Leukemia (1997) 11, 701–708 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Cytokine response profiles of human myeloid factor-dependent leukemia cell lines HG Drexler, M Zaborski and H Quentmeier DSMZ-German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Mascheroder Weg 1 B, D-38124 Braunschweig, Germany Research in cytokine biology has grown exponentially in recent large variety of soluble mediators. Thus, these systems are not years as cytokines (often also termed growth factors) are now always readily amenable for the detailed study of the mech- known to be involved in a wide range of pathological and physiological processes. Continuous human leukemia cell anisms controlling hematopoietic cell processes such as, for lines represent powerful tools to investigate these mech- instance, proliferation. anisms. Most cell lines grow autonomously in standard culture The advent of the cloning and recombinant production of media (containing fetal bovine serum) independent of exter- these soluble effector molecules (variously termed hematopoi- nally added growth stimuli. Over the last 5–10 years a battery etic growth factors or cytokines) and the establishment of of myeloid leukemia-derived cell lines has been established clonal immortalized cell lines have greatly facilitated the that is constitutively dependent on the addition of cytokines to the culture. Such factor-dependent cell lines die rapidly by approaches to in vitro studies. Clearly, continuous leukemia apoptosis when deprived of the appropriate growth factor. We cell lines represent overall a more simple system for the study determined the cytokine response profiles of 19 absolutely of hematopoiesis in vitro. growth factor-dependent leukemia cell lines with myelomono- Until recently all human leukemia cell lines were grown in cytic, erythroid or megakaryocytic phenotypes with regard to medium supplemented only with fetal bovine serum (FBS) and enhanced or suppressed cellular proliferation. Cells were incu- were not dependent on exogenous hematopoietic growth fac- bated in liquid culture with optimal concentrations of various 1 recombinant human cytokines known to have effects on the tors (autonomous growth). Although cell lines dependent on growth of hematopoietic cells. A proliferative or anti-prolifera- interleukin-2 (IL-2) have been previously established, this suc- tive response to these 41 cytokines was assessed by the short- cess was restricted to a particular category of T cell lines, term 3H-thymidine uptake assay. A proliferative response was namely adult T cell leukemia lines. Early attempts to support considered as positive when the stimulation index (SI) was .2; acute/chronic myeloid leukemia (AML/CML) cell growth in , inhibition was regarded as significant with an SI 0.5. The vitro with conditioned media from stimulated lymphocytes response profile of each cell line to these 41 cytokines was different and individual. None of the cell lines responded to one succeeded in sustaining growth only for a limited time period or two factors only (minimum to at least five cytokines). Pro- of several weeks. Thus, growth factors were found to be at liferation of most (n 5 13–17), but not of all cell lines was sig- least permissive for expansion of myeloid leukemia clones. nificantly enhanced by GM-CSF, IL-3, PIXY-321, SCF and IFN- The availability of purified or recombinant cytokines g. TGF-b1 consistently inhibited proliferation (in 11/19 cell enabled further progress in the culturing of leukemia cells. a b a b lines). IFN- , IFN- , TNF- and TNF- had either stimulatory or Over the last 5–10 years a spectrum of factor-dependent cell inhibitory effects. The cell lines responding most often prolifer- atively (to 15–19 different cytokines) were UCSD/AML1, HU-3, lines has been established from patients with various types of TF-1 and M-07e. In summary, these factor-responsive human leukemia that are absolutely dependent on addition of growth leukemia cell lines represent extremely useful model systems factors to the medium for proliferation and survival.2 The cyto- for the analysis of cytokine effects on hematopoietic cells. The kines involved include some of the interleukins (eg IL-2, IL-3, cytokine response profiles of the individual cell lines provide IL-6) and the so-called colony-stimulating factors (CSF) (GM- guidelines for the selection of the appropriate cell culture for CSF, G-CSF, M-CSF).1,2 Many of these cell lines are dependent such experiments. Keywords: leukemia; cytokines; cell lines; proliferation on either IL-3 or GM-CSF, reflecting the ability of these cyto- kines to stimulate early progenitor and immature cells. Further studies also suggested that non-lineage specific hormones such as insulin and insulin-like growth factor (IGF-I) can syn- ergistically stimulate growth of factor-dependent leukemia Introduction cells lines.1 Thus, the establishment of factor-dependent cell lines allowed for the continuous growth of leukemic cells that Mature blood cells have a finite lifespan and must be replen- were previously eliminated during selection in standard ished throughout adult life by the continued activity of hema- culture medium. topoietic stem and progenitor cells that are operationally These factor-dependent cell lines are important models for defined by their capacity to repopulate lymphoid and myeloid cytokine signal transduction as well as proliferative responses lineages. For these stem and progenitor cells to proliferate and and differentiation.2 Withdrawal of the supporting growth fac- mature in multiple lineages, coordinated support by a multi- tor commonly leads to cell death within a few days in most tude of cytokines is required. The complexity of hematopo- cell lines. This rapid cell death occurs by an active cellular ietic cells presents difficulties for the identification of cyto- process (apoptosis) that can be suppressed by new addition kines with specific effects on certain types of cells and at the of growth factors.3 Most of these cell lines showed a growth various stages of activation, proliferation or differentiation. response to several different cytokines; quite heterogeneous Cultures seeded with peripheral blood or bone marrow patterns of response were evident with no two lines exhibiting samples develop adherent layers that are composed of a com- the same response phenotype.2 plex mixture of mesenchymal cell types. These stroma cells Here, we have determined the proliferative or anti-prolifer- provide signals mainly via cell–cell contact or secretion of a ative response of a panel of continuous human myeloid cell lines (derived from different types of AML) to a panel of Correspondence: HG Drexler cytokines known to have mitogenic or inhibitory effects on Received 19 September 1996; accepted 20 January 1997 hematopoietic cells. These cytokine response profiles should Cytokine responses of leukemia cell lines HG Drexler et al 702 facilitate the selection of cell lines for specific experiments cells were seeded in triplicate in 100 ml medium in flat- with regard to elucidating cytokine-related subjects. bottomed 96-well plates and incubated in the absence or presence of cytokines; for the last 4 h of the 48 h incubation period, 1 mCi [methyl-3H]thymidine (Amersham-Buchler, Braunschweig, Germany) was added to each well. Cells were Materials and methods seeded at 2.5 × 105 cells/ml. As the cell lines were constitut- ively dependent on externally added growth factors and nor- Culture of leukemia cell lines mally grown in medium containing such factors, the cells were washed extensively immediately prior to the experi- The continuous cell lines were taken from the stock of the cell ments. The stimulation index (SI) was calculated by dividing bank of the DSMZ (German Collection of Microorganisms and the counts per minute (c.p.m.) in the 3H-thymidine uptake Cell Cultures)4,5 or were generously provided for research pur- assay of the growth factor-containing wells by the c.p.m. of poses by the investigators who established the cell lines. The the media alone control wells of the respective cell lines. characteristics of the cell lines, information on the patient from whom they were derived, growth media and references are provided in Table 1. The establishment of the cell line Cytokines MHH-203 (kindly provided by Prof M Freund, Rostock, The following commercially supplied cytokines were used Germany) has not been published yet. The following cell lines and are listed in alphabetical order (in parentheses: full desig- are available from the DSMZ-German Collection of Micro- nation of factor; used at final concentration; specific activity organisms and Cell Cultures: M-07e (DSM ACC 104); MUTZ- where applicable or known; supplier); all cytokines were the 2 (DSM ACC 271); MUTZ-3 (DSM ACC 295); OCI/AML5 human variant: (DSM ACC 247); TF-1 (DSM ACC 334). Cell lines were grown ° • at 37 C in a humidified atmosphere of air containing 5% CO2. 5637 CM (5637 human bladder carcinoma cell line con- The basal growth media (Table 1; from Gibco BRL, Egg- ditioned medium; 10% vol; containing c. 42 ng/ml G-CSF, enstein, Germany) were supplemented with 5–20% heat- 2.1 ng/ml GM-CSF, 110 pg/ml M-CSF, 120 pg/ml SCF);25 inactivated (at 56°C for 45 min) fetal bovine serum (FBS; • bFGF (basic fibroblast growth factor; 50 ng/ml; 0.4– from Sigma, Deisenhofen, Germany). Freedom of myco- 1.0 × 107 U/mg; R&D Systems, Wiesbaden, Germany); plasma contamination was determined after thawing the cell • CNTF (ciliary neurotrophic factor; 250 ng/ml; R&D); lines by cultivation on agar and by frequent DAPI staining.23 • EGF (epidermal growth factor; 10 ng/ml; .2–4 × 107 U/mg; Cultures were passaged according to standard procedures: Pharma Biotechnologie, Hannover (PBH), Germany); spent culture medium of these suspension cell lines was • EPO (erythropoietin; 5 U/ml; .70 000 U/mg; Boehringer exchanged at regular intervals (usually after 24–48 h).5 All cell Mannheim, Mannheim, Germany); lines were examined daily in their culture vessels under an • FLT3L (flt3-ligand; 100 ng/ml; kindly provided by Dr S inverted microscope.