effect on total or morphologically defined fixation with 5% glutaraldehyde, methanol dehydration, and staining with Luxol's Fast Blue and Harris' hematoxylin (17). CFU-GM proliferation from either normal or leukemic bone Cytogenetic analysis for Ph1 was performed on bone marrow marrow cultures. samples as described (8). Effects of Prostaglandin on CFU-GM from Patients with Reagents. Prostaglandins were generously