Vol.
170,
July
31,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
851-859
PHENOTYPIC REVERSION OF CISPLATIN RESISTANCE IN HUMAN CELLS ACCOMPANIES REDUCED HOST CELL REACTIVATION OF DAMAGED PLASMID 'Chuck C .-K.
Chaol,
Y.-L.
Leel*
and Sue Lin-Chao2
lTumor Biology Laboratory, Departments Medicine, Chang Gung Medical College, 21nstitute
Received
of
June
Molecular
18,
Biology, Republic
of Biochemistry Taoyuan, Taiwan
Academia of China
Sinica,
and 33332
Taipei
11529,
1990
Revertant cell lines were established from cisplatin (CP) resistant HeLa cells. Expression of CP damaged plasmid DNA carrying bacterial chloramphenicol acetyltransferase (CAT) gene after transfection into cells was measured. Revertant cells showed reduced host cell reactivation of damaged plasmid, as compared to resistant cells. Addition of aphidicolin, an inhibitor for DNA polymerase alpha, to resistant cells effectively blocked enhanced plasmid reactivation and acquired resistance. The results are consistent with the notion that cellular ability in repair CP-damaged DNA is a mechanism for CP resistance. ml990 Academic mess, Inc.
SUMMARY:
Cisplatin (CP) is variety of human cancers efficiently
to
resistant
(CPR)
limitation
review, It plasmid *Address University
damage
cancer
to
Substantial decreased intracellular [14-151,
CP
cancer
lines of drug uptake level increased 41.
These
has
been
carrying from
Abbreviations: chloramphenicol
of
a potent drug partly because than
their
normal
cells,
therefore,
treatment
[for
evidence and/or
results shown
that
August 1990: California,
multiple
transient
bacterium
CPR,
that
851
a
CPR
major l-41.
cells show increased
[5-111, metallothioneins removal [recent
mechanisms expression
of Biomedical CA 94720. cisplatin
CP
reviews,
chloramphenicol
Department Berkeley,
CP, cisplatin; acetyltransferase.
recent
[12-131 or and adduct
suggest
of a less
counterparts. constitute
indicate accumulation
of glutathione DNA crosslinks
the
in the treatment cancer cells respond
in of
CPR.
W-damaged
acetyltransferase Sciences, resistance;
CAT,
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
2,
(CAT) gene DNA repair CP damage advantage lesion
is
1990
in human [16-181.
known of
be
CPR in
cells.
when
the human
RESEARCH
with
cells.
Enhanced
represents
role
of
host
DNA
of
the
cell
repair
these features support the in
this
DNA repair
DNA
membrane
repair will
damage
or
observed we attempted
from to
development
of
the
of
detected
in
be lost in DNA repair
mammalian
cell
system
reactivation was
of
in
host
the
assay
in
appear to notion that
development
the
on
cytoplasm that are usually [4]. Using this system, role
In contrast, The results
cells
counted
effects
ability
applied to demonstrate cells [19-221. A major that the level of DNA
addition,
the
COMMUNICATIONS
cellular
into is
In
potential
which
MATERIALS
associated
plasmid
by
in the treatment
significant
BIOPHYSICAL
introduced
cells.
complicated
estimate
is
damaged
tested
alterations traditional
plasmid cells.
and
of
machinery
cells
AND
This system was later and repair in mouse and human of the modified CAT assay is
reactivation not
BIOCHEMICAL
damaged HeLa-CPR revertant plays
a
CPR.
AND METHODS
Dulbecco modified Eagle's medium Chemicals and Medium. and penicillin/streptomycin bovine serum (FBS), (DMW , fetal MD. Platiamine (i.e., were obtained from GIBCO, Gaithersburg, Farmitalia Carloerba Ltd.. cisplatin) was purchased from Aphidicolin and SDS etc. were all purchased from Sigma, St. Louis, MO. [14C]chloramphenicol (Cm) (54 mCi/mmol) was obtained from Amersham, Arlington Heights, Illinois. Selection Revertant Lines and Cell Culture Cell of Conditions. Revertant cells were established by the relief of CP from HeLa cultures that had been selected for surviving 2 p CP for 2 months. CPR cells were established by intermittent exposure of HeLa cells to CP concentration up to 8 pM (see Fig. 1 and below for the determination of drug resistance). Cells were maintained in monolayer culture in DMEM containing 10% FBS, 100 pg/ml streptomycin, 100 units/ml penicillin, with or without CP, and incubated at 37 OC in a humidified atmosphere of 5% CO2 in air. Determination of DrUq Resistance. The fold resistance of cells to CP is defined as the ratio of CP concentration inhibiting colony formation ability by 50% (IC5D) of resistant cells to that of parental cells. Cytotoxicity was quickly measured by the quick MTT colormetric method [23] or assayed by conventional colony forming ability analysis. cp modification of pRSVcat DNA. One 100 pg/ml (3 x 10m4 M nucleotide phosphate) of pRSVcat DNA was treated in the dark with CP in 3 mM NaCl, 1 mM Na phosphate (pH 7.4), at 37OC for 18-24 h [24]. After treatment, NaCl was added to 0.1 M, and the modified DNA was precipitated by standard method [25], washed once in ethanol/O.4 M Na acetate, pH 5.2 (2.5 : 1, v/v) and once in 80% (v/v) ethanol to remove free CP, resuspended in TE buffer (10 mM 852
Vol.
170,
No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Tris-HCl, pH 7.5, 1 mM EDTA), and used for transfection. The molar ratio of free CP to nucleotide phosphate at equilibrium, determines the molar ratio of bound CP to nucleotide r'fr phosphate at equilibrium, rb. Bound CP was calculated from direct measurement by atomic absorption spectroscopy, that reaches the steady-state level, rb = 0.8 rf after 18-24 h [24]. CP bound DNA can also be measured by indirect methods: an electrophoretic mobility shift assay and BamHI cleavage [24]. We monitored CP binding by both mobility shift of CP modified DNA and the loss of sensitivity to BamHI cleavage. DNA-mediated Gene Transfer. A modification of Chu et al. [26] was used to introduce plasmid DNA containing CAT gene into cells. Briefly, cells were seeded at 3 x lo6 cells per loo mm plate one day before eletroporation. A 1 ml cell suspension in Hepes buffer was added to sterile cuvette containing 20 pg pRSVcat plasmid, gently mixed, and subjected to electroporation by GenePulser (BioRad). Conditions with 1000 pF capacity, 250 voltage were routinely used. After 10 min resting at 25 oc following transfection, the cells were returned to the incubator. The following day the cells were fed with fresh medium and incubated for 48 h to allow expression of transfected DNA. The cells were finally harvested into 1 ml phosphate buffered saline and the cell pellet either stored at -800C or (PBS), centrifuged processed for assay of CAT activity. Modified CAT Assav. The assay of CAT enzymatic activity was essentially carried out as Gorman et al. [27-281 with a slight modification. The cell pellet was resuspended in 0.1 ml 0.25 M Tris-HCl, pH7.5 and subjected to sonication at 4OC (120 w, 2 and centrifuged in a microfuge at 4OC for 10 min. min), The supernatant containing the CAT activity was extracted twice into 150 pl ethyl acetate, evaporated to dryness under vacuum, and subsequently resuspended in 20 ul ethyl acetate. The assay for CAT was contained in a total volume of 150 pl: 116.5 pl H20, 25 pl 1 M Tris-HCl, pH7.5, 2 pl [14C]Cm (0.05 pCi), 1.5 pl 40 mM acetyl CloA, and 5 pl cell extract. After incubation at 37OC for 1 derivatives were developed on silica TLC plate h, Cm and its (Macherey-Nagel, Germany). Material corresponding to Cm substracts or products was cut out and the radioactivity counted Activity is calculated as per cent of in totuene/PPO scintillant. [14C]Cm converted into acetylated derivatives.
RESULTS Establishment selection HeLa-CPR
of pressure. cells by
and Methods analyzed by dose-response
revertants
from
CPR
Revertant cells removal of selection
for details). conventional curves
cells
shown
in
removal
of were established from pressure (see Materials
Sensitivity of these colony forming ability are
w
Fig.
cells to CP was assay. Typical 1.
drug I%or concentration causing 50% killing, is 7 x 10m8 M for revertants and parental cells and 2 x low6 M for CPR cells. There is a 30-fold resistance disappearance, as calculated from relative IC50 for revertant cells (see Materials and Methods for details). 853
Vol.
170,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
CP,
RESEARCH
COMMUNICATIONS
M
Drug concentration-viability curves fQr parental and Fisure 1. resistant HeLa cells by colony counting. Vertical bars represent variations from three duplicates for each treatment.
Enhanced
host
pRSVcat introduced expression, representative
cell
reactivation
of
in CPR cells. -plasmids were treated with controlled level of CP and cells by electroporation. Allowing transient into the cell lysates were prepared for CAT assays. A autoradiography
of
CAT activity was reflected figure legends), representing absence
of
CP damage,
there
plasmid
(lanes 4). significant while
nearly
effectively At higher levels of no
CAT
is in
slightly
the
CP concentration, CAT activity were was
(compare lanes 5 in Fig. 2a panel of relative CAT activity (see
is shown migrating
in Fig. spots
chloramphenicol. higher
parental HeLa of CP damage
inhibited
activity
nlasmid
CAT assay fast
from the acetylated
activity in CPR cells than 2 in Fig: 2a). Introduction into
damaued
cells (0.004
CAT
measured
A and B). Materials
in
In
level
of
the CAT
(compare lanes CP/phosphate)
activity
0.008 detected
2a. (see
in
cells
CP/phosphate, in CPR cells, parental
Dose-response
cells curves
and Methods) are presented in Figure 2b. A more sensitive range of dose effect was performed and the calculated results are shown in Fig. 3. IC50, CP concentration inhibiting CAT activity by 50%, for CPR cells is 0.0018 cells. in
CP/phosphate, The results
compared to indicate a 2-3
CPR cells. 854
0.0007 CP/phosphate for HeLa fold increase of CAT activity
Vol.
a
170,
1
No.
2
2,
1990
BIOCHEMICAL
4
3
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
5
b
A Hela
I
A 0 l
Hela-CPR Hela-rev I Hela-rev2
D
Molar
ratio,cP/Phosphate
Dose response curves of relative CAT activity in cisplatin resistant (B), and revertants (C-D) HeLa Fig. 2a shows CAT assay reactions developed on silica TLC lane 1, mock treatment; lane 2, control pRSVcat; lanes 3-5, pRSVcat at CP damage 0.0008, 0.004 and 0.008 CP/phosphate, respectively. Fig. 2b shows relative CAT activity calculated from 2a (see Materials and Methods). The estimated error due to fig. uncertainties in transfection and the CAT assay was about 15% as ascertained from five repeated experiments. Fisure parental cells. paltes:
2.
(A),
Loss of enhanced host cell reactivation of damaqed Dlasmid in CPR revertants. Modified CAT assays were also performed in revertants derived from CPR cells. No detectable CAT activity was seen in two independent revertants transfected with plasmid at CP damage level 0.004 CP/phosphate panels C and D). The relative CAT activity of revertant 1
or greater CAT activity is comparable
855
(lanes 4-5 in Fig. 2a is shown in Fig. 2b. to that of parental
Vol.
170,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
‘-0 0
EeLa-CPR
0
BaLa-CPR
A
HeLa
A
HeLa
+ aphidicolin
CT H~LII-I‘w~
0
Fisure 3. The effect cisplatin resistant was included in the estimated error due assay was about experiments.
HeLa
cells,
indicating
+ aphidicolin
0.002
0.001
Molar
+ aphidicolin
ratio,cP
of aphidicolin and revertant culture medium
0.004 /phosphate
on relative CAT activity cells. Ten j.~g/ml aphidicolin 2 h before drug treament.
was ranges
The
to uncertainties in transfection and the CAT 20% as ascertained from three repeated
that enhanced CPR cells was
host lost
cell in
reactivation this revertant.
damaged plasmid in Revertant 2 showed a more severe loss of CAT activity, at low level CP damage (i.e., 0.008 CP/phosphate). difference sensitive
in
not seen when studies (data not shown).
were
of
especially However, this
performed
at
more
Anhidicolin effectively blocked enhanced host reactivation -cell of damaced olasmid and CPR phenotype. Aphidicolin, a potent inhibitor for DNA polymerase alpha [29] and DNA repair [30-311, was used in the studies of DNA repair. It should be noted that 10 )Ig/ml
has
aphidicolin little cytotoxic
added to effect
the cultures for modified CAT assays on cells (see below). Typical results 856
Vol.
170,
No.
2,
1990
BIOCHEMICAL
Table
AND
1. Drug Resistance
BIOPHYSICAL
RESEARCH
and Revert-ant
Cell
Cisplatin Cell
Lines
uv
Relative resistance
1cs.o M
line
COMMUNICATIONS
I%g
Relative resistance
J/m
HeLa
7 x 10-8
1.0
13
1.0
HeLa-CPR
2 x 10-6
28.6
25
1.9
HeLa-rev1
7 x 10-8
1.0
15
1.2
IC50 or IF50 is the drug concentration or W fluence that caused a 50% inhibition of cell growth. Measurement of the IC50 or IF50 had a 15% estimated error.
are
shown
in
Fig. 3. IC5O's for CAT activity, are shown in Table 1. In relation a 1.3-fold increase of CAT activity
aphidicolin, cells, only cells after parentheses inhibited
of
addition Table
host
cytotoxicity
cell are
A slight
detected
in
cells,
large
cell
in
and by
4 x that
reactivation
damaged
the
table of
revertant of
or
acquired
two The
2 x 10m6 M for cells cells with aphidicolin.
aphidicolin
is
of
damaged
plasmid
CPR was inhibitor
resistance
effective
for
right-hand on
a IC50 of 1O-7 M for
an
in
IC5O's
aphidicolin cells.
HeLa in CPR
indicated effectively
plasmid. (the
without
parental remains
(numbers aphidicolin
of effect
proportion
reflected
aphidicolin and results indicate host
shown
inhibitory
parental a
as
reactivation also
columns). eradicated
of aphidicolin indicating that
l),
with to
in
CPR
without The
inhibitor
for
and CPR.
DISCUSSIQN
Using HeLa-CPR
a
modified
cells
acquired
damaged plasmid. lose this feature.
In
of It
an
assay,
in
we
have
enhanced host CPR revertant
contrast, Expression of was completely
0.002 CP/phosphate significant CAT activity Revertants same treatment. CAT activity, level of reactiva.tion phenotype.
CAT
cell
CAT plasmids blocked in
demonstrated reactivation cells appeared at HeLa
damage cells,
that of to level while
CPR cells was measured following the derived from CPR cells showed parental enhanced host cell suggesting that
damaged plasmid might be responsible is consistant with the idea that 857
for the tolerance
CPR of
Vol.
170,
No.
2,
mammalian
BIOCHEMICAL
1990
cells
cellular
to
ability
[19] have also sensitivity to that acquired enhanced
repair.
reactivation However,
of when
eradicated, completely,
the
are
Using
agents
supported
same
CP
concentration
acquired in
(unpublished not detected CPR in reversion repair. pathway
to
the
the
multidrug
of
(see
development
In
mRNA addition,
data). in CPR cells.
or
In
Similar, can be
but not facilitated
These
studies of
an
Experiments
looking
CP-DNA adducts
are
in
alteration in
is reversible. by the loss of
provide
of
useful
currently
DNA repair. the studies
proteins
in
is
cells.
The
in
of
steady
CPR
cells
minutes were associated Phenotypic enhanced DNA
information
chemotherapeutic
nuclear
It
the reversion CPR cells with for
strategy
for
CPR and revertant the mechanism of
mutated specific proteins this defensive process are for
1).
not
due to overexpression of not likely to be involved
necessary identical, by the treatment
effective
CPR cells. completely
mechansim(s)
chromosomal double conclusion, DNA-repair
cells featuring enhanced will also be valuable for
DNA repair, presumably normally involved in
CPR
cell
but
Table
P-glycoprotein
should
using
host
other
of
resistance,
a1
development
cancer cells
to Berg
significantly,
DNA repair
human cell culture model of this type is paralleled
aphidicolin.
related
Chu and
studies
enhanced
resistance
addition in
by
P-glycoprotein (a drug efflux pump), in the CPR because there was no detectable level
is
and CPR phenotype in CAT activity was
is
state
COMMUNICATIONS
CAT assay,
inhibited
damaged plasmid the enhanced
involved for
is
effectively
that
mechanism
[4].
This
blocked
also
RESEARCH
DNA-damaging
DNA repair
which
hypothesized
other
BIOPHYSICAL
shown that DNA repair plays a role in cellular CP. Our results along with others strongly suggest CPR observed in CPR cells is a conseguense of
DNA
aphidicolin,
CP and
in
AND
which
which are measurable. interact
with
progress.
ACXNOWLEDGMENTS This work was supported in part by research grants from Chang Gung Memorial Hospital (CMRP256) and National Science Council, R.O.C. (NSC79-0412-B182-50). We thank Judith Perry for assistance in manuscript preparation, P.-W. Cheng and W.-C. Yam for technical help, and Dr. Delon Wu for encouragement.
REFERENCES
1.
Loehrer, 704-713.
P.
and Einhorn,
L.H.
858
(1984)
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Intern.
Med.
100,
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13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cancer (Phila.), 55, 2303-2316. Rosenberg, B. (1985) Eastman, A. and Richon, V.M. (1986) In Biochemical Mechanisms of the Platinum Antitumor Drugs (D.C.H. McBrien and T.F. Slater, eds.), pp. 91-119. IRL Press, Oxford, England. Hospers, G.A.P., Mulder, N-H., and De Vries, E.G.E. (1988) Med. Oncol. & Tumor Pharmacother. 5, 145-151. Eicholtz-Wirth, H. and Hietal, B. (1986) Br. J. Cancer 54, 239-243. Hromas, R-A., North, J.A. and Burns, C.P. (1986) Proc. Am. Assoc. Cancer Res. 27, 263. Kraker, A.J., Steinkampf, R.W. and Moore, C.W. (1986) Proc. Am. Assoc. Cancer Res. 27, 286. Richon, V-M., Schulte, N. and Eastman, A. (1987) Cancer Res. 47, 2056-2061. Teicher, B.A., Holden, S.A., Kelley, M.J., Shea, T.C., Cucchi, C-A., Rosowsky, A., Henner, W.D. and Frei III, E. (1987) Cancer Res. 47, 388-393. Waud, W.R. (1987) Cancer Res. 47, 6549-6555. Andrews, P.A., Velury, S., Mann, S.C. and Howell, S.B. (1988) Cancer Res. 48, 68-73. Hamilton, T.C., Winker, M.A., Louie, K.G., Batist, G., Behrens, B.C., Tsuruo, T., Grotzinger, K.R., McKay, W.M., Young, R.C., and Ozols, R.F. (1985) Biochem. Pharmacol. 34, 2583-2586. Bakka, A., Endresen, L., Johnson, A.B.S., Edminson, P.D. and Rugstad, H.E. (1981) Toxicol. Appl. Pharmacol. 61, 215-226. Hromas, R.A., Andrew, P.A., Murphy, M.P. and Burns, C.P. (1987) Cancer Lett. 34, 9-13. Andrews, P.A., Murphy, M.P. and Howell, S.B. (1987) Cancer Chemother. Pharmocol. 19, 149-154. Lehmann, A.R. and Oomen, A. (1985) Nucleic Acids res. 13, 2087-2095. Protic-Sabljic, M. and Kraemer, K.H. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 6622-6626. Klockler, H., Schneider, R., Burtscher, H.J., Auer, B., Hirsch-Kauffmann, M. and Schweiger, M. (1985) Eur. J. Cell Biol. 39, 346-351. Chu, G., and Berg, P. (1987) Mol. Biol. Med. 4, 277-290. Knox, R-J., Lydall, D.A., Friedlos, F., Basham, C. and Roberts, J.J. (1987) Biochim. Biophys. Acta 908, 214-223. Sheibani, N., Jennerwein, M.M. and Eastman, A. (1989) Biochemistry 28, 3120-3124. Chao, C.C.-K., Lee, Y.-L., Cheng, P.-W. ani: Lin-Chao, S. (1989) J. Cell Biochem. 13D, 51. Mosmann, T. (1983) J. Immunol. Meth. 65, 55-63. Ushay, H.M., Tullius, T.D. and Lippard, S.J. (1981) Biochemistry 20, 3744-3748. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory. Chu, G., Hayakawa, H. and Berg, P. (1987) Nucl. Acids Res. 15, 1311-1326. Gorman, C.M., Merino, G.T., Williagham, M.C., Pastan, I. and Howard, B.H. (1982a) Proc. Natl. Acad. Sci. USA. 79, 6777-6781. Moffat, L.F. and Howard, B.H. (198233) Mol. Cell Gorma.n, C.M., Biol. 2: 1044-1051. Ikegami, S., Taguchi, T. and Ohashi, M. (1978) Nature 275, 458-460. Berger, N.A., Kurohara, K.K., Petzold, S.J. and Sikorski, G.W. (1979) Biochem. Biophy. Res. Commun. 89, 218-225. Yamada, K., Hanaoka, F. and Yamada, M. (1985) J. Biol. Chem. 260, 10412-10417. 859
170,
July
31,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
851-859
PHENOTYPIC REVERSION OF CISPLATIN RESISTANCE IN HUMAN CELLS ACCOMPANIES REDUCED HOST CELL REACTIVATION OF DAMAGED PLASMID 'Chuck C .-K.
Chaol,
Y.-L.
Leel*
and Sue Lin-Chao2
lTumor Biology Laboratory, Departments Medicine, Chang Gung Medical College, 21nstitute
Received
of
June
Molecular
18,
Biology, Republic
of Biochemistry Taoyuan, Taiwan
Academia of China
Sinica,
and 33332
Taipei
11529,
1990
Revertant cell lines were established from cisplatin (CP) resistant HeLa cells. Expression of CP damaged plasmid DNA carrying bacterial chloramphenicol acetyltransferase (CAT) gene after transfection into cells was measured. Revertant cells showed reduced host cell reactivation of damaged plasmid, as compared to resistant cells. Addition of aphidicolin, an inhibitor for DNA polymerase alpha, to resistant cells effectively blocked enhanced plasmid reactivation and acquired resistance. The results are consistent with the notion that cellular ability in repair CP-damaged DNA is a mechanism for CP resistance. ml990 Academic mess, Inc.
SUMMARY:
Cisplatin (CP) is variety of human cancers efficiently
to
resistant
(CPR)
limitation
review, It plasmid *Address University
damage
cancer
to
Substantial decreased intracellular [14-151,
CP
cancer
lines of drug uptake level increased 41.
These
has
been
carrying from
Abbreviations: chloramphenicol
of
a potent drug partly because than
their
normal
cells,
therefore,
treatment
[for
evidence and/or
results shown
that
August 1990: California,
multiple
transient
bacterium
CPR,
that
851
a
CPR
major l-41.
cells show increased
[5-111, metallothioneins removal [recent
mechanisms expression
of Biomedical CA 94720. cisplatin
CP
reviews,
chloramphenicol
Department Berkeley,
CP, cisplatin; acetyltransferase.
recent
[12-131 or and adduct
suggest
of a less
counterparts. constitute
indicate accumulation
of glutathione DNA crosslinks
the
in the treatment cancer cells respond
in of
CPR.
W-damaged
acetyltransferase Sciences, resistance;
CAT,
0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
2,
(CAT) gene DNA repair CP damage advantage lesion
is
1990
in human [16-181.
known of
be
CPR in
cells.
when
the human
RESEARCH
with
cells.
Enhanced
represents
role
of
host
DNA
of
the
cell
repair
these features support the in
this
DNA repair
DNA
membrane
repair will
damage
or
observed we attempted
from to
development
of
the
of
detected
in
be lost in DNA repair
mammalian
cell
system
reactivation was
of
in
host
the
assay
in
appear to notion that
development
the
on
cytoplasm that are usually [4]. Using this system, role
In contrast, The results
cells
counted
effects
ability
applied to demonstrate cells [19-221. A major that the level of DNA
addition,
the
COMMUNICATIONS
cellular
into is
In
potential
which
MATERIALS
associated
plasmid
by
in the treatment
significant
BIOPHYSICAL
introduced
cells.
complicated
estimate
is
damaged
tested
alterations traditional
plasmid cells.
and
of
machinery
cells
AND
This system was later and repair in mouse and human of the modified CAT assay is
reactivation not
BIOCHEMICAL
damaged HeLa-CPR revertant plays
a
CPR.
AND METHODS
Dulbecco modified Eagle's medium Chemicals and Medium. and penicillin/streptomycin bovine serum (FBS), (DMW , fetal MD. Platiamine (i.e., were obtained from GIBCO, Gaithersburg, Farmitalia Carloerba Ltd.. cisplatin) was purchased from Aphidicolin and SDS etc. were all purchased from Sigma, St. Louis, MO. [14C]chloramphenicol (Cm) (54 mCi/mmol) was obtained from Amersham, Arlington Heights, Illinois. Selection Revertant Lines and Cell Culture Cell of Conditions. Revertant cells were established by the relief of CP from HeLa cultures that had been selected for surviving 2 p CP for 2 months. CPR cells were established by intermittent exposure of HeLa cells to CP concentration up to 8 pM (see Fig. 1 and below for the determination of drug resistance). Cells were maintained in monolayer culture in DMEM containing 10% FBS, 100 pg/ml streptomycin, 100 units/ml penicillin, with or without CP, and incubated at 37 OC in a humidified atmosphere of 5% CO2 in air. Determination of DrUq Resistance. The fold resistance of cells to CP is defined as the ratio of CP concentration inhibiting colony formation ability by 50% (IC5D) of resistant cells to that of parental cells. Cytotoxicity was quickly measured by the quick MTT colormetric method [23] or assayed by conventional colony forming ability analysis. cp modification of pRSVcat DNA. One 100 pg/ml (3 x 10m4 M nucleotide phosphate) of pRSVcat DNA was treated in the dark with CP in 3 mM NaCl, 1 mM Na phosphate (pH 7.4), at 37OC for 18-24 h [24]. After treatment, NaCl was added to 0.1 M, and the modified DNA was precipitated by standard method [25], washed once in ethanol/O.4 M Na acetate, pH 5.2 (2.5 : 1, v/v) and once in 80% (v/v) ethanol to remove free CP, resuspended in TE buffer (10 mM 852
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BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Tris-HCl, pH 7.5, 1 mM EDTA), and used for transfection. The molar ratio of free CP to nucleotide phosphate at equilibrium, determines the molar ratio of bound CP to nucleotide r'fr phosphate at equilibrium, rb. Bound CP was calculated from direct measurement by atomic absorption spectroscopy, that reaches the steady-state level, rb = 0.8 rf after 18-24 h [24]. CP bound DNA can also be measured by indirect methods: an electrophoretic mobility shift assay and BamHI cleavage [24]. We monitored CP binding by both mobility shift of CP modified DNA and the loss of sensitivity to BamHI cleavage. DNA-mediated Gene Transfer. A modification of Chu et al. [26] was used to introduce plasmid DNA containing CAT gene into cells. Briefly, cells were seeded at 3 x lo6 cells per loo mm plate one day before eletroporation. A 1 ml cell suspension in Hepes buffer was added to sterile cuvette containing 20 pg pRSVcat plasmid, gently mixed, and subjected to electroporation by GenePulser (BioRad). Conditions with 1000 pF capacity, 250 voltage were routinely used. After 10 min resting at 25 oc following transfection, the cells were returned to the incubator. The following day the cells were fed with fresh medium and incubated for 48 h to allow expression of transfected DNA. The cells were finally harvested into 1 ml phosphate buffered saline and the cell pellet either stored at -800C or (PBS), centrifuged processed for assay of CAT activity. Modified CAT Assav. The assay of CAT enzymatic activity was essentially carried out as Gorman et al. [27-281 with a slight modification. The cell pellet was resuspended in 0.1 ml 0.25 M Tris-HCl, pH7.5 and subjected to sonication at 4OC (120 w, 2 and centrifuged in a microfuge at 4OC for 10 min. min), The supernatant containing the CAT activity was extracted twice into 150 pl ethyl acetate, evaporated to dryness under vacuum, and subsequently resuspended in 20 ul ethyl acetate. The assay for CAT was contained in a total volume of 150 pl: 116.5 pl H20, 25 pl 1 M Tris-HCl, pH7.5, 2 pl [14C]Cm (0.05 pCi), 1.5 pl 40 mM acetyl CloA, and 5 pl cell extract. After incubation at 37OC for 1 derivatives were developed on silica TLC plate h, Cm and its (Macherey-Nagel, Germany). Material corresponding to Cm substracts or products was cut out and the radioactivity counted Activity is calculated as per cent of in totuene/PPO scintillant. [14C]Cm converted into acetylated derivatives.
RESULTS Establishment selection HeLa-CPR
of pressure. cells by
and Methods analyzed by dose-response
revertants
from
CPR
Revertant cells removal of selection
for details). conventional curves
cells
shown
in
removal
of were established from pressure (see Materials
Sensitivity of these colony forming ability are
w
Fig.
cells to CP was assay. Typical 1.
drug I%or concentration causing 50% killing, is 7 x 10m8 M for revertants and parental cells and 2 x low6 M for CPR cells. There is a 30-fold resistance disappearance, as calculated from relative IC50 for revertant cells (see Materials and Methods for details). 853
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BIOCHEMICAL
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CP,
RESEARCH
COMMUNICATIONS
M
Drug concentration-viability curves fQr parental and Fisure 1. resistant HeLa cells by colony counting. Vertical bars represent variations from three duplicates for each treatment.
Enhanced
host
pRSVcat introduced expression, representative
cell
reactivation
of
in CPR cells. -plasmids were treated with controlled level of CP and cells by electroporation. Allowing transient into the cell lysates were prepared for CAT assays. A autoradiography
of
CAT activity was reflected figure legends), representing absence
of
CP damage,
there
plasmid
(lanes 4). significant while
nearly
effectively At higher levels of no
CAT
is in
slightly
the
CP concentration, CAT activity were was
(compare lanes 5 in Fig. 2a panel of relative CAT activity (see
is shown migrating
in Fig. spots
chloramphenicol. higher
parental HeLa of CP damage
inhibited
activity
nlasmid
CAT assay fast
from the acetylated
activity in CPR cells than 2 in Fig: 2a). Introduction into
damaued
cells (0.004
CAT
measured
A and B). Materials
in
In
level
of
the CAT
(compare lanes CP/phosphate)
activity
0.008 detected
2a. (see
in
cells
CP/phosphate, in CPR cells, parental
Dose-response
cells curves
and Methods) are presented in Figure 2b. A more sensitive range of dose effect was performed and the calculated results are shown in Fig. 3. IC50, CP concentration inhibiting CAT activity by 50%, for CPR cells is 0.0018 cells. in
CP/phosphate, The results
compared to indicate a 2-3
CPR cells. 854
0.0007 CP/phosphate for HeLa fold increase of CAT activity
Vol.
a
170,
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2
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1990
BIOCHEMICAL
4
3
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
5
b
A Hela
I
A 0 l
Hela-CPR Hela-rev I Hela-rev2
D
Molar
ratio,cP/Phosphate
Dose response curves of relative CAT activity in cisplatin resistant (B), and revertants (C-D) HeLa Fig. 2a shows CAT assay reactions developed on silica TLC lane 1, mock treatment; lane 2, control pRSVcat; lanes 3-5, pRSVcat at CP damage 0.0008, 0.004 and 0.008 CP/phosphate, respectively. Fig. 2b shows relative CAT activity calculated from 2a (see Materials and Methods). The estimated error due to fig. uncertainties in transfection and the CAT assay was about 15% as ascertained from five repeated experiments. Fisure parental cells. paltes:
2.
(A),
Loss of enhanced host cell reactivation of damaqed Dlasmid in CPR revertants. Modified CAT assays were also performed in revertants derived from CPR cells. No detectable CAT activity was seen in two independent revertants transfected with plasmid at CP damage level 0.004 CP/phosphate panels C and D). The relative CAT activity of revertant 1
or greater CAT activity is comparable
855
(lanes 4-5 in Fig. 2a is shown in Fig. 2b. to that of parental
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No.
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RESEARCH
COMMUNICATIONS
‘-0 0
EeLa-CPR
0
BaLa-CPR
A
HeLa
A
HeLa
+ aphidicolin
CT H~LII-I‘w~
0
Fisure 3. The effect cisplatin resistant was included in the estimated error due assay was about experiments.
HeLa
cells,
indicating
+ aphidicolin
0.002
0.001
Molar
+ aphidicolin
ratio,cP
of aphidicolin and revertant culture medium
0.004 /phosphate
on relative CAT activity cells. Ten j.~g/ml aphidicolin 2 h before drug treament.
was ranges
The
to uncertainties in transfection and the CAT 20% as ascertained from three repeated
that enhanced CPR cells was
host lost
cell in
reactivation this revertant.
damaged plasmid in Revertant 2 showed a more severe loss of CAT activity, at low level CP damage (i.e., 0.008 CP/phosphate). difference sensitive
in
not seen when studies (data not shown).
were
of
especially However, this
performed
at
more
Anhidicolin effectively blocked enhanced host reactivation -cell of damaced olasmid and CPR phenotype. Aphidicolin, a potent inhibitor for DNA polymerase alpha [29] and DNA repair [30-311, was used in the studies of DNA repair. It should be noted that 10 )Ig/ml
has
aphidicolin little cytotoxic
added to effect
the cultures for modified CAT assays on cells (see below). Typical results 856
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1990
BIOCHEMICAL
Table
AND
1. Drug Resistance
BIOPHYSICAL
RESEARCH
and Revert-ant
Cell
Cisplatin Cell
Lines
uv
Relative resistance
1cs.o M
line
COMMUNICATIONS
I%g
Relative resistance
J/m
HeLa
7 x 10-8
1.0
13
1.0
HeLa-CPR
2 x 10-6
28.6
25
1.9
HeLa-rev1
7 x 10-8
1.0
15
1.2
IC50 or IF50 is the drug concentration or W fluence that caused a 50% inhibition of cell growth. Measurement of the IC50 or IF50 had a 15% estimated error.
are
shown
in
Fig. 3. IC5O's for CAT activity, are shown in Table 1. In relation a 1.3-fold increase of CAT activity
aphidicolin, cells, only cells after parentheses inhibited
of
addition Table
host
cytotoxicity
cell are
A slight
detected
in
cells,
large
cell
in
and by
4 x that
reactivation
damaged
the
table of
revertant of
or
acquired
two The
2 x 10m6 M for cells cells with aphidicolin.
aphidicolin
is
of
damaged
plasmid
CPR was inhibitor
resistance
effective
for
right-hand on
a IC50 of 1O-7 M for
an
in
IC5O's
aphidicolin cells.
HeLa in CPR
indicated effectively
plasmid. (the
without
parental remains
(numbers aphidicolin
of effect
proportion
reflected
aphidicolin and results indicate host
shown
inhibitory
parental a
as
reactivation also
columns). eradicated
of aphidicolin indicating that
l),
with to
in
CPR
without The
inhibitor
for
and CPR.
DISCUSSIQN
Using HeLa-CPR
a
modified
cells
acquired
damaged plasmid. lose this feature.
In
of It
an
assay,
in
we
have
enhanced host CPR revertant
contrast, Expression of was completely
0.002 CP/phosphate significant CAT activity Revertants same treatment. CAT activity, level of reactiva.tion phenotype.
CAT
cell
CAT plasmids blocked in
demonstrated reactivation cells appeared at HeLa
damage cells,
that of to level while
CPR cells was measured following the derived from CPR cells showed parental enhanced host cell suggesting that
damaged plasmid might be responsible is consistant with the idea that 857
for the tolerance
CPR of
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mammalian
BIOCHEMICAL
1990
cells
cellular
to
ability
[19] have also sensitivity to that acquired enhanced
repair.
reactivation However,
of when
eradicated, completely,
the
are
Using
agents
supported
same
CP
concentration
acquired in
(unpublished not detected CPR in reversion repair. pathway
to
the
the
multidrug
of
(see
development
In
mRNA addition,
data). in CPR cells.
or
In
Similar, can be
but not facilitated
These
studies of
an
Experiments
looking
CP-DNA adducts
are
in
alteration in
is reversible. by the loss of
provide
of
useful
currently
DNA repair. the studies
proteins
in
is
cells.
The
in
of
steady
CPR
cells
minutes were associated Phenotypic enhanced DNA
information
chemotherapeutic
nuclear
It
the reversion CPR cells with for
strategy
for
CPR and revertant the mechanism of
mutated specific proteins this defensive process are for
1).
not
due to overexpression of not likely to be involved
necessary identical, by the treatment
effective
CPR cells. completely
mechansim(s)
chromosomal double conclusion, DNA-repair
cells featuring enhanced will also be valuable for
DNA repair, presumably normally involved in
CPR
cell
but
Table
P-glycoprotein
should
using
host
other
of
resistance,
a1
development
cancer cells
to Berg
significantly,
DNA repair
human cell culture model of this type is paralleled
aphidicolin.
related
Chu and
studies
enhanced
resistance
addition in
by
P-glycoprotein (a drug efflux pump), in the CPR because there was no detectable level
is
and CPR phenotype in CAT activity was
is
state
COMMUNICATIONS
CAT assay,
inhibited
damaged plasmid the enhanced
involved for
is
effectively
that
mechanism
[4].
This
blocked
also
RESEARCH
DNA-damaging
DNA repair
which
hypothesized
other
BIOPHYSICAL
shown that DNA repair plays a role in cellular CP. Our results along with others strongly suggest CPR observed in CPR cells is a conseguense of
DNA
aphidicolin,
CP and
in
AND
which
which are measurable. interact
with
progress.
ACXNOWLEDGMENTS This work was supported in part by research grants from Chang Gung Memorial Hospital (CMRP256) and National Science Council, R.O.C. (NSC79-0412-B182-50). We thank Judith Perry for assistance in manuscript preparation, P.-W. Cheng and W.-C. Yam for technical help, and Dr. Delon Wu for encouragement.
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