Molecular Mechanism Of WAVE1 As An Anti-apoptotic Protein In Leukemia Cells And Role Of HSF1 In The Regulation Of WAVE1 Expression

WASP-family verprolin-homologous protein 1 (WAVE1), a novel member of the actin regulatory protein, is a key regulator of actin polymerization and cytoskeleton reorganization in cell. Leukaemia is one of the most common malignant tumors in children. It is not clear to present scientists yet about the effects of WAVEl on leukaemia.In this paper, we first showed that WAVE1 was overexpressed in human blood cancer cell lines. We studied expression levels of WAVE1 in six leukemia cell lines (the human Jurkat T leukemia cells, U937 histiocytic lymphoma cells, Burkitt's lymphoma cell Raji, acute promyelocytic leukemia cell HL-60, chronic myelogenous leukemia cell K562 and K562/A02) by Western blotting analysis. Levels of WAVE1 expression were high in all six human leukemia cancer cell lines. In contrast, the constitutive expression levels of WAVE1 were noticeably lower in normal human peripheral blood mononuclear cells (PBMCs), or non-blood cancer cell-lines, including human lung A549 cancer cells, Hela cervical cancer cells, MG-63 osteosarcoma cells, CNE2 nasopharyngeal carcinoma cells, human umbilical vein endothelial cell (HUVE), QSG7701 hepatocarcinoma cells, and WI-38 lung fibroblastoma cells. To evaluate the clinical relevance, we measured the relative WAVE1 protein expression levels in bone marrow mononuclear cells (BMMCs) obtained from children with both acute lymphoblastic leukemia and acute nonlymphocytic leukemia. Higher levels of WAVE1 expression were found in BMMCs derived from patients with primary and relapse leukemia. In contrast, consistent with the absence of WAVE1 expression in normal PBMCs, WAVE1 was not detectable in BMMCs derived from normal healthy subjects, or patients with complete remission. It suggests that WAVE1 maybe a marker protein in blood cancer cell lines and a potential monitoring candidate for pathogenetic condition of childhood leukaemia.At second part of this paper, we found that WAVE1 involved in the multidrug resistance (MDR) mechanisms in leukemia. The expression of WAVE1 in K562/A02 cells, the adriamycin (ADM) multidrug-resistance leukemia cell line, went up to a much higher value than that in K562 cells. Overexpression of WAVE1 in K562 cells by transient transfection significantly increased the resistance to ADM. Suppression of WAVE1 in K562/A02 cells by RNA interference resulted in a reversal of MDR to ADM. WAVE1 involved in the MDR mechanisms in K562/A02 leukemia cells through regulation the level of mdrl and Bcl-2. Overexpression of WAVE1 in K562 cells significantly increased the level of mdrl and Bcl-2, and suppression of WAVE1 in K562/A02 cells by RNA interference decreased the level of mdrl and Bcl-2. Our studies provide novel ways to reverse MDR in leukaemia.At third, we demonstrated that WAVE1 was a novel anti-apoptotic protein in human leukemia cells. Enhanced expression of WAVE1 by gene transfection rendered leukemia (K562 and Jurkat) cells resistant to apoptosis induced by hydrogen peroxide; whereas suppression of WAVE1 expression by RNA interference restored the sensitivity of drug-resistant leukemia (K562/A02) cells to apoptosis induced by anti-cancer drugs such as ADM. WAVE1 confers anti-apoptotic activities potentially via: 1) increasing expression of anti-apoptotic proteins (e. g., Bcl-2, Bcl-XL); 2) inhibiting activation and/or mitchondrial translocation of pro-apoptotic proteins, Bid and Bax, respectively; 3) inhibiting mitochondrial release of cytochrome c and Smac/DIABLO, and activation of caspase-3; and 4) inhibiting activation of c-Jun amino-terminal kinase (JNK) MAP kinase and activator protein 1 (AP1) DNA binding activity. Taken together, these experimental data suggest that WAVE1 functions as a novel anti-apoptotic protein in leukemia cancer potentially via sustaining expression of anti-apoptotic proteins, but inhibiting activation of pro-apoptotic proteins.At last, we found that WAVE1 was a novel heat stress protein in cells and heat shock factor 1 (HSF1) mediated regulation of WAVE1. Heat shock increased activation of the WAVE1 promoter and increased WAVE1 gene expression. HSF1 was required for WAVE1 expression. Heat shock induced WAVE1 expression with complete HSF1 structure (e. g. HSF1+/+ mice, HSF1+/+ mouse embryo fibroblasts) or HSF1 overexpression (e. g. HSF1 overexpreeion cell lines) or activated HSF1 transcriptional function (e. g. dominant-positive mutant). However, deletion of HSF1 (e. g. HSF 1-/- mice, HSF1-/- mouse embryo fibroblasts) or inactivated HSF1 transcriptional function (e. g. dominant-negative mutant) inhibited WAVE1 expression when cells were exposed to heat shock. There were four heat shock element (HSE) motifs in the WAVE1 gene promoter by bioinformatics assay. Importanly, we demonstrated that HSF1 was directed bound to HSE of WAVE1 promoter and promoted heat-induced WAVE1 expression by luciferase assays and electrophoretic mobility shift assay (EMSA). In addition, suppression of WAVE1 expression by RNA interference lowered the heat tolerance in K562 cells. It suggests that suppression of WAVE1 expression may be a potential way to decrease the temperature used in cancer thermotherapy.In conclusion, WAVE1 not only play important roles in chilidhood leukaemia including MDR, but also is a novel anti-apoptotic protein and heat stress protein. Our experimental data provides important insights into the novel function of WAVE1 and the mechanisms of leukaemia, and may lead to the development of novel way for treatment of human leukaemia.