Expression of Sialidase Neu2 in Leukemic K562 Cells Induces Apoptosis by Impairing Bcr-Abl/Src Kinases Signaling

Cristina Tringali,Barbara Lupo,Luigi Anastasia,Nadia Papini,Eugenio Monti,Roberto Bresciani,Guido Tettamanti,Bruno Venerando

doi:10.1074/jbc.m700406200

Cristina Tringali, Barbara Lupo + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m700406200

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Abstract

Chronic myeloid leukemia is a hematopoietic stem cell cancer, originated by the perpetually "switched on" activity of the tyrosine kinase Bcr-Abl, leading to uncontrolled proliferation and insensitivity to apoptotic stimuli. The genetic phenotype of myeloid leukemic K562 cells includes the suppression of cytosolic sialidase Neu2. Neu2 transfection in K562 cells induced a marked decrease (-30% and -80%) of the mRNA of the anti-apoptotic factors Bcl-XL and Bcl-2, respectively, and an almost total disappearance of Bcl-2 protein. In addition, gene expression and activity of Bcr-Abl underwent a 35% diminution, together with a marked decrease of Bcr-Abl-dependent Src and Lyn kinase activity. Thus, the antiapoptotic axis Bcr-Abl, Src, and Lyn, which stimulates the formation of Bcl-XL and Bcl-2, was remarkably weakened. The ultimate consequences of these modifications were an increased susceptibility to apoptosis of K562 cells and a marked reduction of their proliferation rate. The molecular link between Neu2 activity and Bcr-Abl signaling pathway may rely on the desialylation of some cytosolic glycoproteins. In fact, three cytosolic glycoproteins, in the range 45-66 kDa, showed a 50-70% decrease of their sialic acid content upon Neu2 expression, supporting their possible role as modulators of the Bcr-Abl complex.

Highlights

Chronic myeloid leukemia is a hematopoietic stem cell cancer cytologically characterized by the Philadelphia chromosome, which results from a reciprocal translocation between chromosome 9 and chromosome 22 [4, 5]
The ability to alter many glycosylation processes is instrumental to cancer cells to bypass the regular checkpoints that control cell life and death [16]
The modification of the sialic acid content of glycoproteins and glycolipids is among the most frequently recognized glycosylation aberrations used by cancer cells to assure proliferation [13, 15]

Summary

EXPERIMENTAL PROCEDURES

Materials—4-Methylumbellyferyl-N-acetyl-D-neuraminic acid (4-MU-NeuAc) and 4-methylumbellyferone, pepstatin A, aprotinin, leupeptin, human fetuin, RPMI 1640 medium, fetal bovine serum (FBS), glutamine, penicillin, streptomycin, Trypan Blue, and Hoechst 33342 were provided by Sigma. The RNeasy mini kit was provided by Qiagen (Milan, Italy), Improm II Reverse Transcription System by Promega (Promega Corporation, Madison, WI), iQ SYBR Green Supermix by Bio-Rad, PVDF membrane, SuperSignal West Pico Chemiluminescent substrate, Coomassie protein assay reagent. 2 ϫ 106 K562 cells were transfected with pcDNA3.1-Neu, pcDNA3.1-Neu2(Ala 3 Asp), or pcDNA3.1 using the DMRIE-C reagent, according to manufacturer’s guidelines. CDNA representing 10 ng of total RNA was used as template for real time PCR, performed using the iCycler thermal cycler (Bio-Rad). Sialidase Activity Assay—Mock, Neu, and Neu (Ala 3 Asp) transfected K562 cells were harvested by centrifugation and resuspended in PBS containing 1 ␮g/ml pepstatin A, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin. The crude extract was, subsequently, centrifuged at 200,000 ϫ g for 20 min on MAY 11, 2007 VOLUME 282 NUMBER 19

Reverse primer

RESULTS

We did not find any significant difference in the ganglioside pattern