Download Expression of drug resistance in Chinese hamster ovary (CHO) cells

278s Biochemical Society Transactions ( 1 99 1 ) 19
Expression of drug resistance in Chinese hamster
ovary (CHO) cells following X-ray pre-treatment in
YitLQ.
SIOBHAN MCCLEAN, RICHARD D. H. WHELAN,
LOUISE K. HOSKING AND BRIDGET T. HILL
Laboratory of Cellular Chemotherapy, Imperial Cancer
Research Fund, P.O. Box 123, Lincoln's Inn Fields, London,
WC2A 3PX. U.K.
Drug resistance is a major cause of failure of cancer treatment.
Clinically, it has been observed in patients, both after treatment
with antitumour agents and after radiotherapy. Multidrug
resistance (MDR) is characterised experimentally by the
expression of cross-resistance to a variety of structurally
different drugs following pulsed or continuous exposure to
only one of them. The classic MDR tumour lines overexpress a
cell surface membrane 170kD glycoprotein, P-glycoprotein
(Pgp), and show cross-resistance to Vinca alkaloids, to
epipodophyllotoxins and to anthracyclines. Studies in this
laboratory have shown that exposure of mammalian tumour
cells in vitro to multiple lethal doses of radiation results in the
expression of a drug resistance phenotype (1). A series of Xray -pre-treated lines has also been established from the Chinese
hamster ovary (CHO) line AuxBl by exposing cells
intermittently to 10 fractions of 9 Gray (a radiation dose which
reduced cell survival to 10% ). These lines have been
designated DXR-101 and DXR-1011 (2). The DXR-10 cells
proved resistant to vincristine (VCR), to etoposide and
colchicine (COL), but not to Adriamycin (ADR). These lines
also overexpressed Pgp, consistent with the MDR phenotype.
However, there was no increase in Pgp message or any
detectable gene amplification.
To investigate this resistance further the cytotoxic effects of a
series of five agents of varied structure and mode of action were
studied on these DXR-10 lines. The drugs include Rhizoxin - a
macrocyclic lactone antimitotic agent which has proved effective
against VCR and ADR-resistant P388 leukaemia cells (3);
Ricin, the castor bean toxin, to which the classic CHO line
CHRCS, also derived from AuxBl cells, has previously been
found to be only 10-fold resistant (4); Actinomycin D, a
chromopeptide antibiotic, was tested to investigate potential
sensitivity similar to ADR; Navelbine, a new Nor-Vinca
alkaloid and taxol. a microtubule stabilising agent, with
potentially interesting preliminary clinical activity (5,6).
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Figure 1. Effects of VCR a n d r v i v a l of Xw nre-tre-
RlClN
V INC RlSTlN E
100
'1: :
10
20
30
40
50
ng per ml
60
70
00 0
5
1
8
0.06
13
15
2.5
10
0.05
6
[3.0]
12.51
[1.3]
[1.0]
[0.46]
15
2
13
0.05
9
[3.01
[2.0]
[1.6]
[I.O]
10.691
IC50: Drug concentration which reduces cell survival to SO %
of control values.
RI: Resistance index, derived from full dose response curves
ratio of ICso values.
~~
We have previously reported that in the AuxB 1 cells the level
of drug resistance, which was detectable after only five 9 Gy
X-ray fractions, increased in cells exposed to 10 fractions, but
was not further enhanced in cells exposed to 20 fractions (2).
To determine whether the resistance in the DXR-10 lines is a
consequence of repeated exposure we derived and studied a
subline which had been pre-treated with a single X-ray dose of
30 Gy. Suspensions of AuxBl cells were exposed to a single
dose of 30 Gy with continuous stimng to minimize cell
protection effects and to ensure linear X-ray dosage. After
exposure cell suspensions were pooled and plated directly onto
plastic. Clones were plucked, propagated and designated line
DXR-30. Colony forming assays were prepared in 0.2%
agarose following 24 hour drug exposures. Cells had a colony
forming efficiency of 50 %. Western blotting was carried out
as described previously from purified membrane preparations
(2) using C219, the anti-Pgp mouse monoclonal antibody, and
a [125I]-labelled anti-mouse IgG.
The DXR-101 and DXR-1011cells showed similar patterns of
response to each of the drugs studied, consistent with their
originally reported comparable responses to VCR, COL and
ADR, (2). Both lines proved marginally resistant to Navelbine,
tax01 and Actinomycin D (table 1). Neither line showed any
resistance to Rhizoxin and both lines proved hypersensitive to
ricin (Fig.]).
The DXR-30 clones proved both VCR resistant (2.8 - fold,
Fig. 1) and overexpressed Pgp, as determined by Western
blotting. This indicates that these alterations are not the result
of a repeated "stress - type" challenge to the cells, but a direct
result of the radiation pre-treatment. DXR-30 clones also show
hypersensitivity to ricin (Fig. 1).
In summary. these X-ray pre-treated cells express resistance to
a wide range of drugs of varied structure, confirming that they
are multi-drug resistant. However, their ricin hypersensitivity,
in contrast to the 10 - fold resistance to ricin shown by the
classic CHRCS MDR cell line, adds weight to our proposal that
different mechanisms may be involved in refractory tumour
cells pre-treated with drugs or with X-rays.
1. Bellamy, AS. & Hill B.T. (1984) J Natl. Cancer Inst.,
72, 411 - 417
10
'0
Navelbine
Tax01
ActinomycinD
Rhizoxin
RiCin
10
20
ng per ml
30
40
2. Hill, B.T. et al (1990) J Natl. Cancer Inst., 82, 607612
3. Tsuruo T. et al. (1986) Cancer Res. 46, 381-384
4. Miyamoto, Y. et al. (1990) Cancer Res. SO, 1572- 1575
5 . Fumoleau P. et al. (1990) Roc. Am. Assoc. Cancer
Res. 31, 1232
6. Einzig, A.I. et al. (1990) Proc. Am. Assoc. Cancer
Res. 31, 1114