Author:

Keywords:

Apoptosis, CDC2 Protein Kinase, Cell Cycle, Cell Survival, Dose-Response Relationship, Drug, Enzyme Inhibitors, G2 Phase, Hela Cells, Humans, Kinetics, Mitosis, Nocodazole, Paclitaxel, Perylene, Photochemotherapy, Protein Kinase C, Proto-Oncogene Proteins c-bcl-2, Research Support, Non-U.S. Gov't, Signal Transduction, Vinblastine, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, ACTIVATED PROTEIN-KINASE, N-TERMINAL KINASE, CYTOCHROME-C, GROWTH-FACTOR, HELA-CELLS, P38 MAPK, MICROTUBULES, MECHANISM, INACTIVATION, INVOLVEMENT, Anthracenes, HeLa Cells, 03 Chemical Sciences, 06 Biological Sciences, 11 Medical and Health Sciences, 31 Biological sciences, 32 Biomedical and clinical sciences, 34 Chemical sciences

Abstract:

The role of Bcl-2 in photodynamic therapy (PDT) is controversial, and some photosensitizers have been shown to induce Bcl-2 degradation with loss of its protective function. Hypericin is a naturally occurring photosensitizer with promising properties for the PDT of cancer. Here we show that, in HeLa cells, photoactivated hypericin does not cause Bcl-2 degradation but induces Bcl-2 phosphorylation in a dose- and time-dependent manner. Bcl-2 phosphorylation is induced by sublethal PDT doses; increasing the photodynamic stress promptly leads to apoptosis, during which Bcl-2 is neither phosphorylated nor degraded. Bcl-2 phosphorylation involves mitochondrial Bcl-2 and correlates with the kinetics of a G(2)/M cell cycle arrest, preceding apoptosis. The co-localization of hypericin with alpha-tubulin and the aberrant mitotic spindles observed following sublethal PDT doses suggest that photodamage to the microtubule network provokes the G(2)/M phase arrest. PDT-induced Bcl-2 phosphorylation is not altered by either the overexpression or inhibition of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal protein kinase 1 (JNK1) nor by inhibiting the extracellular signal-regulated kinases (ERKs) or protein kinase C. By contrast, Bcl-2 phosphorylation is selectively suppressed by the cyclin-dependent protein kinase (CDK)-inhibitor roscovitine, completely blocked by the protein synthesis inhibitor cycloheximide and enhanced by the overexpression of CDK1, suggesting a role for this pathway. However, in an in vitro kinase assay, active CDK1/cyclin B1 complex failed to phosphorylate immunoprecipitated Bcl-2, suggesting that this protein kinase may not directly modify Bcl-2. Mutation of serine-70 to alanine in Bcl-2 abolishes PDT-induced phosphorylation and restores the caspase-3 activation to the same levels of the vector-transfected cells, indicating that Bcl-2 phosphorylation may be a signal to delay apoptosis in G(2)/M phase-arrested cells.