Abstract
It has been shown that changes in spectrin distribution in early apoptosis preceded changes in membrane asymmetry and phosphatidylserine (PS) exposure. PKCθ was associated with spectrin during these changes, suggesting a possible role of spectrin/PKCθ 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 αIIβII spectrin arrangement in Jurkat T and HL60 cell lines revealed the redistribution of spectrin and PKCθ into a polar aggregate in early apoptosis induced by fludarabine/mitoxantrone/dexamethasone (FND). The appearance of an αIIβII 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/PKCθ, and (iii) spectrin aggregation was shown to be partially dependent on PKCθ activity. Our results indicate that spectrin/PKCθ aggregate formation is related to an early stage in drug-induced apoptosis and possibly may be regulated by PKCθ activity. These findings indicate that spectrin/PKCθ 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.
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Acknowledgments
Support for this research was provided by KBN Grant No. 3P04C 097 25 and KBN Grant No. 2P04C 083 30, and by a grant from the Foundation for International Education. We thank Ewa Duszeńko, M.Sc., for her help with the microscopy and Dr. Joanna Miloszewska (Oncology Centre, M. Sklodowska-Curie Institute, Warsaw, Poland) for providing rabbit anti-PKCθ IgG’s.
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Patrycja M. Dubielecka and Michał Grzybek have contributed equally to this study.
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Supplementary Fig. 1.
Morphology and nuclear integrity of Jurkat T (a) and HL60 (b) cells at the 4, 8, and 12 h of apoptosis. The same populations used for the evaluation of the quantitative time course of apoptosis presented in Fig. 1, were used for the preparations presented here. Images were acquired using an Axioskop 2 Zeiss microscope. Magnification ×630 (TIF 1532 kb)
Supplementary Fig. 2.
Thin-section electron micrographs of Triton X-100 extracted peripheral mononuclear blood lymphocytes. Freshly obtained (left) and apoptotic (right) cells were treated with 1% Triton X-100 buffer and pelleted as described in the Materials and methods section. N-nucleus, bar 2 μm. The matrix in the apoptotic cells is filled with some “mesh” (arrowheads), which is probably composed of spectrin subunits (TIF 230 kb)
Supplementary Fig. 3.
Immunofluorescent location of spectrin and PKCθ in Jurkat T (a, b) and HL60 (c, d) cells. Images were acquired after 4 h and after 8 h of incubation with FND after pre-treatment for 1 h with caspase-3, -7, -8, and -10 inhibitors, and calpain I and II inhibitors. Polyclonal rabbit anti-brain spectrin or rabbit anti-PKCθ were used as primary antibodies, and FITC-conjugated swine anti-rabbit IgG was used as the secondary antibody. Images were acquired using a Zeiss LSM. Magnification ×630 (DOC 4476 kb)
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Dubielecka, P.M., Grzybek, M., Kolondra, A. et al. Aggregation of spectrin and PKCθ is an early hallmark of fludarabine/mitoxantrone/dexamethasone-induced apoptosis in Jurkat T and HL60 cells. Mol Cell Biochem 339, 63–77 (2010). https://doi.org/10.1007/s11010-009-0370-4
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DOI: https://doi.org/10.1007/s11010-009-0370-4