- Open Access
Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells
© Wergeland et al; licensee BioMed Central Ltd. 2007
- Received: 13 March 2007
- Accepted: 11 May 2007
- Published: 11 May 2007
Acute myeloid leukemia (AML) cells are characterized by non-mutated TP53, high levels of Hdm2, and frequent mutation of the Flt3 receptor tyrosine kinase. The juxtamembrane mutation of FLT3 is the strongest independent marker for disease relapse and is associated with elevated Bcl-2 protein and p53 hyper-phosphorylation in AML. DNA damage forms the basic mechanism of cancer cell eradication in current therapy of AML.
Hdm2 and pro-apoptotic Bcl-2 members are among the most intensely induced genes immediately after chemotherapy and Hdm2 is proposed a role in receptor tyrosine kinase regulation. Thus we examined the DNA damage related modulation of these proteins in relation to FLT3 mutational status and induction of apoptosis.
Within one hour after exposure to ionizing radiation (IR), the AML cells (NB4, MV4-11, HL-60, primary AML cells) showed an increase in Flt3 protein independent of mRNA levels, while the Hdm2 protein decreased. The FLT3 mutant MV4-11 cells were resistant to IR accompanied by presence of both Mcl-1 and Hdm2 protein three hours after IR. In contrast, the FLT3 wild type NB4 cells responded to IR with apoptosis and pre-apoptotic Mcl-1 down regulation. Daunorubicin (DNR) induced continuing down regulation of Hdm2 and Mcl-1 in both cell lines followed by apoptosis.
Both IR and DNR treatment resulted in concerted protein modulations of Mcl-1, Hdm2 and Flt3. Cell death induction was associated with persistent attenuation of Mcl-1 and Hdm2. These observations suggest that defining the pathway(s) modulating Flt3, Hdm2 and Mcl-1 may propose new strategies to optimize therapy for the relapse prone FLT3 mutated AML patients.
- Acute Myeloid Leukemia
- Ionize Radiation
- Acute Myeloid Leukemia Patient
- Acute Myeloid Leukemia Cell
- Acute Myeloid Leukemia Cell Line
Anthracyclines like daunorubicin (DNR) are used in the induction treatment of acute myeloid leukemia (AML), obtaining short time complete hematological remission for more than 65% of adult AML patients with de novo AML . Successful hematological remission after only one induction cycle is a favorable prognostic parameter and is associated with decreased risk of later AML relapse [1, 2]. Induction therapy causes rapid activation of the tumor suppressor p53 followed by dominating p53-targeted gene expression in vivo . A major mechanism for this p53 induction is DNA damage through anthracycline-stabilization of the DNA:topoisomerase II complex , but cell death induction by anthracyclines may also involve other molecular targets independent of p53 [4–7].
Ionizing radiation (IR) is frequently used in the treatment of solid cancers, in the conditional treatment before allotransplantation of leukemia patients and in radioisotope-conjugated therapeutic antibodies directed against AML cells [8, 9]. IR and anthracyclines induce growth arrest and cell death through DNA-damage, but also involve cell membrane-related effects in regulation of apoptosis [4–7, 10]. We have previously reported that AML patient cells respond with varying sensitivity to IR-induced proliferation arrest , and it may therefore be of interest to determine molecular mechanisms for radioresistance in more detail.
The strongest molecular predictor for AML relapse is internal tandem duplications in the juxtamembrane domain of the receptor tyrosine kinase Flt3 (Flt3-ITD). These mutations are present in approximately one third of the patients . Flt3-ITD are associated with increased DNA repair , an observation suggesting that these cells are able to recover from DNA damage caused by topoisomerase II blockage and thus have a more drug-resistant phenotype. The expression of anti-apoptotic Bcl-2 protein family members is also influenced by the mutational status of Flt3 . We have recently shown that a duplication of Y591 in Flt3-ITDs is associated with elevated Bcl-2 protein and hyper-phosphorylated wild type (wt) p53 in AML, proposing a mechanism for inactivation of p53 .
Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins. High levels of Mcl-1 have been detected in cells from patients with relapsed AML . Therapeutic targeting of Bcl-2 family proteins seems to depend on Mcl-1 to trigger apoptosis . It may therefore be of particular interest to examine the Mcl-1 modulation in DNA damage therapy.
In contrast to solid tumors, more than 90% of the AML cases comprise wild type p53 [17, 18]. On the other hand, the E3 ubiquitin ligase Hdm2 is usually strongly expressed in AML, contributing to block the growth inhibitory and pro-apoptotic effect of p53 . IR induces DNA damage and rapid down regulation of Hdm2 through induction of auto-ubiquitination and subsequent proteasomal degradation . Recent reports indicate that Hdm2 have important p53-independent activities, including regulation of cell membrane receptors like insulin-like growth factor (IGF) 1 receptor and β2-adrenergic receptor through ubiquitination . However, it is not known whether the Flt3 receptor is regulated by Hdm2.
Concerted protein modulation of a receptor tyrosine kinase, the E3 ubiquitin ligase Hdm2 and selected Bcl-2 family members through DNA damage therapy has previously not been reported. Our study indicated that both IR and DNR induced Hdm2 protein down regulation, partly Flt3 protein elevation, and a pro-apoptotic shift in the expression of proteins in the Bcl-2 family. Flt3 and Hdm2 might have a reciprocal regulation at the protein level and FLT3 mutations could be involved in protection against IR-induced apoptosis through a persisting Mcl-1 level.
Ionizing radiation induces reciprocal regulation of Flt3 and Hdm2 protein in NB4 cells
Hdm2 response and stable Mcl-1 in the IR-resistant cell line MV4-11
MV4-11 is characterized by FLT3-ITD, loss of wilt type FLT3 allele, and wild type TP53 [22, 24]. 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-XL 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 .
Attenuation of Hdm2 and Mcl-1 is independent of p53 and Flt3
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).
Daunorubicin induces attenuation of Hdm2 and Mcl-1 independent of TP53 and FLT3 status
Flt3 and Hdm2 protein are reciprocally regulated in vivo
It can not be ruled out that the increase in Flt3 protein after IR is based on mechanisms independent of Hdm2. IR has been shown to increase the mRNA and protein levels of epidermal growth factor (EGF) receptor as well as the cell surface protein expression of IGF 1 receptor [31, 32]. Such mRNA regulation of Flt3 after IR was not observed in our study (Fig. 5).
The NB4 cells, in contrast to the MV4-11 cells, showed IR induced apoptosis (Fig. 5) and a lack of increase in HDM2 mRNA level. Since NB4 cells have non-functional p53 , this suggests that NB4 undergoes a p53-independent apoptosis during IR-exposure. A possible explanation for the IR-resistance of MV4-11 is that AML cells with Flt3-ITD can repair double-stranded breaks in DNA more efficient than in cells with wild type Flt3 , but an anti-apoptotic effect on p53 by the MLL-fusion products may be an alternative mechanism . This makes MV4-11 more protected against apoptosis induced by IR. Other explanations for early IR-induced apoptosis in NB4 cells in contrast to in the MV4-11 cells could include a pro-apoptotic response on the Bcl-2 family members and a lack of Hdm2 induction (Fig. 5). No shift in the balance of Bcl-2/Bax was observed (Fig. 5), thus our data suggest that Mcl-1 is a central player in regulation of DNA-damage induced cell death. A striking feature of IR treated NB4 cells, as well as DNR treated NB4 and MV4-11 cells, was the Mcl-1 down regulation accompanied by apoptosis. These observations emphasize the putative importance of Mcl-1 in regulation of apoptosis in AML, with possible implications for the biology behind disease relapse [15, 16].
MV4-11 cells were resistant to IR while DNR effectively induced apoptosis (Fig. 5). DNR elicited a lasting Hdm2 and Mcl-1 down regulation in contrast to IR. This suggests that DNR ignites apoptosis through more pathways than IR and that the Mcl-1 attenuation is a pre-apoptotic event. In addition to the induction of DNA damage, DNR is known to stimulate the level of the second messenger ceramide by de novo synthesis and thus trigger apoptosis . Anthracyclines may also induce apoptosis via signalling through altered plasma membrane lipid rafts and the death receptor pathway  (for review see ).
The p53-deficient HL-60 cell line demonstrated Hdm2 decrease as well as a putative Flt3 increase in response to IR or DNR (Fig. 5). The FLT3 gene in HL-60 is wild type , confirmed by sequencing of the juxtamembrane region and the kinase activation domain. Interestingly, lack of full length Flt3 protein in HL-60 has previously been reported , and we were not able to detect full length Flt3 in different batches of HL-60 cells from ATCC and DMZS (Fig. 3b). The protein bands between 50 and 100 kDa may be protein products from alternative splicing of FLT3 mRNA, as reported for the closely related platelet-derived growth factor alpha-receptor and KIT [35, 36]. Additional work is clearly needed to address the possibility of alternative splicing of FLT3 in HL-60 and in AML cells in general.
Flt3-ITD is the strongest predictor for relapse of AML in therapy with anthracyclines , and is recently associated with enhanced DNA repair . We demonstrated that the anti-apoptotic protein Bcl-2 was induced in MV4-11, HL-60 cells and primary AML cells during DNA damage therapy (Fig. 5). This could indicate that anthracyclines elicit an anti-apoptotic signal through Flt3. The anti-apoptotic signal may be particular strong in AML cells with a Flt3-ITD mutation including an Y591 duplication .
In this study we show a concerted protein modulation of Flt3, Hdm2 and Mcl-1 after DNA damaging therapy in AML. IR resulted in decreased levels of Hdm2 and elevated levels of Flt3 and may involve p53 independent activities of Hdm2 acting on Flt3 as proposed for other receptor tyrosine kinases. The apoptotic response may depend on a persisting down regulation of Hdm2 and Mcl-1 . Targeting of Flt3, Bcl-2/Bcl-XL and Mcl-1 is proposed to enhance the response of chemotherapy. Preclinical studies and early clinical trials that follow these principles are underway [38, 39], and we believe that relevant biomarker examinations  including the proteins presented in this study may help to pinpoint the patients that will benefit from this enhanced therapy.
Clinical and biological characteristics of AML patients
Previous malignant disease
46 XX, t(9;11), (q22;q23)
Leukemic peripheral blood mononuclear cells (PBMC) were isolated by density gradient separation (Ficoll-Hypaque; Nycomed, Oslo, Norway) and were stored frozen in liquid nitrogen . The percentage of blasts among leukemic PBMC exceeded 95% for all patients as judged by light microscopy of May-Grünwald-Giemsa stained cytospin smears . 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 .
The AML cell line NB4, kindly provided by Dr. Michel Lanotte (INSERM U-301, Hôpital St. Louis, Centre Hayem, Paris, France) , 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  and the IARC TP53 Database . The protein level of Flt3 in NB4 was approximately 50% of the level in MV4-11, estimated by Western blot and flow cytometry.
Irradiation and chemotherapy treatment of cells
For irradiation induced DNA double strand breaks, samples were exposed to 25 Gray (Gy) from a Ce137 source  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 .
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 . 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.
Real time PCR
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).
We thank Anne Døskeland for helpful suggestions and discussions. This work was supported by the Norwegian Cancer Society (Kreftforeningen) and Helse Vest grants 911307 and 911290.
- Smith M, Barnett M, Bassan R, Gatta G, Tondini C, Kern W: Adult acute myeloid leukaemia. Crit Rev Oncol Hematol. 2004, 50 (3): 197-222.View ArticlePubMedGoogle Scholar
- Wheatley K, Burnett AK, Goldstone AH, Gray RG, Hann IM, Harrison CJ, Rees JK, Stevens RF, Walker H: A simple, robust, validated and highly predictive index for the determination of risk-directed therapy in acute myeloid leukaemia derived from the MRC AML 10 trial. United Kingdom Medical Research Council's Adult and Childhood Leukaemia Working Parties. Br J Haematol. 1999, 107 (1): 69-79. 10.1046/j.1365-2141.1999.01684.xView ArticlePubMedGoogle Scholar
- Anensen N, Oyan AM, Bourdon JC, Kalland KH, Bruserud O, Gjertsen BT: A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia. Clin Cancer Res. 2006, 12 (13): 3985-3992. 10.1158/1078-0432.CCR-05-1970View ArticlePubMedGoogle Scholar
- Myers CE, Chabner BA: Antracyclines. Cancer chemotherapy : principles and practice. Edited by: Chabner BA, Collins JM. 1990, 356-381. Philadelphia, Pa , JB Lippincott,Google Scholar
- Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, Kolesnick R: Ceramide synthase mediates daunorubicin-induced apoptosis: An alternative mechanism for generating death signals. Cell. 1995, 82 (3): 405-414. 10.1016/0092-8674(95)90429-8View ArticlePubMedGoogle Scholar
- Dimanche-Boitrel MT, Meurette O, Rebillard A, Lacour S: Role of early plasma membrane events in chemotherapy-induced cell death. Drug Resistance Updates. 2005, 8 (1-2): 5-14. 10.1016/j.drup.2005.02.003View ArticlePubMedGoogle Scholar
- Laurent G, Jaffrezou JP: Signaling pathways activated by daunorubicin. Blood. 2001, 98 (4): 913-924. 10.1182/blood.V98.4.913View ArticlePubMedGoogle Scholar
- Hegenbart U, Niederwieser D, Sandmaier BM, Maris MB, Shizuru JA, Greinix H, Cordonnier C, Rio B, Gratwohl A, Lange T, Al-Ali H, Storer B, Maloney D, McSweeney P, Chauncey T, Agura E, Bruno B, Maziarz RT, Petersen F, Storb R: Treatment for Acute Myelogenous Leukemia by Low-Dose, Total-Body, Irradiation-Based Conditioning and Hematopoietic Cell Transplantation From Related and Unrelated Donors. J Clin Oncol. 2006, 24 (3): 444-453. 10.1200/JCO.2005.03.1765View ArticlePubMedGoogle Scholar
- Pagel JM, Appelbaum FR, Eary JF, Rajendran J, Fisher DR, Gooley T, Ruffner K, Nemecek E, Sickle E, Durack L, Carreras J, Horowitz MM, Press OW, Gopal AK, Martin PJ, Bernstein ID, Matthews DC: 131I-anti-CD45 antibody plus busulfan and cyclophosphamide before allogeneic hematopoietic cell transplantation for treatment of acute myeloid leukemia in first remission. Blood. 2006, 107 (5): 2184-2191. 10.1182/blood-2005-06-2317PubMed CentralView ArticlePubMedGoogle Scholar
- Lewanski CR, Gullick WJ: Radiotherapy and cellular signalling. The Lancet Oncology. 2001, 2 (6): 366-370. 10.1016/S1470-2045(00)00391-0View ArticlePubMedGoogle Scholar
- Bruserud O, Ulvestad E: Effects of gamma-irradiation on acute myelogenous leukemia blasts: in vitro studies of proliferation, constitutive cytokine secretion, and accessory cell function during T cell activation. J Hematother Stem Cell Res. 1999, 8 (4): 431-441. 10.1089/152581699320199View ArticlePubMedGoogle Scholar
- Stirewalt DL, Radich JP: The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer. 2003, 3 (9): 650-665. 10.1038/nrc1169View ArticlePubMedGoogle Scholar
- Seedhouse CH, Hunter HM, Lloyd-Lewis B, Massip AM, Pallis M, Carter GI, Grundy M, Shang S, Russell NH: DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412. Leukemia. 2006, 20 (12): 2130-2136. 10.1038/sj.leu.2404439View ArticlePubMedGoogle Scholar
- Irish JM, Anensen N, Hovland R, Skavland J, Borresen-Dale AL, Bruserud O, Nolan GP, Gjertsen BT: Flt3 Y591 duplication and Bcl-2 overexpression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53. Blood. 2007, 109 (6): 2589-96. 10.1182/blood-2006-02-004234View ArticlePubMedGoogle Scholar
- Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD, Reed JC: Elevated Expression of the Apoptotic Regulator Mcl-1 at the Time of Leukemic Relapse. Blood. 1998, 91 (3): 991-1000.PubMedGoogle Scholar
- van Delft MF, Wei AH, Mason KD, Vandenberg CJ, Chen L, Czabotar PE, Willis SN, Scott CL, Day CL, Cory S, Adams JM, Roberts AW, Huang DCS: The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell. 2006, 10 (5): 389-399. 10.1016/j.ccr.2006.08.027PubMed CentralView ArticlePubMedGoogle Scholar
- Fenaux P, Preudhomme C, Quiquandon I, Jonveaux P, Lai JL, Vanrumbeke M, Loucheux-Lefebvre MH, Bauters F, Berger R, Kerckaert JP: Mutations of the P53 gene in acute myeloid leukaemia. Br J Haematol. 1992, 80 (2): 178-183.View ArticlePubMedGoogle Scholar
- Schottelius A, Brennscheidt U, Ludwig WD, Mertelsmann RH, Herrmann F, Lubbert M: Mechanisms of p53 alteration in acute leukemias. Leukemia. 1994, 8 (10): 1673-1681.PubMedGoogle Scholar
- Bueso-Ramos CE, Yang Y, deLeon E, McCown P, Stass SA, Albitar M: The human MDM-2 oncogene is overexpressed in leukemias. Blood. 1993, 82 (9): 2617-2623.PubMedGoogle Scholar
- Stommel JM, Wahl GM: Accelerated MDM2 auto-degradation induced by DNA-damage kinases is required for p53 activation. Embo J. 2004, 23 (7): 1547-1556. 10.1038/sj.emboj.7600145PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Z, Zhang R: p53-independent activities of MDM2 and their relevance to cancer therapy. Curr Cancer Drug Targets. 2005, 5 (1): 9-20. 10.2174/1568009053332618View ArticlePubMedGoogle Scholar
- Fleckenstein DS, Uphoff CC, Drexler HG, Quentmeier H: Detection of p53 gene mutations by single strand conformational polymorphism (SSCP) in human acute myeloid leukemia-derived cell lines. Leuk Res. 2002, 26 (2): 207-214. 10.1016/S0145-2126(01)00107-2View ArticlePubMedGoogle Scholar
- Song X, Sheppard HM, Norman AW, Liu X: Mitogen-activated protein kinase is involved in the degradation of p53 protein in the bryostatin-1-induced differentiation of the acute promyelocytic leukemia NB4 cell line. J Biol Chem. 1999, 274 (3): 1677-1682. 10.1074/jbc.274.3.1677View ArticlePubMedGoogle Scholar
- Quentmeier H, Reinhardt J, Zaborski M, Drexler HG: FLT3 mutations in acute myeloid leukemia cell lines. Leukemia. 2003, 17 (1): 120-124. 10.1038/sj.leu.2402740View ArticlePubMedGoogle Scholar
- Girnita L, Girnita A, Larsson O: Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor. Proc Natl Acad Sci U S A. 2003, 100 (14): 8247-8252. 10.1073/pnas.1431613100PubMed CentralView ArticlePubMedGoogle Scholar
- Komeno Y, Kurokawa M, Imai Y, Takeshita M, Matsumura T, Kubo K, Yoshino T, Nishiyama U, Kuwaki T, Osawa T, Ogawa S, Chiba S, Miwa A, Hirai H: Identification of Ki23819, a highly potent inhibitor of kinase activity of mutant FLT3 receptor tyrosine kinase. Leukemia. 2005, 19 (6): 930-935. 10.1038/sj.leu.2403736View ArticlePubMedGoogle Scholar
- Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM: Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem. 2000, 275 (12): 8945-8951. 10.1074/jbc.275.12.8945View ArticlePubMedGoogle Scholar
- Linares LK, Hengstermann A, Ciechanover A, Muller S, Scheffner M: HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53. Proc Natl Acad Sci U S A. 2003, 100 (21): 12009-12014. 10.1073/pnas.2030930100PubMed CentralView ArticlePubMedGoogle Scholar
- Cheng TH, Cohen SN: Human MDM2 isoforms translated differentially on constitutive versus p53-regulated transcripts have distinct functions in the p53/MDM2 and TSG101/MDM2 feedback control loops. Mol Cell Biol. 2007, 27 (1): 111-119. 10.1128/MCB.00235-06PubMed CentralView ArticlePubMedGoogle Scholar
- Linares LK, Kiernan R, Triboulet R, Chable-Bessia C, Latreille D, Cuvier O, Lacroix M, Le Cam L, Coux O, Benkirane M: Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2. Nat Cell Biol. 2007, 9 (3): 331-338. 10.1038/ncb1545View ArticlePubMedGoogle Scholar
- Kim KU, Xiao J, Ni HT, Cho KH, Spellman SR, Low WC, Hall WA: Changes in expression of transferrin, insulin-like growth factor 1, and interleukin 4 receptors after irradiation of cells of primary malignant brain tumor cell lines. Radiat Res. 2003, 160 (2): 224-231. 10.1667/RR3040View ArticlePubMedGoogle Scholar
- Schmidt-Ullrich RK, Valerie KC, Chan W, McWilliams D: Altered expression of epidermal growth factor receptor and estrogen receptor in MCF-7 cells after single and repeated radiation exposures. Int J Radiat Oncol Biol Phys. 1994, 29 (4): 813-819.View ArticlePubMedGoogle Scholar
- Gjertsen BT, Cressey LI, Ruchaud S, Houge G, Lanotte M, Doskeland SO: Multiple apoptotic death types triggered through activation of separate pathways by cAMP and inhibitors of protein phosphatases in one (IPC leukemia) cell line. J Cell Sci. 1994, 107 ( Pt 12): 3363-3377.Google Scholar
- Wiederschain D, Kawai H, Shilatifard A, Yuan ZM: Multiple Mixed Lineage Leukemia (MLL) Fusion Proteins Suppress p53-mediated Response to DNA Damage. J Biol Chem. 2005, 280 (26): 24315-24321. 10.1074/jbc.M412237200View ArticlePubMedGoogle Scholar
- Mosselman S, Claesson-Welsh L, Kamphuis JS, van Zoelen EJ: Developmentally regulated expression of two novel platelet-derived growth factor alpha-receptor transcripts in human teratocarcinoma cells. Cancer Res. 1994, 54 (1): 220-225.PubMedGoogle Scholar
- Chen LL, Sabripour M, Wu EF, Prieto VG, Fuller GN, Frazier ML: A mutation-created novel intra-exonic pre-mRNA splice site causes constitutive activation of KIT in human gastrointestinal stromal tumors. Oncogene. 2005, 24 (26): 4271-4280. 10.1038/sj.onc.1208587View ArticlePubMedGoogle Scholar
- Nijhawan D, Fang M, Traer E, Zhong Q, Gao W, Du F, Wang X: Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes Dev. 2003, 17 (12): 1475-1486. 10.1101/gad.1093903PubMed CentralView ArticlePubMedGoogle Scholar
- Baer MR, Sabur KL, O'Loughlin KL, Starostik P, Minderman H: The FLT3 Inhibitor PKC412 Interacts Synergistically with Both Daunorubicin and Cytarabine in Acute Myeloid Leukemia (AML) Cells by Heterogeneous Mechanisms. ASH Annual Meeting Abstracts. 2006, 108 (11): 1378-Google Scholar
- Stone RM, Fischer T, Paquette R, Schiller G, Schiffer CA, Ehninger G, Cortes J, Kantarjian H, DeAngelo DA, Massimini G, Li X, Phillips P, Giles F: Phase IB Study of PKC412, an Oral FLT3 Kinase Inhibitor, in Sequential and Simultaneous Combinations with Daunorubicin and Cytarabine (DA) Induction and High-Dose Cytarabine Consolidation in Newly Diagnosed Adult Patients (pts) with Acute Myeloid Leukemia (AML) under Age 61. ASH Annual Meeting Abstracts. 2006, 108 (11): 157-Google Scholar
- Bruserud O: Effect of dipyridamole, theophyllamine and verapamil on spontaneous in vitro proliferation of myelogenous leukaemia cells. Acta Oncol. 1992, 31 (1): 53-58.View ArticlePubMedGoogle Scholar
- Bruserud O, Gjertsen BT, von Volkman HL: In vitro culture of human acute myelogenous leukemia (AML) cells in serum-free media: studies of native AML blasts and AML cell lines. J Hematother Stem Cell Res. 2000, 9 (6): 923-932. 10.1089/152581600750062372View ArticlePubMedGoogle Scholar
- Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R: NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood. 1991, 77 (5): 1080-1086.PubMedGoogle Scholar
- Olivier M, Eeles R, Hollstein M, Khan MA, Harris CC, Hainaut P: The IARC TP53 database: New online mutation analysis and recommendations to users. Hum Mutat. 2002, 19 (6): 607-614. 10.1002/humu.10081View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.