2-D PAGE-based comparison of proteasome inhibitor bortezomib in sensitive and resistant mantle cell lymphoma.
Although gene expression following bortezomib treatment has been previously explored, direct effects of bortezomib-induced proteasome inhibition on protein level has not been analyzed so far. Using 2-D PAGE in five mantle cell lymphoma cell lines, we screened for cellular protein level alterations following treatment with 25 nM bortezomib for up to 4 h. Using MS, we identified 38 of the 41 most prominent reliably detected protein spots. Twenty-one were affected in all cell lines, whereas the remaining 20 protein spots were exclusively altered in sensitive cell lines. Western blot analysis was performed for 17 of the 38 identified proteins and 70.6% of the observed protein level alterations in 2-D gels was verified. All cell lines exhibited alterations of the cellular protein levels of heat shock-induced protein species (HSPA9, HSP7C, HSPA5, HSPD1), whereas sensitive cell lines also displayed altered cellular protein levels of energy metabolism (ATP5B, AK5, TPI1, ENO-1, ALDOC, GAPDH), RNA and transcriptional regulation (HNRPL, SFRS12) and cell division (NEBL, ACTB, SMC1A, C20orf23) as well as tumor suppressor genes (ENO-1, FH). These proteins clustered in a tight interaction network centered on the major cellular checkpoints TP53. The results were confirmed in primary mantle cell lymphoma, thus confirming the critical role of these candidate proteins of proteasome inhibition.
Weinkauf
Marc
M
CCG Leukemia, Department of Medicine III, University Hospital Grosshadern/LMU, Munich, Germany, in association with Helmholtz Center Munich, Munich, Germany.
Zimmermann
Yvonne
Y
Hartmann
Elena
E
Rosenwald
Andreas
A
Rieken
Malte
M
Pastore
Alessandro
A
Hutter
Grit
G
Hiddemann
Wolfgang
W
Dreyling
Martin
M
D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D020522:Lymphoma, Mantle-Cell; D020411:Oligonucleotide Array Sequence Analysis; D011480:Protease Inhibitors / Q000494:pharmacology*; D025941:Protein Interaction Mapping; D011719:Pyrazines / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001897:Boronic Acids; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D011480:Protease Inhibitors; D011719:Pyrazines; D000069286:Bortezomib