The promyelocytic cell line NB4 is characterized by mutated TP53 and non-functional p53 protein 2223 as well as wild type FLT3 24. DNA damaging 25 Gy IR of NB4 cells resulted in increased apoptosis, but no modulation of FLT3 or HDM2 mRNA was observed (Fig. 1a,b; left panel). Hdm2 responds to IR with protein auto-degradation 20, and it regulates endocytosis of certain receptors like the IGF 1 receptor 25. We examined Flt3 and Hdm2 at different time points after IR (25 Gy) and found highly significant reciprocal regulation at the protein level (Fig. 1c; left panel). This was accompanied by attenuation of the anti-apoptotic Mcl-1, an increase in Bax but unaltered Bcl-2 (Fig. 1d; left panel) and Bcl-X_L (data not shown). Previous studies have shown that DNA-damaging in vivo chemotherapy of AML has no effect on MCL-1 gene induction, but rapidly induces BAX and PUMA mRNA 3 (Øyan et al., manuscript in preparation). p21 protein was not detected in NB4 cells (data not shown), and the p53 protein level was not altered after irradiation (Fig. 1d; left panel), reflecting its non-functional status.

MV4-11 is characterized by FLT3-ITD, loss of wilt type FLT3 allele, and wild type TP53 2224. MV4-11 cells were resistant to IR with regards to apoptosis induction (Fig 1a, right panel), but responded with more than one fold increase in HDM2 mRNA (Fig. 1b), reflecting the functional p53. The level of Hdm2 protein showed a small but significant decrease after 60 minutes before an increase was detected, whereas the Flt3 level increased in response to IR and was not attenuated by the elevated HDM2 level after 180 minutes (Fig. 1c,d). Another striking difference from the NB4 cells with FLT3-wt was that the Mcl-1 level did not change in response to IR (Fig. 1d). Furthermore, MV4-11 responded to IR with increased protein levels of p53, Bax, Bcl-2, (Fig. 1d) and p21 (data not shown) while the level of Bcl-X_L was unaltered (data not shown). The IR induction of p53, Hdm2, Bax and p21 suggests that the p53 transcriptional activation in MV4-11 is intact 3.

The effect of IR was also examined in HL-60 cells, characterized by wild type FLT3 and deleted alleles for TP53 2224. Like inNB4 and MV4-11 cells, Hdm2 was attenuated and Flt3 increased, but the Flt3 protein appeared not to be full length (Fig. 2a; ~150 versus ~60 kDa). The lack of full length Flt3 was confirmed in cells from both ATCC and DSMZ within four passages of culture, and immunoprecipitation of Flt3 in these cells did not reveal any low molecular anti-Flt3 reactive form (data not shown). Flt3 protein has previously been reported non-detectable in HL-60 cells 26.

Bcl-2 family members showed no significant response to IR in HL-60 cells except a late decrease in Mcl-1.

Human primary AML cells (patient 1) were irradiated and examined for Flt3 and Hdm2 modulation (Fig. 2b), indicating that the reciprocal Flt3-Hdm2 response to DNA damage also could be present in primary leukemia cells. In contrast to the HL-60 cells where the p21 response was absent, early increase was present in the primary AML cells. These differences reflects an absence of a p53 response in HL-60 cells and a presence of such in the patient cells (Fig. 2a,b).

Since both DNR and IR induce DNA damage, we examined the effect of DNR in both AML cell lines (NB4, MV4-11 and HL-60) and primary cells (patient 2). The cells were treated in vitro (Fig. 3) with DNR for 5 hours at relevant concentrations 3. The NB4 and MV4-11 cell lines were sensitive to DNR with regards to apoptosis induction, and both Mcl-1 and Hdm2 were down regulated (Fig. 3a). Although DNR increased Flt3 protein in all the AML cells tested (Fig. 3ab), this effect was most prominent in MV4-11 cells, HL-60 and the primary AML cells. HL-60 cells showed an increase in putative short forms of Flt3 protein with low doses of DNR (Fig. 3b).

We have recently demonstrated swift induction of p53 and Bax proteins in AML cells collected from patients undergoing induction chemotherapy with anthracyclines and cytarabine 3. It was therefore of interest to examine the AML cells' protein levels of Flt3 and Hdm2 after in vivo chemotherapy (Fig. 4, one representative patient). AML cells from patients were collected within the first 4 hours of chemotherapy, and showed a strong in vivo decrease in Hdm2, in addition to increase in p53 and Flt3.

All patient studies were approved by the local ethical committee (REK Vest) and the Data Inspectorate, Norway. REK Vest is affiliated with the University of Bergen and Haukeland University Hospital. Samples were collected after informed consent. Patient data is overviewed in Table 1.

Leukemic peripheral blood mononuclear cells (PBMC) were isolated by density gradient separation (Ficoll-Hypaque; Nycomed, Oslo, Norway) and were stored frozen in liquid nitrogen 40. The percentage of blasts among leukemic PBMC exceeded 95% for all patients as judged by light microscopy of May-Grünwald-Giemsa stained cytospin smears 41. PBMC were cultured in serum free conditions in StemSpan (Stem Cell Technologies, Vancouver, BC, Canada) at an average concentration of 2 × 10⁶ cells per ml. Cells collected from patients during therapy followed the procedures as described by Anensen et al. 2006 3.

The AML cell line NB4, kindly provided by Dr. Michel Lanotte (INSERM U-301, Hôpital St. Louis, Centre Hayem, Paris, France) 42, was cultured in RPMI 1640 (Sigma-Aldrich, Inc. St. Louis, MO, USA) with 10% fetal bovine serum (Foetal Calf Serum Gold, PAA Laboratories GmbH, Pasching, Austria) and penicillin/streptomycin 50 IU/50 μg per ml. Sequence analysis of both DNA strands of the NB4 cells used in this study confirmed wild type juxtamembrane region and activation loop of FLT3, and FISH analysis confirmed the presence of t(15;17) translocation. The same culture conditions as for NB4 were used for HL-60, purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Reverse transcriptase PCR of HL-60 confirmed presence of normal length of FLT3 mRNA in the juxtamembranous region. The MV4-11 cell line was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and cultured in IMDM (BioWhittaker, Cambrex Bio Science, Verviers, Belgium) with 10% FBS and penicillin/streptomycin 50 IU/50 μg per ml. The FLT3 gene in MV4-11 comprised a length mutation in the juxtamembrane region, and the t(4;11)(q21;q23) translocation was confirmed by FISH. The TP53 gene in MV4-11 is wild type according to data published 22 and the IARC TP53 Database 43. The protein level of Flt3 in NB4 was approximately 50% of the level in MV4-11, estimated by Western blot and flow cytometry.

For irradiation induced DNA double strand breaks, samples were exposed to 25 Gray (Gy) from a Ce¹³⁷ source 11 and maintained in culture until samples were collected for Western blot analysis at time indicated. To secure that the observed effect was from the irradiation, the control samples were handled the same way as the exposed samples except for the actual irradiation. Collection of cells from AML patients under therapy and in vitro treatment of cells with daunorubicin was performed as previously described 3.

Cells were fixed in 2% paraformaldehyde solution containing the DNA specific nuclear stain Hoechst (Hoechst 33342, Invitrogen, Carlsbad, CA, USA; 10 μg/ml) and examined as previously described 33. The number of normal and apoptotic nuclei was counted in an inverse fluorescence microscope (×400 magnification; Leica IRB, Leica Microsystems GmbH, Wetzlar, Germany). The mean number of three experiments was calculated together with the standard error of mean (standard deviation/√number of experiments). Nuclear staining with Hoechst of the cells treated with daunorubicin was not possible due to the strong fluorescence from the drug. These cells were fixed in 4% paraformaldehyde solution and their forward scatter and side scatter properties were examined by flow cytometry and used to determine the number of living cells. Flow cytometry was performed on a FacsCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and data analyses were carried out using the FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Samples for Western blotting were prepared by pelleting the cells (3–10 millions) and washing them twice in 0.9% NaCl following lysis in the following buffer: 10 mM Tris (pH 7.5), 1 mM EDTA, 400 mM NaCl, 10% glycerol, 0.5% NP40, 5 mM NaF, 0.5 mM sodium orthovanadate, 1 mM DTT, and 0.1 mM PMSF (50–200 μl lysis buffer per sample) and transfered to 1.5 ml tubes. The samples were homogenized by 20 strokes of a plastic mini homogenizer before centrifugation at 14000 × g for 20 minutes. Protein concentrations were determined using the Bradford protein assay, following the manufacturers instructions (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The protein samples were added SDS loading buffer (Final: 1% SDS, 10% Glycerol, 12 mM Tris-HCl pH 6.8, 50 mM DTT and 0.1% Bromophenol Blue) and boiled for 10 minutes.

SDS-polyacrylamide gels, 10 or 12.5 % were loaded with 50–70 μg protein per well. After electrophoresis (100–200 V, 1–3 hours) and electroblotting (200 mA, o/n 4°C) the PVDF-membranes (HybondP, Amersham Biosciences, Oslo, Norway) were blocked for 1 hour in I-Block Blocking agent (Applied Biosystems, Foster City, CA, USA). Primary antibodies were incubated for 1–2 hours in room temperature or over night at 4°C followed by 1 hour washing in TBS-Tween. The antibodies Flt3 S-18, Hdm2 SMP-14, p53 BP53-12, Mcl-1 22, Bcl-2 ΔC 21 and Bax 2D2 were from Santa Cruz Biotechnology, CA, the Actin antibody AC-15 was from Abcam plc, Cambridge, UK and the Hdm2 antibodies 2A10 and IF2 were from Calbiochem, San Diego, CA, USA.

Secondary antibodies conjugated to horse radish peroxidase (Jackson ImmunoResearch laboratories, West Grove, PA, USA) were diluted in 4% fat-free dry milk in TBS-Tween and incubated 1 hour at room temperature. After washing for 1 hour with TBS-Tween, the membranes were developed using Supersignal^® West Pico or West Femto Chemiluminiscence Substate from Pierce Biotechnology Inc, Rockford, IL, USA according to the manufacturers' instructions. The membranes were imaged using a Kodak Image Station 2000R (Eastman Kodak Co., Lake Avenue, Rochester, NY, USA), and bands were quantified using the Kodak analysis software. Data were exported to Excel spreadsheet, corrected for background and loading control intensities and a Student's two-tailed t test was used for determination of significance.

Immediately after in vitro experiments, 5 × 10⁶ cells were dissolved in RNAlater (Ambion Inc.) to stabilize and protect RNA and then stored at -80°C. RNAeasy plus mini kit (Qiagen Inc.) was used for isolation of total RNA. Cells were thawed, centrifuged and resuspended in RTL buffer and further procedures were followed according to manufacturer's instructions. RNA quality was tested on a 2100 Bioanalyzer (Agilent Technologies) and total RNA was quantified with a spectrophotometer for small aliquots (NanoDrop Technologies, Wilmington, DE, USA). cDNA were synthesized using the High-Capasity cDNA Archive Kit (Applied Biosystems, Foster City, CA) running 625 ng RNA in 50 μl total reaction volume. Real Time PCR was performed using assays-on-demand containing primers and FAM dye-labelled probes. Human GAPDH and β-Actin were used as endogenous controls. For Flt3 and Hdm2, assays Hs00174690_m1 and Hs00234753_m1 (Applied Biosystems) were used. TaqMan Universal PCR Master Mix (Applied Biosystems) was run with 2 μl cDNA in 10 μl total reaction volumes. The PCR was performed in a 384-well clear optical reaction plate on a 7900HT real time PCR system (Applied Biosystems). The calibrator sample in each experiment was used for standard curve dilution. All samples were run in three replicates and data were analyzed using the relative standard curve method as described by the manufacturer (Applied Biosystems).