Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
427
of taxane production and cell growth.
Early experiments with callus and cell
cultures of
T. cuspidata
have shown that
growth and Taxol
yield are generally
inversely proportional. The reason for
this phenomenon, also observed with the
production of other secondary metabolites
by a number of plant species, is not well
understood. Possible explanations include
the lack of expression of key enzyme
and/or transcriptional activator genes in
cells with low differentiation, diversion of
substrates for secondary metabolism into
growth processes, unregulated catabolism,
and lack of end product transport systems
and storage sites.
The analysis of Taxol
yields in dif-
ferent
in vitro
cultures of
T. cuspidata
supports the observation that faster growth
rates and/or lower degrees of differentia-
tion are normally associated with lower
Taxol
amounts. Stem-derived explants of
T. cuspidata
after two months of culture
in B5bPVP displayed an average eight-
fold increase in fresh weight and Taxol
yields of 0
.
020
±
0
.
005% of the extracted
dry weight (equivalent to the yield of stems
of the intact plant). Slow-growing, six-
month-old immobilized cell cultures of
T. cuspidata
grown in B5C2 had variable
amounts of Taxol
,upto0
.
012
±
0
.
007%
of the dry weight (equivalent on average
to the bark of the intact plant), whereas
six-month-old immobilized cell cultures
visually selected for faster growth yielded
0
.
0014
±
0
.
0003% of Taxol
on a dry
weight basis. A two-year-old cell suspen-
sion line of
T. cuspidata
g
rowninB5C2
(dry weight doubling time of 17 days)
yielded 0
.
0014
±
0
.
0005% of Taxol
(dry
weight basis) when cultured as 300-mL
suspensions in 1 L Erlenmeyer flasks.
These yields may be altered by precursor
feeding and various other strategies (see
further sections).
Studies on the kinetics of growth and
Taxol
accumulation in cell suspensions
of
T. cuspidata
(cell line FCL1F) showed
a relatively slow growth rate and con-
±rmed the relationship between growth
and Taxol
yield. The doubling time was
approximately 17 days for fresh weight and
21 days for dry weight. Although relatively
long in contrast to data from herbaceous
species, these doubling times are com-
parable to or even shorter than those
of other woody species that grow slowly
(Table 2). The maximum speci±c growth
rates were 0.040 g
1
(fresh weight) and
0.034 g
1
(dry weight). The accumulation
of Taxol
in the cells was not growth
linked. The highest taxol accumulation val-
ues occurred in cells in stationary phase,
and Taxol
amounts in the cells decreased
with the increase in cell biomass (Table 3).
Other taxanes in yew callus and cell
cultures have been detected by HPLC,
such as baccatin III, 10-deacetyl-baccatin
III, 1-dehydroxybaccatin V, 7-epitaxol, and
cephalomannine in both types of culture
of
T. cuspidata
. Baccatin III yields in two-
month-old callus of this species grown
in B5bPVP were 0
.
043
±
0
.
016% of the
extracted dry weight. A two-year-old cell
line suspension of
T. cuspidata
(FCL1) has
been estimated to yield 0
.
018
±
0
.
010% of
baccatin III (extracted dry weight basis).
Tab. 2
Growth parameters of
T. cuspidata
cell
suspensions in comparison to some other plant
species (shake-flask experiments).
Species
Doubling
time
[days]
Specifc
growth rate
[day
1
]
Taxus cuspidata
20.6
0.034
Brucea javanica
32.0
Callitris drummondi
40.0
Catharanthus roseus
3.0
0.32
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