Sequential events of apoptosis involving docetaxel, a microtubule-interfering agent: a cytometric study

Abstract

Background: Despite the great advances in the understanding of programmed cell death, little attention has been paid to the sequence of the events that characterise it. In particular, the course of apoptotic events induced by microtubule-interfering agents such as taxanes is poorly understood. In order to increase such knowledge, we studied a number of independent biochemical and cytological modifications using cytometric methods in a bladder cancer cell line treated with the second generation taxane, docetaxel.

Results: Within a few hours, drug treatment had induced mitochondrial membrane transition, cell shrinkage and a decrease in granularity. Cell cycle was almost completely blocked in G2/M phase within 24 hours. The hypodiploid peak started to become prominent 48 hours after the treatment. At the same time, the appearance of a DNA ladder demonstrated caspase-dependent chromatin fragmentation. Concurrently, specific cell surface modifications took place, involving at first glycoprotein syalilation and later phospholipid asymmetry. DNA fragmentation was subsequently detected by TUNEL assay. Over time, cell membranes became permeable to propidium iodide. A very similar time-course of apoptotic events was found after treatment of a myelomonocytic cell line with the same drug.

Conclusion: After discussing some characteristics of the methods employed and their limitations, a succession of apoptotic events over time is suggested, in which the collapse of mitochondrial transmembrane potential (Deltapsim) is the earliest sign of apoptosis.

Figure 1

Cytotoxic and antiproliferative activity of docetaxel in the HT1376 bladder cancer cell line, evaluated by Sulphorhodamine B assay. The percentage of cell growth is defined in the Methods section. Continuous line: 1-hour treatment with docetaxel followed by a 23-hour wash-out. Dotted line: 24-hour continuous treatment. Experiments were run in octuplet, and each experiment was repeated three times. Standard errors were below 5%

Figure 2

Cell cycle distribution of HT1376 bladder cancer cell line after a 1-hour treatment with 3 μg/mL docetaxel followed by different wash-out times. Cells were permeabilised and stained with propidium iodide, as described in the Methods section. Histograms show a representative experiment.

Figure 3

DNA gel electrophoresis of docetaxel-treated (lanes 1–3) and control (lane 4) HT1376 cells. DNA from HT1376 cells treated for 1 hour with 3 μg/mL docetaxel and cultured for a further 16, 24 and 48 hours was extracted as detailed in the Methods section, then separated in 1.5% agarose gel electrophoresis. DNA from a 72-hour control (untreated) culture was prepared in the same way.

Figure 4

Variation in forward and side scatter of HT1376 bladder cancer cell line after a 1-hour treatment with 3 μg/mL docetaxel followed by different wash-out times. Diagrams show a representative experiment. Owing to a decrease in size and granularity, apoptotic cells progressively accumulate in the lower left area of the diagram, whereas the lower left corner corresponds to cell debris.

Figure 5

Double staining of nuclei (DAPI blue) and mitochondria (JC-1). Left panel: control cells with intact nuclei, where JC-1 aggregates inside healthy mitochondria and fluoresces red (arrows). Right panel: apoptotic HT1376 bladder cancer cells with fragmented nuclei, where the monomeric form of JC-1 diffuses into the cytoplasm and fluoresces green (arrows). Cells in the right panel were treated with docetaxel (1 hour followed by a 96-hour wash-out). The aggregation of JC-1 in the mitochondria is driven by the transmembrane potential. Aggregated extracellular dye deposits (stars) are artifactual.