ICANCERRESEARCH57. I10-I 16, JanuaryI. I997J
Overexpression of DNA Polymerase @J
Sensitizes Mammalian Cells to
2',3'-Deoxycytidine and 3' -Azido-3' .deoxythymidine'
Khalil Bouayadi, Jean-Sébastien Hoffmann, Pascal Fons, Michele Tiraby, Jean-Paul Reynes, and Christophe Cazaux2
Laboratoire de Microbiologic et de Génétique,
UniversitéPaul Sabatier, 118 route de Narbonne, 31062 Toulouse cédex(K. B., P. F., C. C.]; Institut de Pharmacologic et
Biologic Structurale. Centre National de Ia Recherche ScienujIque, UPR 9062, 205 route de Narbonne, 31077 Toulouse cédexfl. S. H.!; and Laboratoire CAYLA, ZI.
Montaudran, 5 rue J. Rodier, 3/400 Toulouse (M. T., J. P. R.J
ABSTRACT
@
nator AZT-MP was also observed (10, 11). In vivo, AZT-MP is
incorporated into cellular DNA (12), and it has been suggested that
polymerase 13may play a role in this process (1 1).
AZT and ddC are antiviral agents currently used in the treatment
of AIDS. These chemotherapeutic drugs target HIV reverse tran
Mammalian
DNA polymerase
@3is a DNA repair enzyme expressed
constitutively at a low level. In vitro, purified DNA polymerase (Pol)
incorporates the nucleotide analogues 2'-3' deoxycytidine (ddC)-triphos
phate and 3'-azido-3'-deoxythymidine (AZT)-triphosphate Into DNA,
causing chain termination. We have tested the possibility ofenhancing the
scriptase
cytotoxicity of these chain terminators
against mammalian cells by in
creasing the level of Pol (I. Chinese hamster ovary AA8 and murine
melanoma B16 cell lines were stably transfected with rat pol fi cDNA
underthe controlof a viral enhancer/promoter.We found that overex
pression of Pol @3
sensitized the cells to ddC and AZT. To confirm the role
of this polymerasein this process,we preparedcell extractsfrom the
control and Pol @3
overexpressing Chinese hamster ovary cell lines and
tested in vitro their capacity to incorporate ddC-triphosphate and AZT
which
incorporates
them
into
HIV
DNA,
terminating
DNA polymerization
(13). In this study, we hypothesized that
overexpression of Pol f3, a cellular target of ddC and AZT, might
render these analogues effective also against tumor cells. To ex
plore this possibility, we stably transfected CHO AA8 and murine
melanoma B 16 cells with rat polf3 cDNA. We found an increased
sensitivity
to ddC
and
AZT
of the
transfected
cells,
and
we
confirmed the involvement of Pol (3 in this process using an in vitro
triphosphate
into DNA. We found that inhibition of DNA replication by
replication assay with cell extracts. To our knowledge, this is the
both chain terminators
was more pronounced
when extracts from pol
first report of a kill or “suicide―
association involving a polymerase
@3-transfectedcells were used, providing a direct evidence of the involve
activity.
ment of Pol @3
in the sensitizationprocess.In addition,we showedthat
The conversion of nucleoside analogues to nucleotide triphosphates
cotransfection
with bacterial or viral thymidine/thymidylate
kinase genes
is
necessary before their incorporation into DNA by DNA poly
enhanced the Pol fl-mediated cytotoxicity of AZT, suggesting that phos.
phorylationandpolymerizationactivitiesmightbecombinedto potentiate merases. Analogues readily enter cells but are often poor substrates
their respective effects. These observations may be useful for improving
for the sequential conversion to the phosphorylated forms by cellular
therapeutic efficiency of DNA chain terminators.
TK and TMK which specifically convert thymidine (14). For instance,
the concentration of AZT-MP is more than 50-fold higher than that of
AZT-TP in human cell lines, suggesting that it is a very poor substrate
INTRODUCTION
for human thymidylate kinase (13). Here, we present data showing
that cells coexpressing both Pol f3 and bacterial or herpes thymidine/
Pol j3@,a single polypeptide of 39 kDa, is one of the five known
thymidylate kinases are more sensitive to AZT than cells expressing
mammalian
DNA polymerases
and is highly conserved among higher
only kinases or polymerase. These data may have particular impor
eukaryotes (I). It is believed to function primarily in the repair of
tance in the perspective of new therapeutic combinations for cancer
damaged DNA (2) but may also have a role in DNA replication (3, 4)
gene therapy.
and in DNA damage processing (5). Pol /3 is expressed at a constant
level throughout the cell cycle (6) and exposure of cells to certain
DNA-damaging agents induces Pol @3
expression (7, 8). Features that
MATERIALS AND METHODS
distinguish Pol @3
from other cellular polymerases are its small size,
the lack of associated exonuclease and endonuclease activities, and its
Materials. All oligonucleotides
were synthesized on a Cyclone Plus
high infidelity in replicating DNA (9).
DNA synthesizer from MilliGenlBiosearch and purified on 20% polyacryl
In vitro, it has been demonstrated that purified Pol (3 efficiently
incorporates
the deoxynucleotide
analogue
ddCMP,
an inhibitor
of
DNA synthesis, with efficiency comparable to that of dCMP when an
oligonucleotide template is used (10). Under similar conditions, but
using a higher amount of enzyme, incorporation of the chain termi
amide
gel. For DNA
purified
calf thymus
charges. This article must therefore be hereby marked advertisement in accordance with
@
18 U.S.C. Section 1734 solely Io indicate this fact.
This research was supported by the Region Midi-Pyrénées
and the Ligue Nationale
Contre Ic Cancer (axe thdrapie génique).
2 To
whom
requests
for
reprints
should
be addressed,
at Institut
de
Pharmacologic
et
de Biologic Structurale, Centre National de Ia Recherche Scientifique UPR A9062, 205
route de Narbonne, 31077 Toulouse cédex,France. Fax: 33-5-61-17-59-94; E-mail:
[email protected].
3 The abbreviations
used are: Pol (3, DNA polymerase
@;
HSV tic, herpes simplex
virus
type-I thymidine kinase gene; TK, thymidine kinase; TMK, thymidylate kinase; Sh, ble
Sh encoded protein conferring resistance to zeocin; ddC, 2'-3' deoxycytidine; AZT,
3'-azido-3'-deoxythymidine;
AZT-MP, AZT monophosphate; AZT-TP, AZT triphos
@
phate; ddCMP, ddC monophosphate; GCV. ganciclovir [(2-amino-1,9-(2-hydroxy-l-hy
droxymethyl)ethoxy)methyl)-6H-purine-6-one]; CHO, Chinese hamster ovary cell; HSV,
herpes simplex virus: HSV-l, herpes simplex virus type I ddCTP, ddC triphosphate.
DNA
ous gifts from Dr. HUbscher
ies against
Received 7/I 5196;accepted 11/1/96.
The costs of publication of this article were defrayed in part by the payment of page
synthesis
assays,
the 60-mer
templates
were hybrid
ized to the 5' 32P-labeled 17-mer primer. Pol 13cDNA harboring plasmid
pRSET was kindly furnished by Dr. Wilson (Galveston, TX). Highly
Pol
@3were
polymerase
(Zurich,
provided
a-primase,
Switzerland)
by Dr. Sweasy
@3,8, and a were gener
and polyclonal
(Yale
antibod
University,
New
Haven, CT). DNA plasmids pUT526& pUT599 (VECT 4991), pZEOSGO,
and pZEOSG1 were obtained from Cayla (Toulouse, France). Analogues
were obtained from Weilcome (AZT), Moravek Biochemicals, Inc. (AZT
TP), Sigma (ddC), and Pharmacia (ddCTP). Restriction and DNA modifi
cation enzymes were obtained from New England Biolabs and Boehringer
Mannheim.
Bacteria and Mammalian Cell Culture. Bacteria were grown in LB broth
complemented
when required with zeocin (Cayla) at 20 p.g/ml. CHO AA8 and
Bl6 BL/6 parental cells were grown in L-glutamine containing a-MEM and
RPMI 1640 (BioWhittaker), respectively, complemented with 10% FCS (Bio
Whittaker), penicillin G/streptomycin sulfate (BioWhittaker), and amphoteri
cm B (Sigma). Media for growth of stably transfected CHO and Bl6 cells were
supplemented by 100 gxg/mland 20 g@g/m1
zeocin (Cayla), respectively.
110
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
CYTOTOXICACTIONOF DNA POLYMERASE@3
Construction
of Expression
Plasmids.
Pol
mm and were disrupted in a Dounce homogenizer. Nuclei were harvested by
@3-overexpressing plasmid
pUTpolIJ was constructed from a pUT687 plasmid4 carrying the thymidine
kinase
gene of Escherichia
coli fused in frame with the bacterial
centrifugation for 10 mm at 3300 X g at 0°C.The nuclei pellet was suspended
in hypotonic buffer containing 350 mtvi NaCI. After 30 mm of extraction at
Sh ble gene
0°C,the nuclear extracts were centrifuged at 15,000 X g for 20 mm. Proteins
from cytosol and nuclear extracts were precipitated by addition of ammonium
conferring resistance to the broad-spectral zeocin xenobiotic of the phleomycin
family (15). The pol @3
gene generated primers TA'VFCCATGOCACTCGTG
GAACFCGCAAAC1TF
and UAAGCTAGCTCACTCCTGTCmGGGC
sulfate, resuspended
TC. Upon restriction enzyme digestion, a NcoI-NheI fragment containing pol
in dialysis
buffer [50 mt@iTris-HC1 (pH 7.5), 1 mM DTT,
100 mt@imono-K glutamic acid, and 10% glycerol], and dialyzed for 2 h at 0°C.
They were used
@3
wasobtainedandthenrecombined
withtheNcoI-AvrIIfragmentof pUT687 Extracts were frozen in liquid nitrogen and stored at —70°C.
to give pUTpolf3(Fig. IA).
The E. coli tmk gene was fused with E. coli tk and Sh ble by using a 4-kb
PCR-generated fragment obtained as already described (16) by amplification
of the acpP-hoIB intergenic region of the Kohara phage AE9G1(17). First, this
tmk-containing PCR product digested with NcoI and MluI (treated with Kle
now enzyme) was inserted into pUT633 (VECT 6331; Cayla) opened by NcoI
and PvuII to give the E. coli tmk::Sh ble harboring pUT832 (Fig. 1B).PUT633
for both Western blotting and in vitro DNA replication assays.
is similar to pUT687 but contains a PvuII site into the genetic link of a
luciferase::Sh-encoding fusion. Second, the MscI-RsrII fragment from the 4-kb
chemiluminescence
PCR-generated fragment containing the E. coli tmk gene was inserted in place
of the Sh ble gene-containing MscI-RsrII fragment of pUT687 to give pUT833
which harbors the E. coli tk::tmk fusion (Fig. 1B). Finally the EcoRV-NotI
fragment from pUT832 was inserted into pUT833 cut with same enzymes to
tures contained 5 ng of labeled primed oligonucleotide
between Bbrpl-SmaI-digested
Sixty @igof cell extracts were electrophoresed
gel. Proteins
phoretically
membrane.
to nitrocellulose
in the gel were transferred
in
electro
Pol (3 was revealed by incubating
the
membrane with purified rabbit anti-PoI (3 polyclonal antibody followed by
incubation with antibody to rabbit IgG conjugated to horseradish peroxidase.
Immobilized
horseradish
peroxidase
activity
was detected
using
enhanced
(Amersham Corp.).
substrates and 3 p@gof
Sh or Pol f3::Shcell extract proteins in reaction buffer J45 mMHEPES-KOH
(pH 7.8), 7 mM MgCl2, 1 mM Dli',
0.4 mM EDTA, 34% glycerol, 50 mM
mono-K glutamic acid, and 18 @xg
of BSAI. The concentrations of the deoxyri
bonucleotides and the amount of ddCTP and AZT-TP used in the reactions are
indicated in the legends of Figs. 1—6.At the end of the reaction, 5 pi of
The pZETkTmk@plasmid carrying polfi and E. coli tk/tmk genes was the
recombination
Blot Analysis.
in Vitro Replication by Cell-free Extracts. Standard 15-pi reaction mix
give pUT834 (Fig. 1B).
result of intermolecular
Western
a 12% SDS-polyacrylamide
stopping
frag
buffer
(90% formamide/0.l%
xylene
cyanol/0.l%
bromphenol
blue/
ments from pUT834 and pZHTk(3DNA (Fig. 1C)carrying pol @3
and the HSV
0.1 mMEDTA) were added. Samples were denatured for 10 mm at 70°Cand
tk::ble
loaded on sequencing gels.
Quantitative Determination
Sh fusion.
EM7-pol
the pZHTk/3
13 fragment
from
plasmid
pUTpolfJ
was constructed
with
primers
by PCR amplifying
a
TFCTCACGTGACCG
Products
GCGCCTAGT and GGGAGCCCAAGGACAGGAGTGAATGAUCGAA
C1TF to create a Bbrpl-BstBI fragment that was inserted into a pZEOSGO
Generated
by Pol
of Protein Overexpression
@l.The autoradiograms
and Replication
were scanned
using
an
plasmid that had been opened with a SpeI-BstBI double cut and then treated
Omnimedia scanner 6cx (Bioimage) and analyzed by a Whole Band Analyser
version 3.2.2, and the bands were quantified with the Bioimage application
with Klenow polymerase
(Roissy-France).
at the SpeI extremity
(Fig. 10.
PZEOSGO is derived
from pZEO SV1 (VECT 2001; Cayla). It harbors the HSV tk gene and a
sequence from the human T-cell lymphotrophic
virus ( I 8) which encodes a
RESULTS
transcriptional activator (Fig. 1C).
The SC18-l2 temperature-sensitive mutant of B/r E. coil polA (from E.
Overexpression of Pol @3
in Pol @3::ShCHO Cells. We have
Witkin, Piscataway, NJ) was transformed with pUTpol@ and pZETkTmkj3 to
transfected
verify the functionality of the polymerase activity. The TK function encoded
by pZETkTmk@3DNA was assessed by using a i/C E. coli mutant,4 TMK
vector pUTpolf3. The plasmid harbors the cDNA encoding the rat Pol
@3
fused in frame with the ble S/i gene conferring resistance to zeocin
(Fig. 1A). This fusion was driven by a strong, constitutive promoter
unit, which is the viral HSV TK promoter coupled to the viral
polyoma pYF44l enhancer. To validate the construct, we showed that
activity being revealed as described previously (16, 19) by complementing
@,ik temperature-sensitive
E.
coli
mutant
named
TD205
(gift
from
J. A.
a
Fuchs,
St. Paul, MN).
DNA Transfection.Parentalcells were transfectedwith plasmidDNA
CHO and melanoma
thermosensitive
BI6 cells. When they appeared, clones were picked, transferred to 35-mm
mammalian
cells, extracts were prepared from cells transfected
with
pUTpol@3 or pUT 526@, the latter carrying the ble Sh gene alone.
Extracts were electrophoresed
and a Western blotting was carried out
In Vitro Sensitivity Assay. Toxicity of AZT and ddC againstparentaland
transfected tumor cells was assessed by inoculating 24-well plates with 3000
cells/ml of serum-supplemented medium containing different concentrations of
nucleoside analogues. Cytotoxic effects were estimated after 5 days of incu
bation by slightly modifying a survival assay described previously (21). Cells
were washed three times with PBS buffer (BioWhittaker), and plated cells
were harvested by trypsinization, counted, and plated in serial dilutions. Ten
were counted
by fixing
mutated
in DNA polymerase
I activity were able to grow at 42°Cwhen transformed by pUTpol@
(data not shown). To assess the expression of Pol @3protein in
dishes, and incubated for 5 days in selective medium before testing for their
sensitivity.
in formation
E. coli bacteria
cells with the DNA expression
using the DMSO/polybrene shock procedure (20). After 48—62 h of pheno
typic expression, cells were split into the appropriate selective medium. One
hundred @.tWmland 20 @xWmlzeocin were added, respectively, to CHO and
days later, colonies
polAl2
Bl6
cells with ice-cold
methanol, washing them twice with PBS, and then staining surviving cells with
using polyclonal antibodies against Pol (3. As seen in Fig. 2, an
immunopositive band corresponding to Pol (.3(39 kDa) was observed
when 60 @g
of proteins of Pol f3::Sh cell extracts were loaded onto a
polyacrylamide gel. We could not see any Pol 13-related immunore
active band after electrophoresis of up to 240 @g
of proteins from the
control Sh cell extracts (data not shown). By scanning the autoradio
graph of the Western blot, the bands corresponding to Pol f3 in the Sh
extract (240 ,.Lg)and Pol @::Shextract (60 pg), we determined a Pol
filtered 10% Giemsa for 15 mm at room temperature. Survival was expressed
13overexpression
ratioof 9.8.In addition,nobandat60 kDacorre
as a percentage
sponding to the Pol
relative
to untreated cells.
@::Shprotein was detected by using Pol @3
Preparation of Cell Extracts. CHO cells were grown to subconfluence as
monolayer cultures on 145-mm plates at 37°Cin a 5% CO2 humidified
antibodies,
suggesting
vivo or during protein
incubator.
bodies against Sh protein showed the same amount of an immunore
active band around 20 kDa corresponding to Sh protein in both Pol
@::Shand Sh CHO extracts (data not shown).
Cells were washed
harvested by centrifugation.
with ice-cold
PBS, scraped off the plates, and
The cell pellet was suspended in hypotonic buffer
[10 mi@tTris-HCI (pH 7.5), 10 mM KC1, 10 mist MgCl2, and I nmi Dill
containing protease inhibitors. The cells were allowed to swell on ice for 10
4 C. Cazaux,
M. Tiraby,
L. Loubieres,
L. Haren,
D. Klatzmann,
that the fusion protein was cleaved either in
extraction. Indeed, Western blotting with anti
Pol @3
Overexpression Enhances Cytotoxicity to ddC and AZT.
Fig. 3 shows the effect of overexpression
and G. Tiraby.
survival
Differential activities toward therapeutic nucleosides of a- and y-herpes virus thymidine
kinases expressed in Escherichia coli, submitted for publication.
when exposed
respectively.
Survival
of Pol f3 on CHO cell
to the DNA chain terminators
of cells
containing
ddC and AZT,
the control
Ill
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
plasmid
@
CYTOTOXIC ACTION OF DNA POLYMERASE
A
@
poll3
pUT
pol@3
Nhe I
Fig. I . Construction of DNA plasmid expression vectors. A, cDNA of
pol 13 was amplified by PCR using restriction site-generating primers
and subcloned into the pUT687 plasmid in place of the E. coli tk gene
@
B
to overexpress only Pol f3.pUT68l harbors E. coli ik- and ble S/i-coding
genes separated by a flexible polypeptide link-encoding sequence; this
pUT633
I
Apr
NcoI
Po.II
\
I
E@h@k@
luc
hybrid gene is driven by both a synthetic strong EM7 bacterial promoter
and the viral HSV TK promoter coupled to the viral polyoma pYF441
@1
J@
zeorl fOnI
/
enhancer (28). B, construction of plasmid pUT834 harboring E. co/i tk,
1 E. co/i
pUT 832
tmk, and ble Sh genes fused in frame. C, for coexpressing HSV TK and
trnk I
Pol f3, we used plasmid pZeoSGO, which is an eukaryotic expression
NcoI
vector carrying the ble S/i resistance and the HSV tk genes. respectively.
@
poly A
MscI
RsrII
poly A
under control of enhancer/promoter sequences from the immediate early
gene of human cytomegalovirus and the SV4Oearly region. cDNA of pol
pUT 687
p wasinserted
intopZEOSGO
togivepZHTh@3.
Plasmid
pZETkTmkfi
I
Apr
E@k@E. coli
Zeo@'(
@I
expressing Pol 13and E. coli TKTI'MKwas constructed by recombining
pUT835andpZHTh/3(seetextfor details).All PCR-amplified
regions
pUT833
were sequenced to ensure that no errors were generated by the PCR
process. MCS is a multicloning site: OR! is the replication origin of the
@
@
E. coli colEl plasmid; ZEO'@ and Ap―
the zeocin and ampicillin
resistance genes; PoIvA is the late polyadenylation signal of SV4O;TX,,,
SV4O,,. and C/PIts'
1E.co/i
onk
E. co/i
pUT834
@y
enhancer-promoter sequences from herpes tk gene
I tk
E. co/i
tink
@or1
and SV4Oand cytomegalovirus (CMV) viruses; and HTLVis a transac
tivating sequence.
Bb,pI
C
@
polo
@
pZHTk@
13si81
EM7
pUT526& which possesses the pUTpolf3 backbone but lacks the pol
13gene,wasslightlyaffectedby bothdrugs,AZT beingmoretoxic
(Fig. 3B). In contrast, pUTpol@3-transfected cells were 30—SOtimes
more sensitive to ddC and AZT than the cell line expressing the
zeocin resistance function alone. Murine melanoma B 16 cells were
also transfected by plasmids pUTS2fiz@and pUTpol/3 and analyzed as
described with CHO cells. We found that survival was very similar to
that obtained with CHO (data not shown). Namely, IC50 and lethal
dose of nucleoside analogues (Table I) were comparable to those
determined for CHO (Fig. 3), AZT being more toxic than ddC. These
results suggest that the data presented here by using the well-known
(Kd)―@@
43
CHO model could be extrapolatedto tumorcell lines.
In Vitro Inhibition of DNA Synthesis by Pol
@::ShCell Extracts
in the Presence of ddCTP. To comparethe effect of ddCTPon DNA
synthesis
catalyzed
by Sh and Pol j3::Sh DNA replication
machineries,
we prepared cell extracts from both cell lines and investigated their
capacity to extend a 5' 32P-labeled phosphorylated 17-mer oligonu
cleotide primer on a G-rich 60-mer template (Fig. 4A). Reactions were
performed
in the presence
of ddCTP
at a ratio of ddCTP:dCTP
equal
to 1. After the substrate was replicated, the newly synthesized DNA
products
were resolved
by a 15% polyacrylamide
gel and visualized
30
Fig. 2. Western blot analysis showing Pol (3protein level in Sh and Pol fl::Sh CHO
cells. Samples of cell extracts (60 @gof protein) or I @sgof purified rat Pol fi were
subjected to a 12% SDS-polyacrylamide
gel, transferred electrophoretically
to nitrocel
lulose membrane, and then probed with purified rabbit anti-Pol @3
polyclonal antibody (see
text for details). Left, size markers.
112
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
CYTOTOXIC ACTION OF DNA POLYMERASE /3
A
B
Co
Co
1@
Ia
CID
Cl)
i04
1
ddC(@ M)
AZT (11 M)
Fig. 3. Toxicity of ddC (A) and AZ'f (B) to CHO cells transfected with DNA plasmid pUT526i@-Sh(0) or pUTpol/3(•).Cytotoxicity was determined from three isolated colonies.
Data given are the means of these three experiments and error is less than 20%. A pool of more than five other independent clones was tested and showed comparable cytotoxicity
results.
with autoradiography (Fig. 4B). With Pol @::Shextracts, most of the
reaction products (61%) migrated as a population of 18-mer, 19-mer,
and 20-mer DNA fragments, showing that DNA synthesis stopped at
the first dG base encountered by the cellular enzymes after ddCTP
incorporation. In contrast to Sh cell extracts, the majority (77%) of the
reaction products migrated after this first dG encountered from the
primer,
indicating
that no or few ddCTPs
were incorporated
with 40 p.M dATP, 40 @M
dGTP, and 50 @tM
or 100 /LMAZT-TP on
an A-rich 60-mer DNA template (Fig. 5A). After the copying of this
template for 30 mm at 37°C,the reaction products were analyzed on
a 20% polyacrylamide gel (Fig. SB). DNA chains migrating as 20mer, arrested opposite to the first dA encountered, were detected for
both Sh and Pol @::Shcell extracts. By scanning autoradiographs, we
evaluated that the band corresponding to this arrest was 2.3 times
more intense for Pol f3-overproducing cell extracts. These observa
tions suggest that, in the absence of dTFP, AZT was preferentially
during
elongation. Therefore, overexpression of Pol (3 in the cell extracts
inhibited in vitro DNA replication by efficient incorporation of
ddCTP. We then performed primer extension experiments by adding
to the Sh cell extracts an excess of highly purified calf thymus DNA
polymerases a, f3, @,or €(Fig. 4B). Severe inhibition of replication
was observed only when Pol @3
was present, confirming its ability to
incorporate ddCTP with a high efficiency in the presence of cellular
proteins. Taken together, these results strongly suggest that sensitiza
tion of the Pol (3::Sh cells to ddC treatment (Fig. 3A) was due to an
enhanced incorporation of the analogue into cellular DNA by DNA
Pol @3.
Capacity
of Pol
@::Sh Extracts
to Incorporate
AZT-TP
incorporated
increasing intracellular pools of phosphorylated analogues on the
sensitivity of cells overexpressing Pol @3.
CHO cells were transfected
with pZETkTmkfJ or pZHTk@ vectors coexpressing Pol @3
and bac
terial or viral TKITMK, respectively, which sequentially promote
monophosphorylation and diphosphorylation of the analogues. The
expression of each activity was assessed by complementing E. coli
into
(mutant
Table 1 Cytotoxic effect of ddC and AZT on murine melanoma B16 cells
cytotoxic
dose'@ (SM)
DNA plasmid
ddC
AZT
ddC
AZT
pUTS29@
pUTpolf3
900
80
500
70
>i04
600
>i0'@
200
a high level of rat Pol
Enhancement ofthe Cytotoxic Effect of Pol fi by Cotransfection
with Nucleoside/Nucleotide Kinases. We investigated the effect of
bacteria
Minimal
containing
to AZT (Fig. 3B) was due to its incorpo
ration into DNA by this DNA polymerase.
DNA in Vitro. To study the effect of AZT-TP on DNA synthesis
catalyzed by Sh and Pol f3::Sh cell extracts, reactions were conducted
IC50a ()
at this site by extracts
@3and that cell sensitivity
a
Inhibitory
concentration
ofanalogue
required
toreduce
cell
survived
by50%.
b Analogue concentration for which no viable cell was detectable with the Giemsa
coloration.
deficient
TD205)
in DNA
polymerase
or TK.4 Cotransfection
I (mutant
SC18l2),
TMK
of CHO cells by pol @3
and E.
coli tkltmk genes led to an increasing sensitization to AZT compared
to cells transfected with each gene alone (Fig. 6A). Similar experi
ments carried out by using plasmid pZHTk@3coexpressing Pol @3
and
HSV TKITMK instead of bacterial TK-TMK fusion led also to an
additive effect, even to a lesser extent (Fig. 6B). ddCMP did not give
a comparable additive effect, probably because HSV TK and E. coli
TK and/or TMK do not efficiently interact with this drug.
113
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
CYTOTOXIC ACTION OF DNA POLYMERASE /3
A
@
DNAsynthesis
17-mer
3'-CATAcGAGAAccAAcAT.s'
5'[email protected]'
60-mer
@
°.,
c@
Ta
•:
D
°*,
@,
dQ@'@
.@(
,c.
‘
S
t
a'.
a@
@
17
Fig. 4. Effect of ddCTP on in vitro replication by Sh and Pol @::ShCHO cell extracts. A, the G-rich 60-mer substrate annealed to the 17-mer primer used for the primer extension
assays. B, 5' 32P-labeled primed 60-mer template was replicated at 37°Cfor 1 h by 3 @gof extract from the indicated cell lines in the presence of ddCTP at a ratio of ddC:dC equal
to I. Two units of each purified DNA polymerase were added to the reaction mixture when indicated. Arrows, position of the 17-mer (primer) and 60-mer (full-size product). To
determine the exact location of the arrest sites, the 60-mer substrate was sequenced using the dideoxy method and migrated on the gel next to the reaction products.
A
DNA synthesis
17-mer
.4—
3'-CATACGAGAACCAACAT-5'
5'-AATAATAATA@CACCAACAATATAGGT1@CAACGCTAACCATCGTATGCI@CTTGGTTGTA-3'
60-mer
B
AZT (jiM)
50
100
I
@
I
;l)
I
III
17
F. I
Fig. 5. Effect of AZT-TP on in vitro replication by Sh and Pol
@::ShCHO cell extracts. A, the A-rich 60-mer substrate annealed to the l7-mer primer used for the primer extension
assays. B, 5' 32-labeledprimed 60-mer template was replicated at 37°Cfor 30 mm by 3 @g
of cell extract in the presence of 40 p@ dATP. 40 j.@M
dGTP, and 50 /LMor 100 ,SMAZT-TP.
The reactions were conducted without dTFP. The printed sequence on the left corresponds to the template.
114
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CYTOTOXIC ACtiON OF DNA POLYMERASE /3
B
A
Co
Co
Ia
Ia
Co
Cl)
Cl)
io@
io@
AZT ( R M)
AZT ( l@M)
Fig. 6. A, toxicity of AZT to CHO cells transfected with DNA plasmid pUTpol@ (•),pZEOSG 1 similar to pZEOSGO but expressing both E. co/i TK and TMK (U), and pZETkTmkf3
coexpressing E. co/i TK!FMK and Pol (3(A). B, toxicity of AZT to CHO cells transfected with DNA plasmid pUTpolf3(•),pUTS99 expressing HSVTK (U), and pZHTk@3
coexpressing
HSV TK and Pol @3
(A). Cytotoxicity was determined from three isolated colonies and a pool of more than five clones. Data given are the means of these four experiments; coors are
less than 20%.
DISCUSSION
metabolization of the drugs by the cellular kinases; (c) the effect of
DNA polymerase a, which is also able to incorporate AZT-MP but
not ddCMP into DNA (10); and (d) higher excision efficiency of ddC
from DNA by the exonuclease proofreading activities, AZT-mono
phosphate termini being 100- and 5-fold more resistant to exonucleo
lytic degradation by the 3' to 5' exonuclease of DNA polymerase €
and t5, respectively, than normal base-paired 3' termini.5
To our knowledge, we present here the first reported kill association
involving a DNA polymerase activity. Thus far, previous suicide
The antiviral action of chain terminators comes from the capacity of
viral polymerases or reverse transcriptases to incorporate these nude
oside analogues into viral DNA, causing termination of their replica
tion (13). The most useful drugs for the treatment of AIDS are
nucleoside analogues, such as ddC and AZT, which target the viral
reverse transcriptase. The poor affinity of these drugs to the cellular
replicative DNA polymerases may explain why this category of
agents has much less effect on cell survival. Because of its high
infidelity in vitro (9) and its ability to catalyze highly error-prone
bypass replication of bulky adducts in vitro (5), Pol @3is probably
mutagenic in vivo. This may explain the constitutive low level of this
enzyme. However, the temporary induction of its expression after
exposure to certain DNA-damaging agents (7, 8) might minimize this
mutagenicity. It has been shown that Pol @3,which is believed to
function primarily in DNA repair (2), is capable of incorporating
chain terminators ddC and AZT into DNA (10), but its concentration
in mammalian cells may be too low for efficient incorporation.
associations
To investigate a potentialcytotoxic association of Pol (3and chain
terminators, we analyzed the effect of overexpression of rat Pol (3 in
CHO andmurinemelanomaB16 cell lines treatedwith ddCandAZT.
We showed that these agents were about 50 times more toxic toward
Pol f3overexpressing cells than control cells (Fig. 3). Using an in vitro
cell extract-based DNA replication assay, we demonstrated a direct
role of the overexpressed Pol (3 in the incorporation of ddC and AZT
into DNA (Figs. 4 and 5). However,
AZT-TP
was incorporated
with
a very low efficiency compared to ddC, whereas the cellular toxic
effect of the two drugs was similar. This discrepancy might be due to
several factors: (a) differential uptake of the analogues; (b) different
using
base
analogues
have
involved
TK activities
be
cause of their efficiency in metabolizing nucleotide analogues into
their active forms (22). The HSV-1 TK has been used in different cell
types, including lymphoma, hepatocellular carcinoma, glioma fibro
sarcoma, and melanoma (23—26),to render targeted cells susceptible
to normally nontoxic chemotherapeutic agents. The viral TK is able to
toxify into the replicating cancer cells systemically administered
antiviral drugs such as GCV, a guanosine analogue normally metab
olized at very low levels by mammalian enzymes. The association of
HSV-1 TK/GCV is presently undergoing clinical trials. The HSV-1
TK/AZT combination was also used as part of a strategy of using
suicide genes for eradicating TK-expressing CD4@ cells in the design
of gene therapy of HIV infection (27). We found here that overex
pression of Pol f3improved the sensitization effect of exogenous HSV
TKI1'MK after treatment with AZT (Fig. 6B). It is worth noting that
the toxic effect of Pol @3
is more important than that of HSV TK when
associated with this agent. No additive effect was observed with GCV,
5 W.
Nickel,
S. Austermann,
G. Bialek,
and
F. Grosse.
Interactions
of azido-thymidine
triphosphate with the cellular DNA polymerases a, b, and a, and with DNA primases. J.
Biol. Chem., 267: 848—854,1992.
115
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CYFOTOXICACTIONOF DNAPOLYMERASE
/3
@
@
probably because GCV-TP is a poor substrate for Pol @3(data not
shown).
In the search for a microorganism which would be naturally
sensitive to low concentrations of analogue added in the growth
medium, which could indicate an efficient drug metabolization, we
have recently found that E. coli K12 was the most sensitive to AZT
of several bacterial and fungal strains tested.4 The E. coli tmk gene
was isolated (16) in such a way that an active gene fusion could be
made with AZT-E. coli tk-metabolizing gene to efficiently promote
the first two steps of AZT phosphorylation, final phosphorylation
of AZT-diphosphate to AZT-TP being carried out by a nonspecific
cellular nucleotide diphosphokinase. We show here that the genetic
fusion of E. coli tic and tmk genes encodes a fusion protein that led
to an additive cytotoxic effect of AZT when associated with Pol @3
expression (Fig. 6A) compared to phosphorylation or DNA polym
erization activity taken alone. This effect is much more marked
than with HSV TK (Fig. 6B), the suicide effect ofthe kinases being still
more marked
than that induced
by Pol (3. TK and TMK activities
were
investigated by measuring the amounts ofanabolic products in extracts of
radiolabeled AZT-treated CHO cells transfected by E. coli tkltmk or HSV
tk (data not shown). In both cases, we found a higher amount of mono
phosphorylated and diphosphorylated forms ofAZT. Similar results were
obtained
with B16 cell extracts
expressing
HSVTK.6
Taken
together,
DNA polymerase 13bypass in vitro d(GpG)-cisplatin adduct placed on codon 13 of
H-rat gene. Proc. NatI. Sci. USA, 92: 5356—5360, 1995.
6. Zmudzka, B. Z., Fornace, A., Collins, J., and Wilson, S. H. Characterisation of DNA
polymerase
mRNA: cell-cycle and growth response in cultured human cells.
Nucleic Acids Res., 16: 9589—9596,1988.
7. Srivastava,D. K., Rawson,T. Y., Showalter,S. D., and Wilson, S. H. Phorbol ester
abrogates up-regulation of DNA polymerase
by DNA-alkylating agents in Chinese
hamster ovary cells. J. Biol. Chem.. 270: 16402—16408,
1995.
8. Fornace, A. J., Zmudzka, B., Hollander, M. C., and Wilson, S. H. Induction of
@-polymerase
mRNAbyDNAdamagingagentsinChinesehamsterovarycells.Mol.
Cell. Biol., 9: 851—853,1989.
9. Kunkel,T. A. Frameshiftmutagenesisby eukaryoticDNApolymerasesin vitro.
J.Biol.
Chem.,
261:13581-13587,
1986.
10. Copeland, W. C., Chen, M. S., and Wang, T. S. F. Human DNA polymerases a and
13areableto incorporateanti-HIVdeoxynucleotides
intoDNA.J. Biol.Chem.,267:
21459—21464,1992.
11. Parker, W. B., White, E. L., Shaddix, S. C., Ross, L J., Buckheit, R. W., Germany,
J. M., Secrist, J. A., Vince, R., and Shannon, W. M. Mechanisms of inhibition of
human immunodeficiency virus type 1 reverse transcriptase and human DNA poly
merases a, @,
and y by the 5'-triphosphates of carbovir: 3'-azido-3'-deoxythymi
dine,2',3'-dideoxyguanosine, and 3'-deoxythymidine. J. Biol. Chem., 266: 1754—
1762, 1991.
12. Sommadossi, J. P., Carlisle, R., and Thou, Z. Cellular pharmacology of 3'-azido-3'deoxythymidine with evidence of incorporation into DNA of human bone marrow
cells. Mol. Pharmacol., 36: 9—14,1989.
13. Furman, P. A., Fyfe, J. A., St Gale, M. H., Weinhold, K., Rideout, J. L, Freeman,
0. A.,Lehrman,S.N.,Bolognesi,D. P.,Broder,S.,andMitsuya,H.Phosphorylation
of 3'-azido-3'-deoxythymidine
and selectiveinteractionof 5'-triphosphatewithhu
man immunodeficiency virus reverse transcriptase. Proc. Ned. Aced. Sci. USA, 83:
8333—8337, 1986.
14. Neuhard, D. J., and Nygaard, P. Biosynthesis and conversions of nucleotides: purines
andpyrimidines.in: F. C. Neidhart,J. C. Ingraham,K. B. Low,B. Magasanik,M.
these assays strengthen the claim that the increased cytotoxicity noted in
cells transfected by nucleoside/nucleotide kinases was due to analogue
Schaecter, and H. E. Umbarger (eds.), Escherichia co/i and Salmonella typhimurium:
CellularandMolecularBiology,pp. 445-473.Washington,DC:AmericanSociety
for Microbiology, 1987.
anabolism.
These data suggest that the use of new suicide molecular combi
nations should be investigated. Pol @3
might also be used to potentiate
the efficacy of novel or existing suicide associations involving TK
activities. This combination ofphosphorylating and replicative suicide
genes may create new opportunities for using chain terminators in
protocols for gene therapy of cancer.
15. Drocourt, D., Calmels, T., Reynes, J. P., Baron, M., and Tiraby, G. Cassettes of the
Streptoal/oteichus hindustanus b/c gene for transformation of lower and higher
eukaryotes to phleomycin resistance. Nucleic Acids Res., 18: 4009, 1990.
16. Reynes, J. P., Tiraby, M., Baron, M., Drocourt, D., and Tiraby, 0. Escherichia co/i
thymidylate kinase: molecular cloning, nucleotide sequence and genetic organization
of the corresponding test locus. J. Bacteriol., 178: 2804—2812,1996.
17. Kohara, Y., Akiyama, K., and Isono, K. The physical map of the whole E. co/i
chromosome: application of a new strategy for rapid analysis and sorting of a large
genomic library. Cell, 50: 495—508,1987.
18. Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida, M., and
Arai, N. SRalpha promoter: an efficient and versatile mammalian cDNA expression
ACKNOWLEDGMENTS
Discussions
with Dr. D. Drocourt from Cayla Laboratory
and Professor
G. Tiraby are acknowledged. We gratefully thank Drs. G. Villani and N.
Johnson for the careful review of the manuscript, Dr. S. Wilson for
providing us with polf3 cDNA, Dr. J. Sweasy for polyclonal antibodies
against Pol f3, and Cayla Laboratory for DNA plasmids pUT526&
pZEOSGO, and pZEOSG1. We also thank N. Tanguy le Gac for scanning
analysis at the Ecole Nationale Vétérinaire
(Service de Biochimie, Tou
louse, France).
system composed of the simian virus 40 early promoter and the R-U5 segment of
human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol., 8:
466—472, 1988.
19. Daws, T. D., and Fuchs, J. A. Isolation and characterization of an Escherichia co/i
mutantdeficientin dl'MP kinaseactivity.J. Bacteriol.,157:440—444,
1984.
20. Kawai, S., and Nishizawa, M. New procedure for DNA transfection with polycation
and dimethyl sulfoxide. Mol. Cell. Biol., 4: 1172—1
174, 1984.
21. Pressacco, J., Mitrovski, B., Erlichman, C., and Hedley, D. W. Effects of thymidylate
synthase inhibition on thymidine kinase activity and nucleoside transporter expres
sion. Cancer Rca., 55: 1505—1508,
1995.
22. Culver, K. W., Van Gilder, 3., Link, C. J., Carlstrom, T., Buroker. T., Yuh, W., Koch,
K., Schabold, K., Doornbas, S., and Wetjen, B. Gene therapy for the treatment of
malignantbrain tumorswith in vivotumortransductionwith the herpessimplex
thymidine kinase gene/ganciclovir system. Hum. Gene Ther., 5: 343-379, 1994.
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Overexpression of DNA Polymerase β Sensitizes Mammalian
Cells to 2 ′,3′-Deoxycytidine and 3′-Azido-3′-deoxythymidine
Khalil Bouayadi, Jean-Sébastien Hoffmann, Pascal Fons, et al.
Cancer Res 1997;57:110-116.
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