Leukemia (1997) 11, 1234–1237 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Expression of FLT4 and its ligand VEGF-C in acute myeloid leukemia W Fielder1, U Graeven1,2, S Ergu¨n3, S Verago, N Kilic3, M Stockschla¨der1 and DK Hossfeld1 Department of 1Hematology/Oncology and 3Institute of Anatomy, University Hospital Eppendorf, Hamburg, Germany FLT4 represents a recently cloned member of class III receptor in angioblasts of head mesenchym, the cardinal vein and tyrosine kinases which include receptors for the angiogenic extra-embryonally in the allantois.10 In late embryos and growth factor VEGF, namely FLT1 and KDR. The ligand of FLT4 has been identified as VEGF-C which shares sequence homo- adults FLT4 expression seems to be restricted to endothelial logy with VEGF and P1GF. In the adult FLT4 shows a restricted cells of lymph vessels and to some high endothelial venules 9 expression pattern that is limited to lymphatic endothelia and (HEV). Additionally FLT4 has been found in pericardium, endothelia of some high endothelial venules (HEV). FLT4 has pleura, lung, kidneys and placenta.7,11 FLT4 is also expressed also been detected in some tumor cell lines including the hema- in a variety of cell lines derived from Wilms’ tumor, retino- topoietic line HEL. We therefore investigated expression of blastoma and teratocarcinoma.12 Since the leukemic hemato- FLT4 and its ligand VEGF-C in fresh samples from patients with 12 AML. Using a sensitive PCR method we detected FLT4 m-RNA poietic cell line HEL is positive for FLT4, we investigated in 15 of 41 patients with de novo AML at diagnosis or relapse expression of FLT4 and its ligand VEGF-C in human bone and in three of 12 patients with secondary AML. FLT4 marrow or peripheral blood samples from normal volunteers expression was confirmed by immunocytochemistry in a sub- and patients with acute myeloid leukemia (AML) with the PCR group of the studied patient population. FLT4 was also found technique and immunocytochemistry. This may lead to in leukemic cell line U937, but not TF-1 and KG1a. VEGF-C possible new insights into autocrine or paracrine growth expression was found in leukemic samples of four of seven FLT4-positive and four of six FLT4-negative patients. U937 cells mechanisms. also produced VEGF-C m-RNA. Interestingly, FLT4 expression was not detected in bone marrow samples of 15 normal volun- teer donors or in CD34-positive cells from three additional Material and methods donors. Possible autocrine and paracrine growth stimulation of leukemic blasts by VEGF-C is currently being investigated in our laboratory. Isolation of peripheral blood or bone marrow Keywords: AML; VEGF-C; FLT4; angiogenesis mononuclear cells Peripheral blood or bone marrow samples from 53 consecu- Introduction tive patients with newly diagnosed or relapsed AML were obtained prior to chemotherapy. Mononuclear cells were sep- Several members of closely related receptor tyrosine kinases arated by density gradient centrifugation on Ficoll–Hypaque (RTK), involved in angiogenesis, have recently been cloned (Pharmacia, Uppsala, Sweden). These preparations contained by virtue of their homology to the FMS oncogene, as has been at least 90% myeloblasts as judged by morphological criteria recently reviewed.1 They include FLT1 and KDR/FLK1 which on Papenheim smears. A control group of 15 healthy volun- are the receptors for vascular endothelial growth factor teer bone marrow donors was included. (VEGF).2 A third RTK, FLT3, has been shown to be expressed by leukemia cells as has been reviewed in this journal.3 FLT4, another member of this class of receptors, forms the high Preparation of CD 34-positive cells affinity binding site for VEGF-C.4 The latter represents a novel angiogenic growth factor with sequence homology to VEGF Mononuclear cells from about 30 ml of bone marrow from and placenta growth factor (P1GF).4 The genes coding for sev- normal human volunteers were recovered from a Ficoll– eral members of this class of RTKs such as FLT4, KIT, FMS, Hypaque gradient, washed twice and counted. To select for FLT3, PDGFRA have presumably been derived from a com- CD34-positive cells about 108 mononuclear cells were mon ancestor by duplications of immunoglobulin-like applied to the MiniMacs column (Miltenyi Biotech, Bergisch domains.5 FLT4, like other RTKs, has been mapped to the long Gladbach, Germany) according to the directions of the sup- arm of chromosome 5.6 plier. Recovered cells were evaluated for purity by FACS Two transcripts of FLT4 with a size of 4.5 kb and 5.8 kb are analysis employing a CD34 monoclonal antibody recognizing expressed in adult and fetal tissues leading to a short and a a different epitope from the one used for the MiniMacs col- long form of the FLT4 protein.7,8 The long form possesses 65 umn (Anti HPCA-2; Becton Dickinson, San Jose, CA, USA). additional amino acids including three tyrosine residues. One Purity of obtained cells was at least 93% (not shown). of these, tyrosine 1337, seems to be necessary for ligand- dependent transformation and signal transduction to the cyto- 9 plasmatic factors GRB2 and SHC. Source of cell lines Expression of FLT4 has been studied in mouse embryos and in adult human tissues. In early embryos FLT4 can be detected All three cell lines, TF-1, U937 and KG-1a, were kindly pro- vided by W Ostertag, Heinrich-Pette Institute, Hamburg, Ger- many. U937 and KG-1a were cultured in RPMI 1640 sup- Correspondence: W Fiedler, Dept Oncology/Hematology, University plemented with 10% fetal calf serum. TF-1 cells were grown Hospital, Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 2Present address: Medizinische Universita¨tsklinik Knappschaftskrank- in the same medium after addition of 100 ng/ml GM-CSF enhaus, In der Schornau 23-25, D-44892 Bochum/Germany (kindly provided by E Henning, Essex Pharma GmbH, Received 21 December 1996; accepted 3 April 1997 Mu¨nchen, Germany). FLT4 and VEGF-C in AML W Fiedler et al 1235 Preparation of c-DNA and RT-PCR activity was visualized by means of the nickel-glucose oxidase technique.15,16 Controls included: blocking of anti-FLT4 anti- Mononuclear cells were washed twice in PBS and collected body by specific peptide, replacement of primary and second- by centrifugation. Total cellular RNA was prepared using ary antibody with PBS, visualization of peroxidase only, incu- Qiagen minicolumns (Qiagen, Hilden, Germany) as described bation of cells with normal rabbit serum (Sigma, Deisenhofen, by the manufacturer. One microgram of RNA was used for c- Germany) in concentrations ranging from 0.1% to 0.01% DNA synthesis employing avian myeloblastosis virus (AMV) instead of primary antiserum. reverse transcriptase and oligo dT as primer. RT reactions were diluted to 100 ml with distilled water and heated to 95°C for 10 min. Results Different aliquots of c-DNA were amplified with specific primers for FLT4, VEGF-C and actin as control for successful FLT4 expression was studied with a sensitive nested RT-PCR c-DNA synthesis. For FLT4 and VEGF-C two rounds and for reaction. The limit of detection is at least one FLT4-positive actin one round of 35 cycles of PCR were performed in a cell in 105 bone marrow cells (data not shown). With this programmable heat block at 94°C for 1.5 min, at 60°C for method c-DNA samples from bone marrow cells of 15 healthy 3 min and at 72°C for 4 min. For the first round 1 ml of the donors were investigated. None was positive for FLT4 RT reaction and for the second round of RCR 1 ml of a 1:100 expression. To confirm further the lack of FLT4 formation in dilution of the first round was used. Five microliters of PCR normal bone marrow cells, CD34-positive cells were prepared products were separated on 1% agarose gels, stained with by use of a mini MACS column from three additional normal ethidium bromide and visualized under UV light. Primer donors yielding a suspension of CD34-positive cells of at least sequences were as follows: FLT4: outer sense primer 59- 93% purity (not shown). FLT4 specific amplification products CTGCTGGAGGAAAAGTCTGG-39; outer antisense primer 59- were not found in the three samples (Figure 1). CTTGCAGAACTCCACGATCA-39; inner sense primer 59- A series of 53 consecutive patients with AML and the leu- CGTATCTGTGCAGCGTGTG-39; inner antisense primer 59- kemic cell lines KG1a, TF-1 and U937 were investigated for GGTTGACCACGTTGAGGTG-39; VEGF-C: outer sense primer expression of FLT4 by PCR analysis. U937, but not TF-1 and 59-AGCTAAGGAAAGGAGGCTGG-39; outer antisense primer KG1a cells were found to be positive for FLT4 m-RNA. Forty- 59-TGTGTTTTCATCAAATTCTCGG-39; inner sense primer 59- one samples originated from patients with de novo AML at TGAACACCAGCACGAGCTAC-39; inner antisense primer 59- diagnosis or at relapse and 12 samples from patients with CATAAAATCTTCCTGAGCCAGG-39. Actin: sense primer 59- secondary AML. From the 53 AML patients overall 34% CGCTGCGCTGGTCGTCGACA-39; antisense primer 59- expressed FLT4. A representative example of a PCR analysis GTCACGCACGATTTCCCGCT-39. The size of the PCR pro- is demonstrated in Figure 1. Table 1 shows the relative per- ducts corresponds to 625 bp for FLT4 outer primer pair, centages of FLT4 positivity of AML samples according to 500 bp inner primer pair, VEGF-C outer primer pair 764 bp, FAB type. inner primer pair 269 bp and for actin 619 bp. To avoid cross- The cell lines U937 and TF-1 and leukemic blasts from contamination the set-up of PCR reactions and gel electro- eight patients were investigated for FLT4 protein expression phoresis were carried out in different rooms using different sets of pipettes.