title: Aggregation of spectrin and PKCtheta is an early hallmark of fludarabine/mitoxantrone/dexamethasone-induced apoptosis in Jurkat T and HL60 cells.
abstract: It has been shown that changes in spectrin distribution in early apoptosis preceded changes in membrane asymmetry and phosphatidylserine (PS) exposure. PKCtheta was associated with spectrin during these changes, suggesting a possible role of spectrin/PKCtheta aggregation in regulation of early apoptotic events. Here we dissect this hypothesis using Jurkat T and HL60 cell lines as model systems. Immunofluorescent analysis of alphaIIbetaII spectrin arrangement in Jurkat T and HL60 cell lines revealed the redistribution of spectrin and PKCtheta into a polar aggregate in early apoptosis induced by fludarabine/mitoxantrone/dexamethasone (FND). The appearance of an alphaIIbetaII spectrin fraction that was insoluble in a non-ionic detergent (1% Triton X-100) was observed concomitantly with spectrin aggregation. The changes were observed within 2 h after cell exposure to FND, and preceded PS exposure. The changes seem to be restricted to spectrin and not to other cytoskeletal proteins such as actin or vimentin. In studies of the mechanism of these changes, we found that (i) neither changes in apoptosis regulatory genes (e.g., Bcl-2 family proteins) nor changes in cytoskeleton-associated proteins were detected in gene expression profiling of HL60 cells after the first hour of FND treatment, (ii) caspase-3, -7, -8, and -10 had minor involvement in the early apoptotic rearrangement of spectrin/PKCtheta, and (iii) spectrin aggregation was shown to be partially dependent on PKCtheta activity. Our results indicate that spectrin/PKCtheta aggregate formation is related to an early stage in drug-induced apoptosis and possibly may be regulated by PKCtheta activity. These findings indicate that spectrin/PKCtheta aggregation could be considered as a hallmark of early apoptosis and presents the potential to become a useful diagnostic tool for monitoring efficiency of chemotherapy as early as 24 h after treatment.
authors.affiliation: Laboratory of Cytobiochemistry, Faculty of Biotechnology, University of Wrocław, S. Przybyszewskiego 63-77, 51-148 Wrocław, Poland. pdubielecka@nybloodcenter.org
authors.forename: Patrycja M
authors.initials: PM
authors.lastname: Dubielecka
authors.forename: Michał
authors.initials: M
authors.lastname: Grzybek
authors.forename: Adam
authors.initials: A
authors.lastname: Kolondra
authors.forename: Bozena
authors.initials: B
authors.lastname: Jaźwiec
authors.forename: Anna
authors.initials: A
authors.lastname: Draga
authors.forename: Paulina
authors.initials: P
authors.lastname: Aleksandrowicz
authors.forename: Monika
authors.initials: M
authors.lastname: Kołodziejczyk
authors.forename: Anna
authors.initials: A
authors.lastname: Serwotka
authors.forename: Barbara
authors.initials: B
authors.lastname: Dolińska-Krajewska
authors.forename: Jerzy
authors.initials: J
authors.lastname: Warchoł
authors.forename: Kazimierz
authors.initials: K
authors.lastname: Kuliczkowski
authors.forename: Aleksander F
authors.initials: AF
authors.lastname: Sikorski
mesh_terms: D000199:Actins / Q000378:metabolism; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D003907:Dexamethasone / Q000008:administration & dosage; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D019169:Jurkat Cells; D008942:Mitoxantrone / Q000008:administration & dosage; D017830:Octoxynol; D011493:Protein Kinase C / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D055503:Protein Multimerization; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013049:Spectrin / Q000378:metabolism*; D014740:Vidarabine / Q000008:administration & dosage / Q000031:analogs & derivatives; D014746:Vimentin / Q000378:metabolism
chemical_list: D000199:Actins; D061945:Caspase Inhibitors; D007527:Isoenzymes; D012333:RNA, Messenger; D014746:Vimentin; D013049:Spectrin; D003907:Dexamethasone; D017830:Octoxynol; D008942:Mitoxantrone; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D053148:Caspase 3; D014740:Vidarabine; C024352:fludarabine