Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
433
cell wall regeneration. Immobilizing pro-
toplasts is akin to providing them with an
artifcial cell wall. This concept has mer-
its in that various useFul Functions can
be Freely incorporated in protoplasts by
using artifcial cell walls with such Func-
tions. Production oF Taxol
using
Taxus
protoplasts with artifcial cell walls was
Feasible.
The
optimal
condition
For
isolating
large amounts oF viable protoplasts From
T. cuspidata
cultured cells was investi-
gated. With a combination oF cellulase,
pectin lyase, polygalacturonase, hemicel-
lulase, potassium citric acid buFFer solu-
tion, bovine serum albumin (BSA), and
potassium dextran sulFate and degassing
treatments were Found to be eFFective in
increasing the yield oF protoplasts isolated
From
T. cuspidata
cultured cells. Proto-
plasts yields (3.2–6
.
4
×
10
6
number/g-
Fresh weight cells) were achieved with
specifc culture and cell phases. This con-
dition and these Factors are thought to
be useFul For isolation oF various
Taxus
species protoplasts.
The localization oF Taxol
in cultured
T. cuspidata
cells was investigated using
protoplast isolation reaction. The amount
oF Taxol
released by protoplasts isolation
reaction was considered to be located
in
the
cell
wall
and/or
between
cell
wall
(CW)
and
cell
membrane.
The
amount oF paclitaxel in the protoplasts was
considered to be located in the cytoplasm.
About 30% and 35% oF total Taxol
was
located in CW oF suspension cells in
the growth phase and in the stationary
phase, respectively (Table 1). It was also
Found that about 30% and 43% oF total
Taxol
was located in CW oF the cells
g
rowninso
l
idcu
l
tu
r
ea
tg
row
thph
a
s
e
and at the stationary phase, respectively
(Table 4).
Since some oF the Taxol
is located in
CW and the accumulation oF Taxol
in
the medium was low, it was assumed that
the cell wall limits the excretion oF Taxol
and that the amount oF Taxol
in CW may
aFFect production oF the same compound.
It should be noted that Taxol
production
by
Taxus
cells is controlled by Feedback
inhibition due to the accumulation oF
Taxol
in CW. ThereFore we considered
that the removal oF cell walls would en-
hance Taxol
production and developed
a novel production system For isolated
protoplasts. ±igure 4 shows the Taxol
Tab. 4
Localization of Taxol
in
T. cuspidata
cells (1.0-g fresh weight) in suspension and solid
cultures.
Phase
Taxol
in CW
a
[
µ
g]
Taxol
in cytoplasm [
µ
g]
Ratio
b
[%]
Suspension culture
Growth phase (15 days)
6
.
6
±
0
.
81
5
.
0
±
1
.
43
0
.
5
±
1
.
9
Stationary phase (22 days)
11
.
7
±
0
.
82
1
.
6
±
1
.
63
5
.
1
±
1
.
0
Solid culture
Growth phase (20 days)
9
.
9
±
0
.
92
3
.
0
±
2
.
23
0
.
1
±
1
.
3
Stationary phase (35 days)
22
.
8
±
0
.
83
0
.
0
±
1
.
84
3
.
2
±
1
.
4
a
CW; cell wall and/or between cell wall and cell membrane.
b
Ratio; [Taxol
in CW/(Taxol
in CW
+
Taxol
in cytoplasm)]
×
100.
Numbers represent mean value
±
S.E.
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