1. ¹Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Segrate, Milan, Italy
2. ²IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy
3. ³Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, Milan, Italy
4. ⁴Department of Biomedical Science and Biotechnology, University of Brescia, Brescia, Italy

Correspondence: B Venerando, Department of Medical Chemistry, Biochemistry and Biotechnology, Faculty of Exercise Science, University of Milan, via F.lli Cervi 93, Milan (Segrate) 20090, Italy. Tel: +39 02 50330361; Fax: +39 02 50330365; E-mail: bruno.venerando@unimi.it; G Tettamanti, IRCCS Policlinico San Donato, via Morandi 30, San Donato Milanese, Milan 20097, Italy. Tel: +39 02 52774679; Fax: +39 02 52774336; E-mail: guido.tettamanti@unimi.it

Received 22 January 2008; Revised 25 July 2008; Accepted 13 August 2008; Published online 26 September 2008.

Abstract

In chronic myeloid leukemia K562 cells, differentiation is also blocked because of low levels of ganglioside GM3, derived by the high expression of sialidase Neu3 active on GM3. In this article, we studied the effects of Neu3 silencing (40–70% and 63–93% decrease in protein content and activity, respectively) in these cells. The effects were as follows: (a) gangliosides GM3, GM1, and sialosylnorhexaosylceramide increased markedly; (b) cell growth and [³H]thymidine incorporation diminished relevantly; (c) as mRNA, cyclin D2, and Myc were much less expressed, whereas cyclin D1 was expressed more like its inhibitor p21; (d) as mRNA, pro-apoptotic proteins Bax and Bad increased with concurrent decrease and increase in the anti-apoptotic proteins Bcl-2 and Bcl-XL, respectively; (e) the apoptosis inducers etoposide and staurosporine were active on Neu3 silencing cells but not on mock cells; (f) as mRNA, the megakaryocytic markers CD10, CD44, CD41, and CD61 increased similar to the case of mock cells stimulated with PMA; (g) the signaling cascades mediated by PLC-2, PKC, RAF, ERK1/2, RSK90, and JNK were largely activated. The induction of a GM3-rich ganglioside pattern in K562 cells by treatment with brefeldin A elicited a phenotype similar to that of Neu3 silencing cells. In conclusion, upon Neu3 silencing, K562 cells show a decrease in proliferation, propensity to undergo apoptosis, and megakaryocytic differentiation.