G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis

Abstract

Previous studies have demonstrated that G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases (CDKs) prevent the death of nerve growth factor (NGF)-deprived PC12 cells and sympathetic neurons, suggesting that proteins normally involved in the cell cycle may also serve to regulate neuronal apoptosis. Past findings additionally demonstrate that DNA-damaging agents, such as the DNA topoisomerase (topo-I) inhibitor camptothecin, also induce neuronal apoptosis. In the present study, we show that camptothecin-induced apoptosis of PC12 cells, sympathetic neurons, and cerebral cortical neurons is suppressed by the G1/S blockers deferoxamine and mimosine, as well as by the CDK-inhibitors flavopiridol and olomoucine. In each case, the IC50 values were similar to those reported for inhibition of death induced by NGF-deprivation. In contrast, other agents that arrest DNA synthesis, such as aphidicolin and N-acetylcysteine, failed to block death. This suggests that the inhibition of DNA synthesis per se is insufficient to provide protection from camptothecin. We find additionally that the cysteine aspartase family protease inhibitor zVAD-fmk inhibits apoptosis evoked by NGF-deprivation but not camptothecin treatment. Thus, despite their shared sensitivity to G1/S blockers and CDK inhibitors, the apoptotic pathways triggered by these two causes of death diverge at the level of the cysteine aspartase. In summary, neuronal apoptosis induced by the DNA-damaging agent camptothecin appears to involve signaling pathways that normally control the cell cycle. The consequent death signals of such deregulation, however, are different from those that result from trophic factor deprivation.

Fig. 1.

The CDK inhibitors flavopiridol and olomoucine prevent camptothecin-induced death of neuronally differentiated PC12 cells. The neuronal PC12 cell phenotype was attained by treatment with NGF in serum-free medium for 8 d. Replicate cultures were treated with camptothecin (10 μm) as indicated. A, Effect of flavopiridol (1 μm), olomoucine (200 μm), and iso-olomoucine (200 μm) on the time course of survival of neuronally differentiated PC12 cells after treatment with camptothecin. B, C, Effect of various concentrations of flavopiridol and olomoucine on survival of camptothecin-treated neuronally differentiated PC12 cells. Each data point is the mean ± SEM (n = 3) and is expressed relative to the number of cells initially plated.

Fig. 2.

Induction of apoptotic chromatin condensation in neuronal PC12 cells by camptothecin. PC12 cells were neuronally differentiated by treatment with serum-free RPMI 1640 medium containing NGF (100 ng/ml) for 10 d. Cells were then cultured with serum-free RPMI medium containing NGF in the presence or absence of 10 μm camptothecin for 72 hr, with or without flavopiridol (0.5 μm) or olomoucine (200 μm), then fixed and stained with DAPI to visualize nuclear chromatin. Images were captured under differential interference contrast (A,C, E, G, I,K) or fluorescence (B,D, F, H, J,L) optics. Condition depicted are no additives (A, B), camptothecin alone (C, D), camptothecin and flavopiridol (E, F), camptothecin and olomoucine (G, H), flavopiridol alone (I, J), and olomoucine alone (K, L). Camptothecin produced a significant degree of cell death, characterized by blebbing (C, arrow) and apoptotic chromatin condensation (D, arrow), which could be inhibited by both flavopiridol (E,F) and olomoucine (G,H). However, both flavopiridol and olomoucine, in combination with camptothecin or alone, were able to induce partial changes in chromatin structure (H,arrowhead). Scale bar (shown in L), 20 μm.

Fig. 3.

Phase-contrast micrographs of neuronally differentiated PC12 cells maintained in serum-free medium containing NGF and treated for 4 d with the following: 10 μmcamptothecin (A); no additives (B); 10 μm camptothecin + 1 μm flavopiridol (C); 10 μm camptothecin + 200 μm olomoucine (D); 1 μmflavopiridol (E); 200 μm olomoucine (F).

Fig. 4.

CPT-cAMP and the G1/S blockers deferoxamine and mimosine suppress camptothecin-induced death of neuronally differentiated PC12 cells. Replicate cultures were treated with camptothecin (10 μm) as indicated. Effect of deferoxamine (1 mm) (A), mimosine (400 μm) (B), and CPT-cAMP (100 μm) (C) on the time course of survival of neuronally differentiated PC12 cells after treatment with camptothecin. Each data point is the mean ± SEM (n = 3) and is expressed relative to the number of cells initially plated.

Fig. 5.

Aphidicolin (A) and NAC (B) do not block camptothecin-mediated death of neuronally differentiated PC12 cells. Replicate cultures were treated with and without 10 μm camptothecin in the presence and absence of aphidicolin (10 μm) or NAC (60 mm) as indicated. Each data point is the mean ± SEM (n = 3) and is expressed relative to the number of cells initially plated.

Fig. 6.

Effect of cell cycle blockers on camptothecin-induced death of naive PC12 cells. Cells were cultured in the presence of serum with and without 10 μm camptothecin as indicated. Replicate naive PC12 cell cultures were grown as indicated in the presence of 1 mm deferoxamine (A); 400 μm mimosine (B); 100 μm CPT-cAMP (C); or 10 μm aphidicolin (D). Each data point is the mean ± SEM (n = 3) and is expressed relative to the number of cells initially plated.

Fig. 7.

The CDK inhibitors flavopiridol and olomoucine inhibit the camptothecin-induced death of rat sympathetic neurons. Primary cultures of neonatal rat superior cervical ganglion neurons were grown in the presence of NGF for 3 d before drug treatment. Replicate cultures were treated with camptothecin (10 μm) as indicated. Each data point is the mean ± SEM (n = 3) and is expressed relative to the number of neurons present in each culture at the time of drug treatment.A, Effects of flavopiridol (1 μm) on the time course of survival of sympathetic neurons treated with camptothecin. Actinomycin D (10 μm) treatment is included to control for death induced by inhibition of transcription.B, Effects of various doses of flavopiridol on the survival of camptothecin-treated sympathetic neurons at 5 din vitro. C, Effects of olomoucine and iso-olomoucine (200 μm) on the time course of survival of sympathetic neurons treated with camptothecin. Actinomycin D (10 μm) treatment is included to control for death induced by inhibition of transcription. D, Effects of various doses of olomoucine on the survival of camptothecin-treated sympathetic neurons at 5 d in vitro.

Fig. 8.

Phase-contrast micrographs of primary sympathetic neurons maintained in medium containing NGF and treated for 5 d with the following: 10 μm camptothecin (A); no additives (B); camptothecin + 1 μm flavopiridol (C); camptothecin + 200 μm olomoucine (D); camptothecin + 200 μm iso-olomoucine (E).

Fig. 9.

The ICE family protease inhibitor zVAD-fmk (100 μm) does not inhibit camptothecin-promoted death of sympathetic neurons but does promote survival of neurons deprived of NGF. Primary cultures of neonatal rat superior cervical ganglion neurons were grown in the presence of NGF for 3 d before experimentation. A, NGF withdrawal; cultures were deprived of NGF and treated as indicated for 3 d before assessment of neuronal survival. B, Camptothecin treatment; cultures were treated with or without camptothecin for the indicated times and with or without zVAD-fmk as indicated. Each data point is the mean ± SEM (n = 3) and is expressed relative to the number of neurons present in each well at the time of drug treatment.

Fig. 10.

Inhibition of camptothecin-induced cortical neuronal apoptosis by flavopiridol and olomoucine. Mixed cultures were treated with (C–H) or without (A, B) 10 μm camptothecin in the presence of no additional additives (A—D), 0.5 μm flavopiridol (E, F), or 200 μmolomoucine (G, H) for 18 hr. The cells were then fixed and stained with DAPI to visualize nuclear chromatin, and images were captured under differential interference contrast (A, C, E,G) or fluorescence (B, D,F, H) optics. Scale bar (shown inH), 20 μm.

Fig. 11.

Dose–response relationships for inhibition of camptothecin-induced cortical neuronal death by flavopiridol and olomoucine. Mixed cultures were treated with or without 10 μm camptothecin in the presence or absence of flavopiridol (A), olomoucine (B), or iso-olomoucine (C), and neuronal survival was evaluated at 24 hr by the CFDA assay. Data are expressed as percent control of the mean ± SD (n = 3–4 per condition). The effects of both flavopiridol and olomoucine on neuronal survival were significant (p < 0.0001 by one-way ANOVA).