Publication Date:
2008-11-16
Description:
Background: TRKA (Tropomyosin Receptor Kinase A) is a member of the neurotrophin tyrosine kinase receptor family. Nerve Growth Factor (NGF) binds specifically to TRKA triggering multiple downstream pathways which regulate proliferation and differentiation in a number of neural, epithelial and epidermal cell types. TRKA is also expressed in hematopoietic progenitor cells and NGF has been demonstrated to promote the colony growth and differentiation of myeloid progenitor cells. Interestingly, Pralle et al. found that 44% of patients with acute myeloid leukemia (AML) express TRKA mRNA and up to 30% encode activating TRKA mutations. Furthermore, a constitutively-active TRKA variant has been shown to contribute to myeloid leukemogenesis. Purpose: To evaluate the role of NGF/TRKA signaling on AML proliferation and survival through effects on downstream signaling pathways, and evaluate the effects of TRK inhibition on human AML. Methods: TRKA protein expression was measured in a panel of human AML cell lines (U937, K562, THP1, NB4, HL60, KU812). AlamarBlue (MTT) assays were used to measure proliferation/viability. For proliferation assays, cells were stimulated with NGF (5 ng/ml) for 1–3 days with or without the addition of a TRK inhibitor AZ-23 (25nM). Cell counts, cell cycle and survival were measured using trypan blue cell counts, and propidium iodine-based flow cytometry assays. Downstream signaling events were measured following a 15 minute exposure to recombinant human NGF. Antibodies for phosphorylated and total AKT, ERK, JNK and MEKK were used. In a human AML xenograft model, mice were treated with 100 mg/kg (2.5 mg) of AZ-23 by gavage for 2 weeks without toxicity. Weekly tail vein blood collection and flow cytometry were used to evaluate leukemia tumor burden. Kaplan-Meier curves were used to assess effects on morbidity. Results: Five of six human AML cell lines showed detectable TRKA protein levels by Western. When stimulated with NGF, all five cell lines which expressed TRKA demonstrated enhanced proliferation within 24 hours with mean value of 20% (10 to 45%, p=
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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