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.