Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

Abstract

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed.

• Authors: Laura Poliseno, Laura Bianchi, Lorenzo Citti, Sabrina Liberatori, Laura Mariani, Alessandra Salvetti, Monica Evangelista, Luca Bini, Vitaliano Pallini, Giuseppe Rainaldi
• Journal: The Biochemical journal (Biochem J) Vol. 379 Issue Pt 3 Pg. 823-32 (May 01 2004) ISSN: 1470-8728 [Electronic] England
• PMID: 14748742 (Publication Type: Journal Article)

Chemical References

• Chromatin
• HMGB1 Protein
• Proteome
• Proto-Oncogene Proteins c-bcl-2
• RNA, Catalytic
• RNA, Messenger
• hammerhead ribozyme
• Staurosporine

Topics

• Amino Acid Sequence
• Apoptosis (drug effects)
• Base Sequence
• Breast Neoplasms (genetics, metabolism, pathology)
• Cell Line, Tumor
• Chromatin (metabolism)
• Clone Cells (metabolism)
• Flow Cytometry
• HMGB1 Protein (metabolism)
• Humans
• Molecular Sequence Data
• Necrosis
• Peptide Mapping
• Proteome (chemistry, genetics, metabolism)
• Proto-Oncogene Proteins c-bcl-2 (deficiency, genetics)
• RNA, Catalytic (chemistry, genetics, metabolism)
• RNA, Messenger (genetics, metabolism)
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• Staurosporine (pharmacology)
• Transfection