rAAV producing cultures treated with nocodazole have an increased proportion of cells in G2/M phase
Nocodazole is an anti-mitotic agent, used as both a chemotherapeutic and as a common agent of cell cycle synchronisation[18],[33]–[35]. Nocodazole exerts its effect by reversibly inhibiting the polymerisation of β-tubulin, destabilising microtubules and preventing the formation of mitotic spindles, thus arresting cells within the G2/M phase of the cell cycle. Cells treated with nocodazole typically enter mitosis but are unable to progress through cytokinesis, leading to either apoptosis in cells remaining in arrested mitosis for an extended period of time, or subsequent “mitotic slippage” into G0/G1 phase followed by apoptosis[35]–[38]. In addition to its use as a cell synchronisation agent, nocodazole has previously been shown to increase transient recombinant protein expression in a mammalian cell system[39].
Flow cytometry was carried out to determine the cell cycle status of rAAV8-producing cultures treated with nocodazole. Untreated rAAV8 producing cells were found to be almost entirely in either G1 or S phase at 72 HPT (Fig. 4A). Cells treated with 4 μM nocodazole at the point of transfection (0 HPT) resulted in a very significant proportion (38%) arresting in G2/M phase (Fig. 3B). The ratio of cells in G2/M : G1 phase was found to decrease the later nocodazole was added (Fig. 4C). Of note is the observation that a significant number (26%) of cells remain arrested in G2/M phase at 72 HPT when nocodazole is added as late as 24 HPT, yet the positive effect on rAAV8 genome titre is substantially lessened compared to earlier treatment. This would suggest a critical temporal component to the addition of nocodazole, such that later additions to cell culture are sub-optimal in terms of producing high rAAV titres, likely due to the rapid assembly process of cap proteins that largely occurred within 24 HPT[31].
The nucleolus is a dynamic compartment within the nucleus which undergoes extensive remodelling during cell cycle progression, particularly during mitosis[40]. Previous immunocytochemical examination of nucleolar localisation throughout normal cell cycle progression shows a distinctive dispersal of nucleolar protein staining throughout the cytoplasm during prometaphase and metaphase – concomitant with the breakdown of the nuclear membrane during mitosis [41]–[43]. Nucleolar proteins in cells in interphase and prophase are typically found located within the nucleus itself. Of note, the nucleolus is considered to be a likely site for AAV capsid assembly and Rep-mediated loading of AAV genome into capsids [44]–[47], as well as being linked more generally to viral replication in other human viruses[48].
Cells from rAAV8 producing cultures - both with and without nocodazole addition 4 HPT - were harvested and fixed at 24 HPT. Cells were stained with a nucleolar marker (fibrillarin - a protein component of the nucleolus associated with ribosomal RNA processing[42]), and DAPI (a nuclear DNA stain). Widefield fluorescent microscopy was used to visualise the phenotypic changes induced by nocodazole addition. Untreated cells displayed highly localised or punctate staining of fibrillarin within the area of nuclear DNA staining (Fig. 4D), indicative of cells in interphase or prophase. In contrast, cells treated with nocodazole exhibited a proportion displaying a disorganised nuclear phenotype - with fibrillarin distributed broadly throughout the cytoplasm, and with condensed nuclear DNA (Fig. 4E) –indicative of cells either progressing through mitosis or arrested within G2/M phase.