Abstract
BackgroundDespite 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.ResultsWithin 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.ConclusionAfter 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 (Δψm) is the earliest sign of apoptosis.
Highlights
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
CeFniegtlluwcrayeschl2e-oduistttrimbuetsion of HT1376 bladder cancer cell line after a 1-hour treatment with 3 μg/mL docetaxel followed by differCell cycle distribution of HT1376 bladder cancer cell line after a 1-hour treatment with 3 μg/mL docetaxel followed by different wash-out times
Despite the considerable amount of information on apoptosis in tumour cell lines acquired from cytometrybased studies, little attention seems to have been paid to the time course of the main events that characterise the apoptotic process, and in particular those induced by taxanes
Summary
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 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. Apoptosis is the most widespread form of cell death, characterised by distinguished biochemical and morphological events triggered by signalling cascades and finely regulated by the products of oncogenes, tumour suppressor genes, and apoptosis-specific genes. Induction is controlled by a wide range of mechanisms that involve either surface (extrinsic) signalling and subsequent signal transduction mechanisms, or internal (intrinsic) pathways leading to the release of mitochondrial components. Both pathways (page number not for citation purposes).
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