Leukemia (1997) 11, 479–484 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 LEADING ARTICLE Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells M Towatari1, H Iida1, M Tanimoto1, H Iwata2, M Hamaguchi2 and H Saito1 1First Department of Internal Medicine, and 2Laboratory of Molecular Pathogenesis, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Japan Mitogen-activated protein (MAP) kinase appears to be one of c-Raf protein kinase; Raf has been shown to phosphorylate the key regulators of cell proliferation and differentiation. Very and activate MAPK kinase (MAPKK), also known as MEK, a little, however, has been revealed as to how MAP kinase is dual specificity kinase that phosphorylates MAPKs on both involved in leukemogenesis. We have studied the activation of 18 the MAP kinase pathway in 100 human primary leukemia cells threonine and tyrosine residues. Recently, it has been shown including 73 acute myelogenous leukemias (AMLs). Forty acute that the activation of MAPKK is necessary and sufficient for leukemia samples (40% of the total), including 37 AML samples PC12 differentiation and for transformation of NIH3T3 (51% of AML), showed activation of MAP kinase as revealed by cells.19,20 Although dysregulation of Ras gene has been the mobility shift of the phosphorylated form of the protein and reported in a variety of human cancers, including leuke- by in vitro kinase assay. This activation was correlated with 21–24 MAP kinase kinase activity in these cells. In contrast, none of mias, little is known concerning the role of this cascade 14 chronic myelogenous leukemia samples showed the acti- in primary human neoplasms. The possible involvement of the vation of MAP kinase. These results suggest that the MAP kin- abnormal activation of these components in the hematopo- ase pathway is constitutively activated in a subset of primary ietic cell transformation has been of interest. The kinases in acute leukemias, and thus indicate the possible role of the the cytoplasm, for example, may work not only as transporters constitutively activated MAP kinase in leukemogenesis. of the signal but also as so-called oncogenes. In the light of Keywords: MAP kinase; MAP kinase kinase; phosphorylation; human leukemia; AML these speculations, we examined the activation of one of the most important pathways for cell proliferation, the MAPK pathway. Here we show the constitutive activation of this pathway in a comparatively large proportion of patients with Introduction acute leukemia. Mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs),1–5 are serine/ Materials and methods threonine kinases that appear to convert a variety of extra- cellular signals such as growth factors and cytokines to cell Leukemia cells growth or differentiation. MAPKs translocate to the nucleus upon activation6–8 and a number of transcription factors We used 40 fresh heparinized leukemia samples which were including c-jun,9 Elk-1,10 c-myc,11 and NF-IL612 have been separated by Ficoll–Hypaque, washed twice with phosphate- revealed to be phosphorylated and activated by MAPKs. buffered saline (PBS) followed by the extraction of whole cell MAPKs are therefore key kinases in the intracellular signal lysates. The other 60 samples, previously frozen by a con- transduction pathways. trolled-rate freezer to −80°C and stocked in liquid nitrogen, A large number of leukemia-specific cytogenetic abnormali- were subjected to quick thawing and extraction of whole cell ties have been identified and the genes involved cloned.13,14 lysate. The viability was assessed by the trypan blue exclusion Some of them, including receptor abnormalities, appear to test. All acute leukemia samples contained over 90% viable involve the signal transduction pathways, which have drawn blast cells. Hematological malignancies analyzed included 73 attention as another possible mechanism for leukemogenesis; acute myelogenous leukemias (AMLs) (five M0, 16 M1, 20 one such pathway is the receptor tyrosine kinase-mediated M2, 10 M3, 11 M4, 10 M5 and one M6 in the French–Amer- pathway. Mutations of c-fms,15 M-CSF receptor, or c-kit recep- ican-British classification), 13 acute lymphoblastic leukemias tor tyrosine kinase16 have been revealed to activate hemato- (ALLs) (four T-ALL and nine B-lineage ALL), and 14 chronic poietic cell proliferation. A frameshift mutation in the erythro- myelogenous leukemias (CML) (six chronic phase and four poietin receptor was reported in primary familial myeloid/four lymphoid blastic crisis). Whole cell lysates were polycythemia.17 Continuous activation of these cytokine extracted as reported previously.25 Concisely, the cells were receptors activates the shared signal transduction pathways lysed in RIPA buffer (10 mM Tris-HCl, pH 7.5, 0.4 M NaCl, 1% resulting in the constitutive activation of the downstream Nonidet P-40, 0.1% sodium deoxycholate, 0.1% sodium lau- components. The upstream components of MAPK pathway ryl sulfate (SDS), 1 mM EDTA) containing 20 mg/ml aprotinin, contain the GTP-binding protein p21Ras which binds to the 20 mg/ml leupeptin, 1mM benzamidine, 1 mM phenylmethyl- sulfonyl fluoride (PMSF), 3 mM sodium vanadate and 40 mM sodium pyrophosphate. After incubating for 1 h at 4°C, an Correspondence: M Towatari, First Department of Internal Medicine, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, equal volume of RIPA buffer without NaCl was added to the Nagoya 466, Japan extracts followed by centrifugation at 17 000 g for 20 min. The Received 30 September 1996; accepted 11 December 1996 concentration of the lysates was examined by a commercial MAP kinase activation in leukemia M Towatari et al 480 kit (Bio-Rad Protein Assay; Bio-Rad, CA, USA). Equal amount samples examined by comparing freshly prepared with frozen- of the protein was used for Western blotting. thawed MAPK (data not shown). MAPKs (ERKs) are activated when they are phosphorylated at both threonine and tyrosine residues by MEKs,18 and the phosphorylated form of MAPK Western blot analysis migrates slower than its unphosphorylated form due to this gel system.19,25 The activity in each sample was measured by the The SDS polyacrylamide gel for resolving the mobility shift relative intensity of each band estimated by a phosphoimage between the phosphorylated form and unphosphorylated form analyzer (BAS2000, FUJIX, Tokyo, Japan). The relative ratio of of MAPK contained low bisacrylamide as we described pre- the intensity of the phosphorylated form to the total intensity viously.19,25 The bands were detected by either polyclonal of the phosphorylated and unphosphorylated forms was calcu- anti-ERK2 antibody or anti-ERK1 antibody (Santa Cruz lated; samples with more than 5% phosphorylation were arbi- Biotechnology, Santa Cruz, CA, USA) which is not cross- trarily defined as positive, because a large portion of the sam- reactive with ERK1 p44 or ERK2 p42, respectively, followed ple with visible phosphorylated band showed a much higher by ECL kit (Amersham International, Buckinghamshire, UK). number, while the negative sample always showed a much smaller one. Normal bone marrow samples invariably exhib- ited less than 1% ERK2 phosphorylation. Representative data MAPK activity assay were shown in Figure 1a. In 18 of 40 freshly extracted samples and 22 of 60 frozen-thawed samples, the phosphorylated form Activity of MAPK was assessed by the kinase activity for the of MAPK was enhanced by more than 5%. A summary of the synthesized peptide using a commercial kit (BIOTRAK MAP results were presented in Table 1. In AMLs, all subtypes of the kinase assay; Amersham). The peptide contains the sequence FAB classification, except M0 and M6, contained a relatively PLS/TP, a recognition sequence for p42/p44 MAPK,26 and no high percentage of the positive samples: 37 out of 73 samples other phosphorylation sites. Therefore, this assay reflects the were positive in AMLs in total (average of phosphorylated MAPK activity more specifically than a standard MBP phos- form ratio, 22%). Three of nine B-lineage ALL and none of phorylation assay. three CLL had positive phosphorylation of MAPK. Interest- ingly, neither the chronic phase nor the acute (blastic) phase of CML showed any detectable phosphorylated form of ERK2. Kinase activity for GST-ERK2 The marrow cells in complete remission showed minimal ERK2 phosphorylation even at the recovery phase after con- Full-length human ERK2 cDNA was cloned into the pGEX-2T solidation chemotherapy (Figure 1a, lanes 7 and 13). Phos- vector (Pharmacia Biotechnology, Uppsala, Sweden) and phorylation of ERK1, an isoform of ERK2, was also examined GST-ERK2 was generated bacterially as recommended by the in 20 leukemia samples by the same gel shift assay, and the manufacturer (Pharmacia). The reaction mixture contained representative data are shown in Figure 1b. All 10 ERK2-phos- 10 mg of glutathione-S-transferase (GST)-ERK2 coupled with phorylated samples exhibited enhanced phosphorylation glutathione agarose, 1 mg of whole cell lysate, 10 mM HEPES (average of phosphorylated form ratio, 17%), while the other g32 (pH 7.6), 10 mM MgCl2,1mM DTT, and 370 kBq of P-ATP. ° The reaction was censored after incubation at 30 C for Table 1 Summary of patient characteristics and elevated phos- 30 min. The precipitant was washed five times by RIPA buffer phorylation of ERK2 and subjected to 8% SDS-PAGE. Phenotype No. of Elevated phosphorylation of patients ERK2a DNA preparation and Southern blotting No. % Extraction of genomic DNA from the mononuclear cells and Southern blotting were performed by the standard methods.27 AML M0 5 0 0 The regions centering on codon 12 of K-ras and N-ras, and M1 16 5 31 codons 13 and 61 of n-ras gene were amplified selectively M2 20 16 80 M3 10 3 30 using PCR. Ras mutations were detected by an algorithm M4 11 5 45 based on allele-specific oligonucleotide hybridization.