A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells

Abstract

Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIPL, a protein that blocks caspase 8 activation. cFLIPL levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIPL in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.

Detection of endoplasmic reticulum stress and the unfolded protein response in naturally-occurring endocrinopathic equine laminitis

“…As a positive control for immunoblots and to test antibody specificity, lysates were prepared from the human Hela cell line as described previously [66]. Where noted, Hela cells were incubated for 24 h in either Brefeldin A (BFA; 3.6 μM; Sigma-Aldrich) or tunicamycin (TM; 2 μg/ml; Sigma-Aldrich) to induce ER stress prior to lysate preparation [45].…”

Exploring stathmin control of cell survival through negative feedback of a JNK-dependent pathway  

“…We were unable to detect increased phosphorylated (active) JNK in stathmin-depleted cells by western blots. It is possible that increased active JNK occurs asynchronously in a population of cells, much like the asynchrony in the onset of cell death [12], and, therefore, is undetectable by western blots from cell populations. In order to assess JNK activity in individual cells over time, we used a fluorescently tagged JNK kinase translocation reporter (KTR), a localization-based biosensor of JNK kinase activity [2].…”

“…In the absence of stathmin and loss of a negative feedback loop, active JNK levels should rise and eventually activate apoptosis. HeLa cells are a convenient model system to test this hypothesis since they are easily transfected, and previous studies demonstrated that stathmin depletion induces apoptosis in these cells[7] [6][12]. Using a specific JNK inhibitor or siRNA knockdown to abolish JNK activity, we addressed whether JNK is necessary for induction of cell death in stathmin-depleted cells.…”

Relevant coexpression of STMN1, MELK and FOXM1 in glioblastoma and review of the impact of STMN1 in cancer biology

“…101 Corroborating these observations, STMN1 inhibition in cancer cells harboring TP53 mutation has decreased cell proliferation and viability, increased apoptosis and suppressed tumorigenicity, suggesting that STMN1 is required for the survival of p53--mutant cells. [102][103][104][105] Recently, it was suggested that overexpressed STMN1 interacts with p53 and contributes to the gain-of-function of p53. 105 Altogether, these data suggest that targeting STMN1 may be an interesting approach to treat different types of cancers with aberrant p53 function.…”