| Abstract |
In mammalian cell culture, serum deprivation inhibits cell growth and
induces apoptosis. Adapting host cells to serum-free medium is difficult
and time-consuming, therefore anti-apoptosis engineering of mammalian cells
were used to account the problems caused by serum deprivation. The
X-linked inhibitor of apoptosis protein (XIAP) is the most effective member
of the inhibitor of apoptosis protein (IAP) family, where it inhibits
apoptosis by retarding the downstream caspase activity in the apoptosis
chain cascade. In an attempt to investigate the ability of XIAP to delay
serum-deprived-induced-apoptosis, stable CHO-K1 cell lines expressing the
XIAP proteins were established by introducing the XIAP gene into the host
genome. Transfected cells with high expression of XIAP were selected by
conducting the MTT assay and flow cytometric analysis. Selected cell line
was cultured and closely examined under several serum-deprived
conditions. During exposure to serum deprived medium, cells expressing
XIAP showed decreased level of apoptosis and a higher number of viable
cells compared to the control cell lines. The viability of control cells
dropped to 40% after 2 days of serum deprivation, while the XIAP
expressing cells still maintained at a viability over 90%, where caspase-3
activity was found to be inhibited. Meanwhile, the study also showed that
CHO-K1-XIAP cells exhibited a slower growth profile, where most of the
cells were found to be located in the G0/G1 phase. The results suggest
that the over-expression of XIAP induces G0/G1 growth arrest, where
proliferation was retracted, leading to a 50% reduction in maximum cell
density. In the following attempt to investigate the productivity of the
established cell line, the CHO-K1-XIAP cell line was then co-transfected
with cycle-3 green fluorescent protein (GFP), as a model protein to access
the productivity of the cells. Cells were cultured under serum-deprived
condition with addition of sodium butyrate (NaBu) treatment in various
concentrations. NaBu is widely used to boost up the commercial production
of recombinant proteins, but was found to be cytotoxic and causes rapid
apoptotic cell death in mammalian cell cultures. Upon subjecting the cells
to serumdeprived and NaBu-induced condition, the control significantly
declines in cell viability and GFP production. The CHO-K1-GFP expressing
XIAP remained robust and continued to express GFP for the initial 2
days, having a 52% increment of high GFP producers. Moreover, the results
indicated that the intracellular levels of GFP were further increased
while cells became progressively growth-arrested, shifting 33% of the low
GFP producers to high GFP producers. Although XIAP over expression could
not effectively inhibit NaBu-induced apoptosis, however, a 35% reduction
of caspase-3 activation was observed in CHO-K1-XIAP-GFP cells at its
termination point. Taken together, host cells solely expressing a single
type of anti-apoptotic protein are found to be inadequate in regard to
extension of culture longevity and enhancement
of protein production.
Therefore, for a better and more effective apoptosis inhibition, a
combination of caspases inhibitor and mitochondrial dysfunction should
be considered in future studies.
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