calpain and Leukemia--Myeloid--Acute

Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Calcium; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Ikaros Transcription Factor; Immunologic Factors; Lenalidomide; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Peptide Hydrolases; Receptors, G-Protein-Coupled; RNA Interference; Thalidomide; Ubiquitin-Protein Ligases

To identify novel anti-cancer agents, we created and screened a unique nutraceutical library for activity against acute myeloid leukemia (AML) cells. From this screen, we determined that glucopsychosine was selectively toxic toward AML cell lines and primary AML patient samples with no effect toward normal hematopoietic cells. It delayed tumor growth and reduced tumor weights in mouse xenograft models without imparting toxicity. Glucopsychosine increased cytosolic calcium and induced apoptosis through calpain enzymes. Extracellular calcium was functionally important for glucopsychosine-induced AML cell death and surface calcium channel expression is altered in AML cells highlighting a unique mechanism of glucopsychosine's selectivity.

Topics: Animals; Antineoplastic Agents; Apoptosis; Calcium; Calcium Channels; Calpain; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Psychosine; Signal Transduction; Time Factors; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Beta carotene (BC) is a nutritional compound widespread in foods which can influence vital cellular functions--differentiation, proliferation and apoptosis of normal and cancer cells. However its role in the carcinogenesis remains controversial. We performed a microarray expression analysis in three human acute leukemia cell lines (HL-60, U937 and TF-1) exposed to 10mM BC and found that BC stimulated the apoptosis in all studied cell lines. This effect was most evident in the HL-60 cell line and correlated with increased expression of proapoptotic BAX and CAPN2 genes. The micro-array findings were replicated by the quantitative BAX and CAPN2 expression analysis using real-time PCR and by Western Blot on protein level. The biological tests (TUNEL method) for apoptosis showed consistent proapoptotic effects in all studied cell lines. In this paper the stimulatory effect of BC on apoptosis (enhanced expression of proapoptotic genes and proteins) in human acute myeloid leukemia cells was confirmed. The most potent activation of apoptosis in the HL-60 cells is in line with other investigators observations suggesting distinct molecular mechanism of apoptosis stimulation by BC in different human acute myeloid leukemia cells.

Topics: Apoptosis; bcl-2-Associated X Protein; beta Carotene; Calpain; Gene Expression; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Real-Time Polymerase Chain Reaction; Tumor Cells, Cultured; U937 Cells

Topics: Animals; Animals, Genetically Modified; Calpain; Cells, Cultured; Core Binding Factor Alpha 2 Subunit; Disease Models, Animal; DNA-Binding Proteins; Drosophila melanogaster; Drosophila Proteins; Gene Expression Regulation, Leukemic; Genes, Lethal; Hemocytes; Leukemia, Myeloid, Acute; Oncogene Proteins, Fusion; Phenotype; Preleukemia; Protein Processing, Post-Translational; Pupa; RUNX1 Translocation Partner 1 Protein; Transcription Factors