Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.
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.
Poliseno
Laura
L
Laboratorio di Terapia Genica e Molecolare, Istituto di Fisiologia Clinica, Area della Ricerca del CNR, Via G. Moruzzi, 1, 56124 Pisa, Italy.
Bianchi
Laura
L
Citti
Lorenzo
L
Liberatori
Sabrina
S
Mariani
Laura
L
Salvetti
Alessandra
A
Evangelista
Monica
M
Bini
Luca
L
Pallini
Vitaliano
V
Rainaldi
Giuseppe
G
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D002999:Clone Cells / Q000378:metabolism; D005434:Flow Cytometry; D024243:HMGB1 Protein / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009336:Necrosis; D010449:Peptide Mapping; D020543:Proteome / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000172:deficiency* / Q000235:genetics*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019311:Staurosporine / Q000494:pharmacology*; D014162:Transfection
D002843:Chromatin; D024243:HMGB1 Protein; D020543:Proteome; D019253:Proto-Oncogene Proteins c-bcl-2; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D019311:Staurosporine