Unique effects of KIT D816V in BaF3 cells: induction of cluster formation, histamine synthesis, and early mast cell differentiation antigens - PubMed (original) (raw)

. 2008 Apr 15;180(8):5466-76.

doi: 10.4049/jimmunol.180.8.5466.

Karoline V Gleixner, Andrea Hoelbl, Stefan Florian, Gregor Hoermann, Karl J Aichberger, Martin Bilban, Harald Esterbauer, Maria-Theresa Krauth, Wolfgang R Sperr, Jack B Longley, Robert Kralovics, Richard Moriggl, Jacques Zappulla, Roland S Liblau, Ilse Schwarzinger, Veronika Sexl, Christian Sillaber, Peter Valent

Affiliations

Unique effects of KIT D816V in BaF3 cells: induction of cluster formation, histamine synthesis, and early mast cell differentiation antigens

Matthias Mayerhofer et al. J Immunol. 2008.

Abstract

Oncogenic tyrosine kinases (TK) usually convert growth factor-dependent cells to factor independence with autonomous proliferation. However, TK-driven neoplasms often are indolent and characterized by cell differentiation rather than proliferation. A prototype of an indolent TK-driven neoplasm is indolent systemic mastocytosis. We found that the D816V-mutated variant of KIT, a TK detectable in most patients with systemic mastocytosis, induces cluster formation and expression of several mast cell differentiation and adhesion Ags, including microphthalmia transcription factor, IL-4 receptor, histamine, CD63, and ICAM-1 in IL-3-dependent BaF3 cells. By contrast, wild-type KIT did not induce cluster formation or mast cell differentiation Ags. Additionally, KIT D816V, but not wild-type KIT, induced STAT5 activation in BaF3 cells. However, despite these intriguing effects, KIT D816V did not convert BaF3 cells to factor-independent proliferation. Correspondingly, BaF3 cells with conditional expression of KIT D816V did not form tumors in nude mice. Together, the biologic effects of KIT D816V in BaF3 cells match strikingly with the clinical course of indolent systemic mastocytosis and with our recently established transgenic mouse model, in which KIT D816V induces indolent mast cell accumulations but usually does not induce a malignant mast cell disease. Based on all these results, it is hypothesized that KIT D816V as a single hit may be sufficient to cause indolent systemic mastocytosis, whereas additional defects may be required to induce aggressive mast cell disorders.

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Figures

FIGURE 1

FIGURE 1

Establishment of BaF3 cell lines with conditional expression of wt KIT and KIT D816V. A, Ton.Kit.D816V.27 cells and Ton.Kit.wt cells were kept in control medium (−Doxy) or doxycycline (1 _μ_g/ml (+Doxy)) for 24 h. After incubation, cells were spun on cytospin slides and analyzed by indirect immunoalkaline phosphatase staining technique using an anti-KIT Ab. B, Ton.Kit.D816V cells (clones 3, 6, and 27) and Ton.Kit.wt cells (wt) were cultured in the presence of doxycycline (1 _μ_g/ml) for 24 h. Thereafter, the presence or absence of the D816V mutation was determined by RFLP as described in Materials and Methods. C, Ton.Kit.wt cells and Ton.Kit.D816V.27 cells were incubated in control medium or doxycycline (Doxy) for 24 h, and then were kept with or without SCF (100 ng/ml) for 15 min at 37°C. After immunoprecipitation (IP) for KIT, lysates were split and subjected to immunoblotting (IB) using anti-pTyr (p-tyr) or anti-KIT Abs. The blot shows one representative experiment; identical results were obtained in two other independent experiments. D, Ton.Kit.D816V.27 cells were grown in the absence or presence of doxycycline for 24 h and were then treated with PKC412 (200 nM to 1 _μ_M), imatinib (1 _μ_M), or were kept in control medium for 4 h. Phosphorylation of KIT was determined as described above. One typical experiment (out of three independent experiments with identical results) is shown.

FIGURE 2

FIGURE 2

Effects of KIT D816V on growth and survival. A, Comparative effects of KIT D816V and BCR/ABL on growth of BaF3 cells. Ton.Kit.D816V.27 cells (left) and Ton.B210 cells (right) were cultured in control medium (Co) or medium supplemented with doxycycline (Dox) (1 _μ_g/ml) for 3 days. Then, the number of viable cells was determined. B, Ton.Kit.D816V cells were cultured in IL-3 (▲), IL-3 plus doxycycline (□), IL-3 plus doxycycline plus SCF (100 ng/ml) (●), doxycycline (◆), doxycycline plus SCF (100 ng/ml) (○), or in the absence of growth factors (=starved, △) for various time periods. Results show cell counts and represent the means ± SD of three independent experiments. C, Ton.Kit.wt cells were cultured in the presence of doxycycline plus SCF (100 ng/ml) (▲), doxycycline plus SCF (100 ng/ml) plus IL-3 (□), IL-3 (●), doxycycline (◇), or in the absence of growth factors (=starved, ■) for various time periods. Results show cell counts and represent the means ± SD of three independent experiments. D, Ton.Kit.D816V cells (107) (left) or Ton.B210 cells (107)(right) were injected subcutaneously into the flanks of nude mice given water with doxycycline (400 _μ_g/ml). Sites of Ton.B210 injections resulting in tumors are indicated by arrows. Control mice receiving water without doxycycline did not develop any tumors (not shown). E, Rag2−/−c_γ_−/− mice were transplanted with KIT D816V-transduced bone marrow cells or BCR/ABL-transduced bone marrow cells. After 6 wk, mice transplanted with BCR/ABL-transduced bone marrow cells developed a lethal leukemia, whereas mice transplanted with KIT D816V-transduced bone marrow cells remained healthy. On day 46, mice were sacrificed and the percentage of GFP-positive cells in the spleen of mice was determined by FACS analysis. The figure shows typical examples of mice transplanted with KIT D816V-transduced bone marrow cells (left panel) and mice transplanted with BCR/ABL-transduced bone marrow cells (right panel). SSC, Side scatter.

FIGURE 3

FIGURE 3

Effects of KIT D816V on growth of UT7 cells. A, UT7 cells with inducible expression of KIT D816V (UT7-tK.D816V) were cultured in the absence or presence of doxycycline (1 _μ_g/ml) for 24 h. Thereafter, expression and phosphorylation of KIT was analyzed by immunoprecipitation/immunoblotting using the 1C1 and 4G10 Abs. One typical experiment (out of three independent experiments with identical results) is shown. B, UT7-tK.D816V cells were cultured in GM-CSF (■), GM-CSF plus doxycycline (▼), doxycycline (○), or in the absence of GM-CSF (= starved, ◆) for various time periods. Results show the number of viable cells and represent the means ± SD of three independent experiments.

FIGURE 4

FIGURE 4

Effects of the D816V mutation on signaling of the KIT receptor. A and B, Ton.Kit.D816V.27 and Ton.Kit.wt cells were kept in the absence or presence of doxycycline (Doxy) for 24 h at 37°C. Then, cells were incubated with or without SCF (100 ng/ml) for 15 min at 37°C. Phosphorylation of Akt and ERK (p42/44) was determined by immunoblotting using Abs against phosphorylated forms (p-) or total Akt and total ERK, respectively. Densitometric quantification showed 68 ± 14-fold induction of pAkt in Ton.Kit.wt cells in the presence of doxycycline and SCF, whereas pAkt was induced 127 ± 51-fold (in the presence of doxycycline) and 129 ± 36-fold (in the presence of doxycycline and SCF) in Ton.Kit.D816V cells (three independent experiments, means ± SD). C, Ton.Kit.wt and Ton.Kit.D816V.27 cells were kept in control medium (0) or were cultured in the presence of doxycycline (1 _μ_g/ml) for 24 h. Then, doxycycline-treated cells were stimulated with SCF (100 ng/ml (Doxy + SCF)) for 15 min or were left untreated (Doxy). Extracts were analyzed using blunt-ended annealed oligonucleotides. For supershift reactions of STAT-containing complexes, Abs specific for the C-terminal transactivation domains of STAT1 and STAT5 were added before EMSA was performed. One out of three independent experiments is shown.

FIGURE 5

FIGURE 5

Effects of KIT D816V on cluster formation. A, Examples of cluster formation of Ton.Kit wt cells and Ton.Kit.D816V cells exposed to control medium or doxycycline (Doxy) with or without SCF (100 ng/ml) under inverted microscope (magnification ×20). B, Ton.Kit.D816V cells were cultured without (Co) or with (Doxy) doxycycline (1 _μ_g/ml) for 24 h. Doxycycline-exposed cells were kept in the presence of various concentrations of PKC412 as indicated. Cluster formation is expressed as cluster per field. Results represent the means ± SD of three independent experiments. C, Ton.Kit.D816V cells were cultured in the absence or presence of doxycycline as indicated. KIT D816V-expressing cells were treated with the MEK inhibitor PD98059 (50 _μ_M) or with the PI3K inhibitor LY294002 (20 _μ_M) for 24 h. Cluster formation is expressed as cluster per field. Results represent the means ± SD of three independent experiments.

FIGURE 6

FIGURE 6

Effects of KIT D816V on differentiation. A, Ton.Kit.D816V.3 cells were exposed to control medium (left panel), doxycycline (middle panel), or doxycycline and murine mast cell growth factors (IL-3, IL-4, and IL-9, each 100 ng/ml) (right panel) for 2 days. Cells were spun on cytospin slides and stained by Wright-Giemsa. B, Histamine levels determined by RIA in Ton.Kit.D816V.27 (left panel) cells and Ton.B210 cells (right panel) after exposure to control medium (Co) or doxycycline (left panel, KIT D816V; right panel, BCR/ABL) for 24 h. C, Induction of HDC mRNA in BaF3 cells by KIT D816V. Ton.Kit.D816V.27 cells (left panel) or Ton.Kit.wt cells (right panel) were cultured in the absence (Co) or presence of doxycycline (Doxy) with or without SCF (100 ng/ml) for 24 h as indicated. Expression levels of HDC were quantified by real-time PCR and were normalized to _β_-actin mRNA. Results represent triplicates ± SD from one representative experiment. D, Ton.Kit.D816V cells induced to express KIT D816V were treated with various drugs (PD98059, 50 _μ_M; LY294002, 20 _μ_M; PKC412, 1 _μ_M; imatinib, 1 _μ_M) as indicated for 24 h. Thereafter, histamine levels were determined by RIA. Results represent the means ± SD of three independent experiments. E, Ton.Kit.D816V cells transduced with the empty MSCV vector were cultured in the absence or presence of doxycycline (1 _μ_g/ml), PD98059 (50 _μ_M), or LY294002 (20 _μ_M) as indicated. Additionally, Ton.Kit.D816V cells transduced with MSCV-STAT5aΔ749 or with MSCV-STAT5bΔ754 were induced to express KIT D816V. After 24 h, cells were harvested and the expression of HDC mRNA levels was quantified by real-time PCR. Results represent triplicates ± SD from one representative experiment.

FIGURE 7

FIGURE 7

Effects of KIT D816V on expression of differentiation-associated Ags. A, Gene expression profiles determined by Affymetrix technology in two independent experiments (Expt. 1 and Expt. 2) employing Ton.Kit.D816V.3 cells. Cells were exposed to IL-3 alone (Control) or IL-3 and doxycycline (1 _μ_g/ml (Doxy)) for 24 h and then subjected to gene array analysis. The scatter blots show the following patterns: nonexpressed (yellow dots), constantly expressed (red dots), and switched on or switched off (blue dots) as determined by default settings by the MAS 5.0 algorithm for detection calls. Lines indicate 3-, 10-, and 30-fold changes (up- or down-regulation) in mRNA expression levels. The black arrow shows up-regulation of the reference mRNA (= HDC) (21-fold and 11-fold in experiments 1 and 2, respectively). B, Ton.Kit.D816V.27 cells were exposed to control medium or doxycycline (1 _μ_g/ml) for 24 h (37°C). Expressions of mast cell-related differentiation and activation Ags were quantified by real-time PCR. Expression levels were normalized to _β_-actin mRNA and are given as “fold of control”. Results represent the mean ± SD of three independent experiments. C, Flow cytometric evaluation of expression of ICAM-1/CD54 on Ton.Kit.D816V.27 cells after induction of KIT D816V. Ton.Kit.D816V.27 cells were cultured in control medium (gray graph) or doxycycline (1 _μ_g/ml; black graph) for 24 h. The Ab omission control is also shown (dotted lines).

FIGURE 8

FIGURE 8

Cluster formation and growth of mast cells in ISM. A, Cluster formation of MC in the bone marrow of patients with ISM (KIT D816V+) as opposed to patients with MML (D816V− MC disease) or the normal bone marrow. Neoplastic MC (in focal aggregates) in ISM express tryptase, KIT, HDC, and CD63, but do not express Ki67. B, Serum tryptase levels in ISM patients (n = 10) exhibiting KIT D816V (typical ISM, n = 8; smoldering SM (SSM), n = 2) (left panel), and in 7 patients with tryptase+ (Ki67+) leukemias (KIT D816V+ MC leukemia (MCL), n = 2; tryptase+ acute myeloid leukemia (AML), n = 5) (right panel). In healthy individuals, serum tryptase levels average ~5 ng/ml (range 0–15 ng/ml).

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