Paclitaxel Induces Primary and Postmitotic G1 Arrest in Human Arterial Smooth Muscle Cells

Abstract

Paclitaxel (PTX), a microtubule-active drug, causes mitotic arrest leading to apoptosis in certain tumor cell lines. Here we investigated the effects of PTX on human arterial smooth muscle cell (SMC) cells. In SMC, PTX caused both (a) primary arrest in G 1 and (b) post-mitotic arrest in G 1 . Post-mitotic cells were multinucleated (MN) with either 2C (near-diploid) or 4C (tetraploid) DNA content. At PTX concentrations above12 ng/ml, MN cells had 4C DNA content consistent with the lack of cytokinesis during abortive mitosis. Treatment with 6-12 ng/ml PTX yielded MN cells with 2C DNA content. Finally, 1-6 ng/ml of PTX, the lowest concentrations that affected cell proliferation, caused G 1 arrest without multinucleation. It is important that PTX did not cause apoptosis in SMC. The absence of apoptosis could be explained by mitotic exit and G 1 arrest as well as by low constitutive levels of caspase expression and by p53 and p21 induction. Thus, following transient mitotic arrest, SMC exit mitosis to form MN cells. These post-mitotic cells were subsequently arrested in G 1 but maintained normal elongated morphology and were viable for at least 21 days. We conclude that in SMC PTX causes post-mitotic cell cycle arrest rather than cell death.

Mechanism of G1-like arrest by low concentrations of paclitaxel: next cell cycle p53-dependent arrest with sub G1 DNA content mediated by prolonged mitosis

“…Proteins were resolved with 12% SDS-polyacrylamide gel electrophoresis for detection of p21 and p53, as previously described (Giannakakou et al, 2001;Blagosklonny et al, 2004).…”

“…Although traditionally a 4C DNA peak is called 'G2/M arrest', PTX does not cause G2 arrest. In fact, the G2/M peak consists of both mitotic cells (mitotic arrest) and tetraploid cells, which escape mitotic arrest without division (Andreassen et al, 2001;Giannakakou et al, 2001;Blagosklonny et al, 2004). The exact nature of this G1-like state is not clear.…”

Mitotic Arrest and Cell Fate: Why and How Mitotic Inhibition of Transcription Drives Mutually Exclusive Events

“…21,22 Generally, cells could be either apoptosis-prone (with 'ready-to-go caspase cascades') or apoptosis-reluctant. [23][24][25][26] For example, leukemia cell lines such as Jurkat, HL60 and U937 readily undergo apoptosis. 11,24,27,28 HeLa cells are also relatively apoptosis-prone.…”

“…Such apoptosisreluctant cells exit mitosis without division (mitotic slippage), forming tetraploid MN cells. 17,24,26,[39][40][41] Thus, inhibitors of transcription (actinomycin D and flavopiridol) and mitotic inhibitors (e.g., paclitaxel) cause apoptosis in the same types of cells, implying a common mechanism. As we will see, this is not coincidental.…”

Selective Killing of Adriamycin-Resistant (G2 Checkpoint-Deficient and MRP1-Expressing) Cancer Cells by Docetaxel

“…Cells washed with PBS, pelleted onto glass slides in a cytocentrifuge, fixed with 90% ethanol/10% glacial acetic acid. Nuclei were stained with 1 Ag/mL of 4V ,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Inc., Eugene, OR) in PBS and inspected under UV microscope (Nikon Microphot) as previously described (41,42).…”