Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis.
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.
Vantieghem
Annelies
A
Division of Biochemistry, Faculty of Medicine, Catholic University of Leuven, B-3000 Leuven, Belgium.
Xu
Yan
Y
Assefa
Zerihun
Z
Piette
Jacques
J
Vandenheede
Jackie R
JR
Merlevede
Wilfried
W
De Witte
Peter A M
PA
Agostinis
Patrizia
P
D000873:Anthracenes; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology*; D010569:Perylene / Q000031:analogs & derivatives* / Q000494:pharmacology*; D010778:Photochemotherapy; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014747:Vinblastine / Q000494:pharmacology
D000873:Anthracenes; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D010569:Perylene; D014747:Vinblastine; C004965:hypericin; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase; D017239:Paclitaxel; D015739:Nocodazole