[Cancer Research 61, 50-52, January 1, 2001]
© 2001 American Association for Cancer Research
Highly Elevated Ultraviolet-induced Mutation Frequency in Isolated Chinese Hamster Cell Lines Defective in Nucleotide Excision Repair and Mismatch Repair Proteins1
Ken-ichiro Nara2,
Fumio Nagashima2,3 and
Akira Yasui4
Department of Molecular Genetics, Institute of Development, Aging and Cancer [K-i. N., F. N., A. Y.], and The Third Department of Internal Medicine [F. N.], Medical Faculty, Tohoku University, Sendai, 980-8575 Japan
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ABSTRACT
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We have isolated
N-methyl-N'-nitro-N-nitrosoguanidine-resistant
cell lines from 43–3B Chinese hamster ovary cells, which are deficient
in the ERCC1 gene involved in nucleotide excision
repair. By Western blotting analysis, we found cell lines that are
deficient or decreased in the amount of MSH6, or PMS2, or MSH2
proteins. Cell extracts of these cell lines show reduced efficiency of
G:T mismatch repair activity. Compared with 43–3B, these cell
lines exhibit highly elevated UV-induced mutation rates, indicating
that mammalian mismatch repair can suppress UV-induced mutagenesis and
may play a role in the fidelity of DNA replication at the sites of UV
damage.
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Introduction
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From bacteria to human cells, there is a set of very
well-conserved proteins involved in MMR, which is able to detect and
correct mispairing between DNA double strands (reviewed in Refs.
1
and 2
). A defect in MMR causes an increased
mutation rate in bacterial as well as in human cells. Cells derived
from patients with the cancer syndrome hereditary non-polyposis
colon cancer contain mutations in genes that are involved in MMR and
are defective in MMR activity. Mismatched DNA base pair are recognized
by protein complexes, which initiate the repair. hMutS
consists of
hMSH6 (or
GTBP,5
a G:T mismatch-binding protein) and hMSH2, and binds mainly to
single-base DNA mismatches. hMutSß is a complex between hMSH3 and
hMSH2 and is able to bind to DNA loops. hMutL (or ß) complex
consisting of hMLH and hPMS2 (or hPMS1) binds to the MutS complex and
results in a large complex, which is thought to lead to the subsequent
DNA mismatch excision reaction. MMR-deficient mammalian cells express a
methyl-tolerant phenotype, which is characterized by the resistance of
the cells to several mono-alkylating agents including MNNG (3
, 4)
. This resistance is thought to be attributable to the lack of
cell-killing ability of MMR in the mutant cells (5)
.
Methyl tolerance suggests that the MMR complex recognizes not only
mismatches between normal bases but also bases paired with modified or
damaged DNA. Besides a pair between mono-alkylated and normal bases,
MMR complex also binds to mispaired UV-induced DNA damage (6
, 7)
. To understand the distinct role(s) of MMR in DNA damage
recognition after DNA replication, we used a NER-deficient Chinese
hamster ovary cell line, 43–3B, and isolated a number of
methyl-tolerant cell lines, in which MSH2-, MSH6-, and PMS2-deficient
cells were found. Because 43–3B is deficient in the Chinese hamster,
ERCC1, a protein necessary for incision at 5' to the sites of DNA
damage in NER, the isolated cells are double mutants defective in both
MMR and NER. Using these mutant cells, we found that MMR is able to
decrease the level of UV-induced mutation.
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Materials and Methods
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Chemicals and Antibodies.
MNNG, methylnitrosourea, and 6-thioguanine were purchased from
Sigma. They were dissolved in PBS (pH 3.5) at 10 mg/ml.
Anti-GTBP antibody was a kind gift from Dr. J. Jiricny
(University of Zurich, Zurich, Switzerland). Anti-hMSH2 antibody,
anti-hMLH1, and anti-hPMS2 antibodies were obtained from Santa Cruz
Biotechnologies. ACNU (Sankyo, Tokyo, Japan) was freshly prepared in
PBS (pH 3.5) at 1 mg/ml.
Cell Lines and Culture.
43–3B is a NER-deficient CHO cell line (8)
that has a
point mutation in ERCC1 gene (9)
. Colorectal
carcinoma cell lines, LoVo and HCT-15, were purchased from American
Type Culture Collection. HCT116 was obtained from Dr. S. Fukushige
(Tohoku University, Sendai, Japan). HeLa, CHO-9, 43–3B and isolated
MNNG-resistant cell lines were cultured in MEM (Nissui) with 8% FCS.
LoVo was cultured in Ham’s F12 medium (Nissui) + 10% FCS
medium. HCT-15 was cultured in McCoy’s 5A medium (Sigma) + 10% FCS. All of the cells were cultured at 37°C with 5%
CO2.
Isolation of MNNG-resistant Clones.
Exponentially growing 43–3B cells were treated with 0.05 µg/ml MNNG
in MEM + 8% FCS for 2 h. After the treatment, cells
were cultured in MEM for 4 days. Surviving cells were collected and
treated with MNNG again. After seven treatments with MNNG, one colony
from each flask was isolated for further characterization.
Measurement of Survival.
On the day after seeding of 200 or 2000 cells on 6-cm dishes containing
4 ml of MEM, cells were treated with MNNG or methylnitrosourea at
different concentrations. After 2 h of treatment, cells were
washed with Hanks buffer (Nissui) and further cultured in fresh
MEM for 10–14 days. Surviving colonies were fixed and stained with 2%
crystal violet solution in ethanol before counting. In UV survival
experiments, seeded cells were washed twice with Hanks buffer before UV
irradiation (254 nm, Toshiba UV lamp). The survival data presented in
this paper are the results of three independent experiments using three
to four dishes for each point.
Assay for in Vitro MMR Activity.
Heteroduplex open circular M13 phage DNA containing a G:T
mismatch in the lacZ gene was treated with the cell free
extracts from various cell lines. MMR activity in the extracts was
measured by introducing the phage DNA into the Escherichia
coli indicator strain NR9162 (mutS) and the rate of
blue/(white + blue) plaques corresponds to the MMR activity
in the cell extracts (detailed in Ref. 10
).
Determination of MNNG- or UV-induced Forward Mutation Rates of
Ouabain Resistance.
One day after seeding 200 cells in 6-cm dishes and 50,000 cells in
10-cm dishes, cells were treated with MNNG at different concentrations
for 2 h. In UV irradiation experiments, cells were washed twice
with Hanks buffer before UV exposure. Cells in 10-cm dishes were
then cultured in fresh MEM for one day, followed by 20 days culture
with selection medium containing 1 mg/ml ouabain (Sigma). Cells in 6-cm
dishes were cultured with fresh MEM, and surviving colonies were
counted 10 days after treatment. The mutation frequency rates presented
in Table 1
are the mean values of two independent experiments
using five dishes for each point.
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Results
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Isolation of Clones Resistant to Alkylating Agents.
To isolate MNNG-resistant CHO cell lines, 25 individual cultures of
43–3B cells were repeatedly treated with MNNG, and a single surviving
clone was isolated from each culture. Twenty-two clones were thus
isolated, and these turned out to be over 10-fold more resistant to
MNNG than the parental cell (Fig. 1)
. All of the isolated clones are much more resistant to
N-methyl-N-nitrosourea or 6-thioguanine than the
parental cells, but show the same sensitivity to X-rays and ACNU as the
parental cells (not shown). These results suggest that the acquired
resistance is not attributable to the increased
O6-methylguanine-DNA methyltransferase
activity, but attributable to methyl-tolerance, which is explained by
defective MMR.
Absence of MMR Proteins in Five Isolated Cell Lines.
Using antibodies raised against peptide sequences from the mammalian
MMR proteins, MSH2, MSH6 (GTBP), MLH1, MSH3 and PMS2, we identified
cell lines defective in the expression of MSH2, MSH6, and PMS2 (Fig. 2a)
. In extracts of the N5–2 cell line, the MSH6 signal at
Mr 160,000 is completely absent,
whereas the other four clones showed its expression of the parental
cell line level. The MSH2 signal in N3–2 cell line is very much
reduced as compared with the signals in 43–3B and the other isolated
cell lines. Finally, in N4–14, N1–9 and H5 cell lines, PMS2 protein
is completely absent in their cell extracts, whereas in N5–2 and N3–2
cell lines, PMS2 protein was expressed as in the parental cell line.
These five clones show the MLH1 expression of the parental clone level.
Defects in MMR Activities.
We determined the MMR activity of the isolated clones using an in
vitro assay for double stranded DNA substrate containing a single
G:T mismatch. As shown in Fig. 2b
, in vitro repair
activities of extracts prepared from the isolated cell lines are much
lower than that of the parental 43–3B. 43–3B showed a similar level
of activity to that found in extracts of HeLa cells, whereas cell
extracts of the isolated CHO cells are comparable with the activity in
HCT15 and LoVo, which are human cell lines established from hereditary
non-polyposis colon cancer patients. These data indicate that all of
the five cell lines are MMR mutants in this assay.
UV Sensitivity and UV-induced Mutation in the Putative MSH2, MSH6,
and PMS2 Mutant Cell Lines.
To analyze the involvement of MMR in the processing of UV damage,
double mutants in NER and MMR are extremely useful. Firstly we
determined the UV sensitivity of the five isolates. As shown in Fig. 3
, N5 –2 cells are more UV sensitive than the parental cell, whereas the
other four clones show almost the same sensitivity as 43–3B. We next
examined frequency of UV-induced mutation to ouabain resistance in
N5–2, N3–2, and N4–14 compared with 43–3B and wild-type CHO9. In
contrast to the UV sensitivity, all of the three isolated cell lines
showed extremely high mutation rates on UV irradiation as compared with
the parental 43–3B cell, which exhibited a slightly higher UV-induced
mutation rate than CHO9 (Table 1)
. These data suggest that MMR is able to suppress UV-induced mutation
and may play a role in increasing the fidelity of replication at the
sites of UV damage.
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Discussion
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From a NER-deficient CHO cell line we isolated a number of
independent MNNG-resistant clones. The parental cell line 43–3B has a
point mutation in the ERCC1 gene in a region necessary for
interaction of ERCC1 protein with the xeroderma pigmentosum group A
(XP-A) protein (9)
. Because this interaction is essential
for the initiation of NER, 43–3B is completely defective in NER and
hypersensitive to UV. By Western blot analysis, five clones of the
twenty-two MNNG-resistant clones were found to be deficient in one of
the known MMR proteins. The results of an in vitro MMR assay
support the Western data, and the five cell lines show almost no MMR
activity against TG mismatched substrate. A Western blot analysis
showed that one clone, N5–2, is deficient in MSH6, whereas MSH2 is
greatly reduced in another clone and three further clones have no PMS2
protein. There was no difference in the expression of MLH1 protein
among the cell lines. Thus, as far we know, this is the first isolation
of MSH6- and PMS2-deficient mammalian cells by screening of
MNNG-resistant cells.
Among the isolated cell lines, N5–2 (MSH6-deficient) is mildly more
UV-sensitive than the parental cell line, whereas the other four cell
lines showed similar UV-sensitivity to that of 43–3B. Because MMR
defects do not increase UV-sensitivity of NER-deficient mammalian cells
(11)
, we think that the isolated clones are the first
double mutants harboring mutations in one of the three MMR genes and in
the ERCC1 gene of NER. The reason for the increased
UV-sensitivity in the N5–2 cell line remains to be elucidated.
We are very surprised by the fact that, although the UV
sensitivities do not differ greatly among the isolated double-mutant
cell lines and the parental cell 43–3B, the frequency of UV-induced
ouabain-resistance differs significantly. As shown in Table 1
, in all
of the three isolated mutant cell lines defective in either MSH2, MSH6,
or PMS2, the numbers of UV (3.9-J/m2)-induced
mutants per 106 cells are about 500- to 1000-fold
more than that in the parental cell line. Although UV-induced sister
chromatid exchange was measured in XP-A single and XP-A/MSH2 double
mutant, which showed no difference after UV irradiation
(11)
, UV-induced mutagenesis has not been compared between
NER-deficient and NER/mismatch double mutant cells. Even in yeast
cells, only spontaneous mutation frequency in MSH2-deficient has been
shown to be epistatic to the mutator phenotype observed in
NER-deficient mutants (12)
. Our data suggest that the
fidelity of replication after UV irradiation is greatly dependent on
the activity of MMR in mammalian cells, especially when NER is
deficient. If NER is proficient, UV-induced mutation is not much
influenced by the absence of MMR (13)
. This is explained
by the activity of NER, which repairs premutagenic DNA lesions almost
completely. Although we could not test MLH1-deficient cell for
UV-induced mutation, our data suggest that both the hMutS and the
hMutL MMR complexes are necessary for the suppression of UV-induced
mutation in NER-deficient cell.
Recently, an increased UV-induced mutation frequency of 2- to 3-fold
over that in the NER-deficient single mutant was reported in an
E. coli strain defective in both NER and MMR
(14)
. This may suggest the presence of similar mechanisms
in bacterial and mammalian MMR for suppressing UV-induced mutation.
However, the highly elevated UV-induced mutation frequency in
mammalian cells may reflect significant differences in the cellular UV
response from that of bacteria. In E. coli, in the absence
of NER, UV-induced DNA damage is mainly processed by a
recA-dependent recombination pathway. In contrast, in
mammalian cells translesion synthesis including DNA pol 
is now
thought to take over replication at the site of DNA damage. pol
introduces deoxyadenosine opposite the UV-induced pyrimidine dimer
(15)
. However, it is not well known yet, how replication
fidelity is ensured, when pol encounters UV-induced
thymine-cytosine or cytosine-cytosine dimers. One possible model to
explain our data is that MMR plays a role in proofreading for
translesion synthesis at the site of DNA damage. It has been reported
that MMR complexes can recognize compound DNA lesion (base damage and
mismatch) involving UV photoproducts (6)
. Compound DNA
lesion produced by pol may be also the substrate for MMR. Because
MMR cannot complete the repair replication because of the presence of
DNA damage, translesion synthesis may take over the replication again.
This process may repeat until the correct nucleotide is incorporated by
translesion synthesis. Furthermore, MMR may function as a general
proofreading machinery for pol (and other polymerases), which
possesses an extremely low fidelity in replication (16)
.
Further analysis will elucidate a possible interaction between the two
postreplicative repair systems, MMR and translesion synthesis.
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ACKNOWLEDGMENTS
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We thank Drs. M. Takao and S. Nakajima (Tohoku University,
Japan) for discussion and Dr. S. J. McCready (Oxford, United Kingdom)
for editing the manuscript. We thank Dr. T. A. Kunkel (NIEH,
NC) for providing us with the materials necessary for in
vitro MMR assay.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by a Grant in
Aid 08280101 for Scientific Research on Priority Areas (to
A. Y.). 
2 K-i. N. and F. N. contributed equally to the
work.
3 Present address: Department of Gastrointestinal
Oncology/Gastroenterology, National Cancer Center Hospital East 6-5-1
Kashiwanoha, Kashiwa, Chiba, 277-8577 Japan.
4 To whom requests for reprints should be
addressed, at Department of Molecular Genetics, Institute of
Development, Aging and Cancer, Tohoku University, Seiryomachi 4-1,
Aobaku, Sendai, 980-8575 Japan.
5 The abbreviations used are: GTBP,
G:T mismatch binding protein; MNNG,
N-methyl-N'-nitro-N-nitrosoguanidine;
NER, nucleotide excision repair; ACNU,
1-[(4-amino-2-methylpyrimidin-5-yl)methyl]-3-(chloroethyl)-3-nitrosourea
hydrochloride; MMR, mismatch repair; pol , polymerase .
Received 6/21/00.
Accepted 11/ 7/00.
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ABSTRACT
Introduction
