Mechanisms of staurosporine induced apoptosis in a human corneal endothelial cell line

Abstract

Background: Apoptosis very probably plays a key part in endothelial cell loss during corneal storage in organ culture as well as hypothermic storage. However, the mechanisms underlying endothelial apoptosis are poorly understood. The response of a human corneal endothelial cell (HCEC) line to staurosporine, a known inducer of apoptosis, was investigated to gain insights into the intracellular modulators that participate in endothelial cell death.

Methods: Immortalised HCECs were studied after 3, 6, 12, and 24 hours of incubation with 0.2 micro M staurosporine. Cell shedding was monitored. Hoechst 33342 fluorescent DNA staining combined with propidium iodide was used for apoptosis/necrosis quantification and morphological examination. The caspase-3 active form was assessed using western blot, proteolytic activity detection, and immunocytochemistry. The cleaved form of poly(ADP-ribose) polymerase (PARP) was assessed using immunocytochemistry and western blot. The ultrastructural features of cells were screened after 12 hours with staurosporine or vehicle.

Results: The specific apoptotic nature of staurosporine induced HCEC death was confirmed. The ultrastructural features of staurosporine treated cells were typical of apoptosis. HCEC shedding and DNA condensation increased with time. Caspase-3 activity was detected as early as 3 hours after exposure with staurosporine, peaking at 12 hours of incubation. The presence of cleaved PARP after 3 hours confirmed caspase-3 activation.

Conclusions: These data suggest strongly that HCEC cell death induced by staurosporine is apoptosis. The main consequence of HCEC apoptosis is shedding. Staurosporine induced apoptosis of endothelial cells involves activation of caspase-3, and could be a useful model to study strategies of cell death inhibition.

Figure 1

Progressive shedding of human corneal endothelial cells induced by 0.2 μM staurosporine after 3, 6, 12, and 24 hours (B, C, D, and E respectively), compared with untreated cells (A) or cells incubated for 24 hours with the vehicle (F). Scale bar = 100 μm.

Figure 2

Percentage of shedding of endothelial cells after exposure to staurosporine. Staurosporine induced the progressive shedding of endothelial cells. In our model, 0.2 μM staurosporine triggered the shedding of nearly half the cells within 24 hours, which appeared to be sufficient to study large number of cells engaged in cell death but without excessive cell toxicity related to a too high dose. Results expressed the mean (SD) of each experiment done in triplicate.

Figure 3

Ultrastructural features of untreated corneal endothelial cells (A) compared with 0.2 μM staurosporine treated cells for 12 hours (B). Morphological changes observed in treated cells were typical of apoptosis and comprised cell shrinking and chromatin condensation at the periphery of the nucleus. Asterisks indicated the nuclei. Scale bar: 2 μm.

Figure 4

Double staining of cells with (A) Hoechst 33342 (H) and (B) propidium iodide (PI) distinguished between the typical features of apoptosis (fragmented bright H+/PI−) (arrowheads), necrosis (H−/PI+ or weak H+/PI+) (asterisk) and rare mitosis (H+/PI−) (arrow). Superposition (C) of the two images allowed detection of double stained cells probably indicating late apoptosis. Here, an example of staining after 6 hours’ incubation with 0.2 μM staurosporine (original magnification ×40)

Figure 5

Quantification of apoptosis and necrosis and caspase-3 activity after incubation with 0.2 μM staurosporine for 0–24 hours. Double staining with Hoechst 33342 (H) and propidium iodide (PI) was applied on adherent cells. Apoptotic cells were defined as cells with bright blue nuclei (H+) and intact membranes (PI−). All PI+ nuclei were deemed necrotic. Apoptosis increased during the first 12 hours. Afterwards, necrosis (true necrosis and/or late apoptosis) was the main phenomenon. Bars represent the mean (SD) of the three separate experiments and were expressed as percentages of, respectively, apoptotic and necrotic cells among the remaining adherent cells. For caspase-3 activity, the bars represent the mean (SD) of a triplicate. Caspase-3 activity started at 3 hours and peaked at 6–12 hours.

Figure 6

Western blot analysis of procaspase-3 (A), cleaved caspase-3 (B), poly (ADP-ribose) polymerase (PARP) (C), and cleaved PARP (D) expression in human corneal endothelial cells incubated with 0.2 μM staurosporine for 3, 6, 12, and 24 hours. Ct: control (untreated cells). Each lane was loaded with 40 μg of protein (total cell extract). Blots were probed with monoclonal antibody to human pro-caspase-3 (32 kDa), or polyclonal antibodies against cleaved caspase-3 (19 and 17 kDa), PARP (116 kDa), or cleaved PARP (89 kDa).

Figure 7

Immunostaining of cleaved caspase-3 (A, B) and cleaved poly (ADP-ribose) polymerase (PARP) (C, D). (A) and (C) were untreated cells and (B) and (D) were stained after 12 hours’ incubation with 0.2 μM staurosporine. This time corresponded to the peak of caspase-3 activity. Normal culture exhibited very few positive cells. At 12 hours, numerous cells dysplayed a strong cytoplasmic red staining either for cleaved caspase-3 or for cleaved PARP. Positive cells had a condensed nucleus, a shrunken cytoplasm, and membranes blebbing consistent with morphological features of apoptosis.