Mutator Phenotype in Msh2-deficient Murine Embryonic Fibroblasts Armin H. Reitmair, Robert Risley, Robert G. Bristow, et al. Cancer Res 1997;57:3765-3771. Published online September 1, 1997.
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ICANCER RESEARCH57, 3765-3771. September I. 19971
Mutator Phenotype in Msh2-deficient
Murine Embryonic Fibroblasts'
Armin H. Reitmair,2 Robert Risley,2 Robert G. Bristow,2 Teresa Wilson,2 Ami Ganesh, Anne Jang, James Peacock, Samuel Benchimol, Richard P Hill, Tak W. Mak, Richard Fishel, and Mark Meuth3 Ontario Cancer Institute/Amgen Institute, Departments of Medical Biophysics and Immunology, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada [A. H. R., T. W. M.]; Departments of Oncological Sciences and Radiation Oncology. Eccles Institute of Human Genetics, Universirv of Utah, Salt Lake City, Utah 84! 12 [R. R., A. G., M. MI: Ontario Cancer Insitute, MSG 2M9, Canada JR. G. B., A. J., R. P. Hi;
Departments of Medical Biophysics and Radiation Oncology, University of Toronto, 610 University DNA Repair and Molecular Carcinogenesis Program, Kimmel Cancer Center, Thomas Jefferson
Avenue, Toronto, Ontario University, Philadelphia,
Pennsylvania 19107 (T. W., R. Fl; and Ontario Cancer Institute, Department of Medical Biophysics. University of Toronto, 6/0 University Avenue. Toronto, Ontario MSG 2M9, Canada (J. P., S. B.J
ABSTRACT
copy is mutated or lost as an early event in the development of a tumor. This second event renders cells mismatch repair deficient (7, Embryonic fibroblast cell lines were established from mice deficient, 8), presumably leading to the rapid accumulation of mutations that heterozygous, or proficient for Msh2, one of the three known DNA mis drive tumor development. Two other genes encoding homologues of match repair genes involved in hereditary nonpolyposis colon cancer (HNPCC). Cell lines were established by transfection of primary mouse the E. coli mutS gene (hMSH3 and hMSH6) appear to be involved in the repair of some types of DNA replication errors or damage (9—11). embryo fibroblasts with E7 and Ras oncogenes or mutantp53. Spontane ously immortalized cells derived from the primary cultures were also Mutations in these genes have been found in some tumor cell lines, studied.To determinewhetherthesecellsdevelopeda mutatorphenotype but not HNPCC patients (12—14).Finally a fifth homologue of the similar to that found in colon cancer cells deficient in mismatch repair, we mismatch repair genes, hPMSJ, was identified based on its homology measured mutation rates, microsatellite instability, and sensitivities to a with MutL. However only one germline alteration of this gene has range of DNA-damaging agents. The mutator phenotype detected in the been identified and no apparent relationship to tumorigenesis has been E7 and Ras or mutantpS3-lmmortalized Msh2'-/mouse cells was sim reported (6). liar to that found in humanmismatchrepair-deficientcolorectalcard Cell lines originating from tumors deficient in mismatch repair aetna cell lines. Mutation rates to ouabain resistance were increased have a distinctive mutator phenotype. The rate of mutations that result 8-42-fold relative to lines from Msh2+/+ mice, and microsatellite mats in the loss or gain of repeat units in microsatellites is dramatically bility was detectable in 12—18%of subclones derived from the Msh2—/— increased (7, 15, 16). Studies from several laboratories provide strong line but was undetectable in subclones developed from the Msh2+I+ line. evidence that this microsatellite instability is diagnostic of the mis Furthermore, E7 and Ras or spontaneously immortalized Msh2—/- cells were significantly more resistant to the cytotoxic effects of 6-thioguanine match repair deficiency in colorectal carcinoma cell lines (7, 8). This relative to Msh2+/+ cells. In contrast, these lines showed various re DNA sequence instability is not limited to simple sequence repeat spouses to UV light and cis-platinum, suggesting that mismatch repair structures. Mismatch repair-deficient cell lines have a similar (60— deficiency was not the sole determinant for sensitivity to these DNA 600-fold) increase in the rate of spontaneous mutations at selectable damaging agents. Particular attention was paid to the properties of cells loci such as those encoding the punne salvage enzymes HPRT (16— heterozygous for the Msh2 mutant gene, which would mimic the situation 18) or APRT (19). of an HNPCC carrier. However, our studies failed to reveal any properties In addition to an increased rate of spontaneous mutation, mismatch of these cells that might provide a growth advantage or predispose them repair-deficient tumor cell lines are altered in their response to a wide for the acquisition of further mutations. This observation is consistent range of DNA-damaging agents. Mismatch repair-deficient colorectal with the model that Inactivation of the wild-type Msh2 allele is a critical step for tumorigenesis
in HNPCC
carcinoma
patients.
INTRODUCTION Recent work revealed a distinctive class of colorectal carcinomas having a high level of microsatellite repeat instability (1). In heredi tary forms of these cancers (HNPCC4 and Muir-Tone syndrome), microsatellite instability is largely associated with mutations in one of two human homologues of the Escherichia coli mismatch repair genes MutS and MutL (hMSH2, hMLHJ; Refs. 2—5).Rare germline muta tions of the MutL homologue hPMS2 also have been found associated with HNPCC (6). HNPCC patients inherit a mutation of one of the alleles encoding a mismatch repair gene, and the second wild-type
proteins Received 12/18/96; accepted 7/2/97. 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. I A. H. R.
was
supported
by
a stipend
of
the
German
Academic
Exchange
Service
(DAAD-Doktorandenstipendium aus Miueln des zweiten Hochschulsonderprogramms). This work was funded in part by grants from the Medical Research Council of Canada and
the National Cancer Institute of Canada (to R. B., S. B., R. P. H., and T. W. M.). National Cancer Institute Grants ROl CA62244 and ROl CA22188 supported the work of M. M.;
Grants ROl CA56542 and ROl CA 67007 supported the work of R. F. 2 These
authors
3 To whom
contributed
requests
equally
for reprints
to this
should
work.
be addressed.
Phone:
(801) 585-5258;
Fax:
(801) 585-3501; E-mail:
[email protected]. 4 The
abbreviations
used
are:
HNPCC,
hereditary
nonpolyposis
colon
cancer:
HPRT.
hypoxanthine guanine phosphoribosyl transferase; APRT, adenine phosphoribosyl trans ferase; 6-tg, 6-thioguanine.
cell lines are resistant
to DNA-alkylating
agents
or base
analogues, such as 6-tg. More recently, evidence has been presented that cis-platinum-resistant cell lines may also be deficient in mismatch repair (20—22).Lesions produced by these agents are recognized both by the hMSH2 protein alone and the hMSH2/hMSH6 heterodimer (22—24)and may induce a futile mismatch repair cycle that ultimately leads to cell death (17, 25). The loss of mismatch repair would prevent the initiation of this futile repair and therefore increase cell survival. In contrast, some mismatch repair-deficient colorectal carcinoma cell lines are more sensitive to the cytotoxic effects of UV irradiation (26). This increased sensitivity is accompanied by the loss of the preferen tial repair of UV-induced lesions on the transcribed strand of coding sequences in human cells and suggests that the mismatch repair also play some role in the transcription
coupled
repair
path
way (26). Unfortunately, the colon cancer cell lines used in these character izations
have
numerous
alterations
of proto-oncogenes
and tumor
suppressors that may contribute to the final mutator phenotype. The assignment of a mutator phenotype to a specific mismatch repair gene is further complicated by the recent observation that a single tumor cell line may harbor mutations of more than one mismatch repair gene (14) or of other genes that govern mutation rates (27). The recently described mice containing knockouts of mismatch repair genes (28— 32) potentially offer a powerful approach for determining the effects of mismatch repair gene deficiencies in the absence of other alter ations. Homozygous mice deficient in Msh2, Pms2, or MIhI are viable
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MUTATOR PHENOTYPE IN M5H2-DEFICIENT MOUSE FIBROBL.ASTS
but primarily develop lymphomas between 2 and 12 months of age (28—31). Intestinal tumors are also observed in the majority of Msh2—I— mice that survive to >6 months (33). About 7% of animals develop a variety of skin neoplasms similar to those found in humans suffering from Muir-Torre syndrome. In the work reported here, we characterized immortalized embryo fibroblasts obtained from Msh2+/+, +1—, and —I— mice to determine whether they develop a mutator phenotype similar to that found in human colorectal carci noma cell lines. Dc Wind et al. (29) showed that mouse ES cells with knockouts of Msh2 developed an increased frequency of Hprt mutants and resistance to DNA alkylating agents. We have made more quan titative measurements of mutation rate at two genetic loci that allow us to compare the level of mutation in different cell lines. We show that mouse embryo fibroblasts derived from Msh2—I-- cells develop a mutator phenotype similar to that found in human tumor cell lines deficient in mismatch repair. However, important differences in sen sitivity to DNA-damaging agents in the immortalized cell lines were detected. Considering the high penetrance of HNPCC (34), we paid particular attention to embryo fibroblasts derived from Msh2 het erozygous mice for evidence of effects on spontaneous mutation rates or sensitivity to DNA-damaging agents, but no significant changes were detectable.
A
+1+ +1-. -1-
208
-
118
-
86 -
51 -
B
+1++1--I-‘5 10 20'‘510 20'‘510 20 @.tg protein
@
.
7
e
‘@‘
MATERIALS AND METHODS Recombinant Vectors. Mp53pro193contains a 16-kbEcoRI fragmentin pUCl8 carrying the entire mouse p53 gene and bearing a single point mutation at codon 193 that converts an arginine residue to a proline (35). The HPVI6 E7
coding sequence is present on the pJ4fll6.E7 plasmid (36). Activated c-Ha-ras sequences are present on pEJ6.fl (37). Plasmid pHMR272 contains the hygro mycin resistance gene (38).
Embryonic
Fibroblasts. Heterozygote (Msh2@)
..
mice of mixed
C57BIJ6J and 129/Ola genetic background (Fl generation) were mated. A timed pregnant female was sacrificed after 13 days of gestation, and individual fetuses were removed and washed repeatedly with PBS. Murine embryonic fibroblasts from these littermate embryos were prepared as described previ ously (35), and their Msh2 status was determined using a PCR-based geno typing assay (33). Cells to be transfected were pooled according to genotype and plated
from primary
cell cultures
at a cell density
of 3.0 X 10' cells per
60-mm dish, giving rise to secondary cultures and transfected the following
day. Transformations were performed as described previously (39) and con tamed 10 iLgof NIH3T3 carrier DNA, I @g of pHMR272, and either 10 @g of pEJfl.fl with 5
[email protected]
or 20 @.tgofMpS3prol93.
Approximately
24 h
after transfection, the cells were trypsinized and plated in fresh medium. Selection in a-MEM containing 15 @g/mlhygromycin commenced 48 h after transfection. Colonies were identified visually by microscope approximately 2 weeks later. After 14 days in selective medium, cells from individual hygro mycin-resistant
colonies
were isolated
with cloning
cylinders
and transferred
to 24-well plates containing 2 ml of selective medium. When cultures reached confluency they were passaged at a ratio of 1:5, and clones that survived the 10th passage were considered immortal.
The immortalized mouse fibroblast lines were subsequently maintained on a-MEM supplemented with 10% FCS. Drug or radiation sensitivity was examined in three E7- and ras-immortalized fibroblast lines derived from three different colonies obtained from the transformation of Msh2+/+ embryo fibroblasts, one line derived from Msh2+/— embryo fibroblasts, and two lines
Fig. I. Western blot and Gff binding activity analysis of E7/Ras-immortalized cells derived from Msh2+/+ , +1—,and —I— mice. A, Westem blot analysis of Msh2 content in the three cell lines. B, GiT binding activity. Various amounts of extracts prepared from
the three mouse lines were incubated with the 32P oligoduplex containing the G/T mismatch. The resulting protein-DNA complexes were analyzed using a gel mobility shift assay.
derived from Msh2—/— fibroblasts. Cells were treated 16—18h after being seeded at densities that would yield 25—70surviving colonies at the time of fixation. Graded doses of ionizing radiation (i.e., 0—10Gy) were delivered @
fixed and stained with a methylene blue solution made up in 50% ethanol.
lamp at a dose rate of 8
Colonies with more than 50 cells were scored, and survival was expressed as the relative plating efficiency of treated cells to the control. in vitro parameters
J/m2 per mm. For treatment with cis-platinum, cells were exposed to various
were intercompared between the cell lines with the nonparametric Mann
concentrations of drug mixed in culture medium for 72 h. Following exposure, cells were washed in PBS, and fresh medium was added. To determine 6-tg sensitivity, cells were exposed continuously to various concentrations of the drugs in growth medium. For all treatments, after 8—10days, colonies were
Whitney test using the MINITAB
using
37Cs -y-rays under
irradiation
aerobic
of cells was performed
conditions
and at room
using a germicidal
temperature.
UV
general statistics program (Minitab Inc.,
PA). Western Blot and Mismatch Binding Analysis. Approximately5 X 106 cells were harvested by treatment with trypsin followed by two washes with
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MUTATOR PHENOTYPE IN MSH2-DEFICIENTMOUSE FIBROBLASTS
(5.6%) Msh24' and 3 of 15 (20%) Msh2@ colonies survived the tenth passage. Therefore, the immortalization frequency by a mutant
Table 1 Microsatellite instability in E7/Ras-immortalized mouse embryofibroblast cell lines
(EF2O1)cell DNA was purified from subclones derived from Msh2+/+ (EFIOO)or —I— ofclones lines and used for amplification of the four microsatellite loci. The proportion showing presented.Locus novel alleles is EF2O1D3mit203
p53 allele did not appearto depend upon Msh2 status in this expert
EF100
2/18D6mit59 3/18D7mit62 2/18DlPmit36
0/17 0/14 0/16 0/17
3/17
PBS. The cell pellets were resuspended in 150 pi of high-salt buffer (50 rust potassium phosphate, pH 7.5, 300 mM NaCl, 0. 1 rust EDTA, 1 mM DTT, and 10% glycerol) and subjected to three freeze-thaw cycles. The samples were
centrifuged, and the supernatant dialyzed against the above buffer containing 50 mM NaCI. Protein
concentrations
of the total cell extracts
were determined
using the Bradford method (Bio-Rad). Ten @gof +1+, +/—, and —I— total cell extracts were electrophoresed on a 7% SDS-polyacrylamide gel and
transferred to nitrocellulose. Anti-hMSH2 polyclonal antibody (40) was used at a concentration of 3 ,.@g/ml,and Msh2 was detected by chemiluminescence as directed by the manufacturer
(Boehringer-Mannheim,
Indianapolis,
IN).
For the mismatch binding assay, 32P-labeledDNA duplex containing a G@ mismatch was prepared as described previously oligonucleotides
used
to construct
(41). The sequences of the
this substrate
were
as follows:
top, 5'-
CGGCGAATfCCACCAAGCTFGATCGCTCGAGGTACCAGG-3'; bottom, 5'-CCTGGTACCTCGAGCGATCGAGCTFOGTGQAATFCGCCG-3'. Mis matched DNA binding assays were carried out in 30-id reactions containing 5, 10, or 20 i.@gof whole cell protein extract, DNA binding buffer [50 mivi potassium phosphate, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM DTF, 10%
glycerol, 200 ng of poly(dI'dC)poly(dFdC)J, 1 ng of 32P-labeledDNA duplex and incubated at 37°Cfor 10 mm. The binding reactions were loaded onto a 5% polyacrylamide gel (1.5 mm thick, 22-cm-long, and 14 cm wide) contain ing 2.5% glycerol
that was run at 35 mA for 3 h. The gel was then dried and
analyzed
on a Molecular
obtained
from the Kimmel
Measurement
Dynamics
Phosphorlmager.
Cancer
of Mutation
Institute
Rates.
Nucleic
Oligonucleotides Acids
were
Facility.
Mutation rates were measured by Luria
Delbruck fluctuation tests as described previously (16). Replica cultures were inoculated with 100 cells and then grown to a final density of 1—4X 106. All
of the cells from each replica were then plated in selective medium containing either 5 !.Lg/ml6-tg or 2 inst ouabain. Colonies growing after 10 days were fixed, stained, and scored. Mutation rates were calculated by the method of the mean. Microsatellite MSH2+/+
Instability.
or MSH2—/—
microsatellite
Subclones
mice
loci were analyzed
were
grown
derived
from cell lines derived
out for purification
for each subclone.
Primers
of DNA.
from Four
for the amplifi
cation of these microsatellites were obtained from the mouse genome data base.
RESULTS
ment. Several hygromycin-resistant colonies that were cotransfected with activated Ras and HPVJ6 E7 survived at least 10 passages. The transfected clones used in these experiments were carried as long as 40 generations with no decrease in growth rate or plating efficiency. Msh2 Expression and Mismatch Binding. E7/Ras-transfected cell lines were first examined for Msh2 expression by Western blot analysis to be certain that the immortalized cells maintained the expected levels of this protein. Fig. 1A shows that Msh2 was absent in cells derived from the —I— mice and that cells derived from the +1— mice had lower levels of Msh2 relative to the +1+ cells. Off mis match binding activity in the three lines showed a similar pattern (Fig. 1B). The E7/Ras-immortalized cell line derived from the Msh2—/— mice showed a loss of binding activity. The line derived from the +1— mice had lower levels of binding relative to that found in the Msh2+/+ line. Microsatellite Instability. The frequency of cells containing novel microsatellite alleles at four different loci was determined using DNA purified from cultures grown out from single cell clones. Four loci were examined in 14—18subclones isolated from +1+ or —I--cells obtained from the E7/Ras-transfected lines. Novel alleles were de tected at each of the loci tested in 12—18%of the subclones isolated from the Msh2-deficient line (Table 1). In contrast, no novel alleles were detected in subclones isolated from the Msh2+/+ cells (overall frequency, 2n DNA content. Although the number of active X chromosomes may not be altered in these cells, this problem was avoided by measuring the rate of mutation to ouabain resistance. Such mutations act dominantly, so they are not obscured in polyploid cells (42). Furthermore, recovery of 6-tg-resistant cells in the Msh2 proficient and deficient fibroblasts may be affected by the differing sensitivity of these cells to the selective agent (see below). Table 2 shows that the rate of mutation to ouabain resistance in the Msh2—/—line was elevated 12-fold relative to the Msh2+/+ line and 25-fold relative Msh2+I— line. The lower
p53-l5 +1+ 3.8X l0@
+1— 4.4 X lO_8
3.1X
forplating Mutation rates are expressed as mutations/cell/generation and are not corrected efficiency. b
not tested.
3767
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MUTATOR PHENOTYPE IN MSH2-DEFICIENTMOUSE FIBROBLASTS
A
B
C
.1
.91
U La
‘U be
be
.E
.5 a :a
Fig. 2. Sensitivity of E7/Ras-immortalized mouse embryo fibroblasts derived from Msh2+/+, +1—, and —I— mice to DNA-damaging agents. A, sensitivity to 6-tg; B, sensitivity to UV light; C, sensitivity to cis-platinum; D, sensitivity to ioniz
ing radiation. The results represent mean survival 6-thioguanine
values based on 2—6independent experiments. The
@g/m1)
Uv (j/m2)
range of plating efficiencies of the E7/Ras-immor talized mouse embryo fibroblasts was 33—I10% for
the Msh2+/+ cells, 8—52%for the Msh2+/— cells, and 29—130%for the Msh2—/— cells. , values for the Msh2—/— cells; L@,Msh2+/—cells; ., Msh2+/+ cells. Data presented are for one
C
D
E7/ras-transformed line derived from Msh2+/+, +1—, or —I— mice. Two additional clones from Msh2+/+ mice and one additional clone from Msh2—/—mice gave similar results (data not
shown). C .@
LI
‘U be .@
.5 a
.0001 0.0
0.2
0.4 0.6 0.8 cisplatinu.m (IIM)
rate of mutation in the Msh2 +1—line may be a reflection of the lower plating efficiency of these cells. Plating efficiencies ranged from 33 to 110% for the Msh2+/+ cells, from 8 to 52% for the Msh2+/— cells, and from 29 to 130% for the Msh2—I—cells. Mutant p53-transfected embryo fibroblasts were also examined for mutation
rates
to ouabain
resistance
to determine
whether
the gene(s)
used for immortalization influenced mutation rates. Mutant p53-trans fected embryo fibroblast clones derived from —I— mice showed a very similar response in mutation rate: an 8-fold increase in the rate of mutation to ouabain resistance relative to p53-transfected fibroblasts derived from Msh2+/+ mice and a 70-fold increase compared to cells derived from the +1—mice (Table 2). Sensitivity to Damaging Agents. To determine whether cells de rived from the Msh2—/—,+1—, or +1+ mice displayed altered sensitivity to DNA-damaging agents similar to that shown by the mismatch repair-deficient human colorectal carcinoma cell lines, we determined response of these cells to 6-tg, UV light, cis-platinum, and ionizing radiation. In these experiments, both E7/Ras-transfected fi broblasts and spontaneously immortalized lines growing out of the primary
mouse
embryo
fibroblast
cultures
were
used.
Both
types
1.0
1.2
4
6
8
10
12
Radiation(Gy)
levels of resistance found in human colon cancer cell lines. Sponta neously and E7/Ras-immortalized cells derived from Msh2+/— mice showed no significant change in sensitivity to 6-tg relative to lines derived from the +1+ mice. Different results were obtained for spontaneously immortalized and E7/Ras-transfected mouse embryo fibroblasts in response to UV, ionizing
radiation,
or cis-platinum
treatment
(Figs.
2 and
3). Sponta
neously immortalized cultures derived from Msh2—I—mice were significantly more resistant (more than 2-fold) to the cytotoxic effects of UV compared with cells derived from +1+ or +1— mice (P = 0.02). E7/Ras-immortalized cells from the —I— mice showed no difference in UV sensitivity compared to those derived from +1+ mice. Spontaneously immortalized mouse fibroblasts derived from Msh2—/—and +1—embryos were significantly more resistant to cis-platinum than those from +1+ embryos (P = 0.02 and 0.05, respectively). E7/Ras-transfected fibroblasts from +1+ or —I— em bryos showed no difference in sensitivity to cis-platinum. E7/Ras and spontaneously
immortalized
cell lines derived
from all three types of
mice showed similar responses to ionizing radiation.
of
Msh2—/—lines showed a statistically significant (3—50-fold; DISCUSSION P = 0.04 and 0.01 for the E7/Ras and spontaneously immortalized cells, respectively) resistance to the cytotoxic effects of 6-tg relative We examined embryonic fibroblast cell lines derived from to the Msh2+/+ or +1—lines (Figs. 2 and 3). This is similar to the Msh2—/— , +1—,and +1+ mice to determine whether they developed 3768
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MUTATOR PHENOTYPE IN MSH2-DEHCIENT MOUSE FIBROBLASTS
A
B
I
@
.1 .@
.01
a. 4' ‘4
.5
Fig. 3. Sensitivity of spontaneously immortal ized mouse
embryo
cells derived
from Msh2+/+,
.001
+1—,and —I—mice to DNA-damaging agents.
0
The resultsrepresentmeansurvivalvaluesbased on 3—6independent experiments. A, sensitivity to 6-tg; B, sensitivity to UV light; C, sensitivity to cis-platinum;
4
8
6-thioguanine
12
16
20
UVQ/m2)
(jag/mi)
D, sensitivity to ionizing radiation.
The results represent mean survival values based on 2—6independent experiments. The range of plating efficiencies of the spontaneously immortal ized mouse embryo fibroblasts was 0.27—6.2%for the Msh2+/+ cells, 0.31—3.5%for the Msh2+/— cells, and 0.4—31%for the Msh2—/— cells. U, values for the Msh2—/— cells;
C
D
@,Msh2+/— cells;
., Msh2+/+ cells.
.1
.1
.@
.5
I
.01
.001
0
2
4
6
8
10
12
Radiation (Gy) cisplatinum
a mutator phenotype similar to that found in human colorectal carcinoma cell lines deficient in mismatch repair. Colon cancer cell lines have numerous alterations of proto-oncogenes and tumor suppressors that could influence this phenotype. The use of the mouse embryo cell lines appears to circumvent this difficulty, although the lines used have been immortalized to facilitate the measurements of mutation rate and sensitivity to agents that induce the futile repair cycle. Our data indicate that the E7/Ras-immor talized mouse line derived from Msh2—/-—mice develops micro satellite instability, resistance to the cytotoxic effects of 6-tg, and a 6—12-fold increase in mutation rate. Thus, by these criteria the mutator phenotype in the immortalized Msh2—/—embryo fibro blast lines is similar to that found in the repair-deficient human tumor cell lines (7, 8, 16, 18). There are quantitative differences in the elevation of mutation rates in the mouse Msh2-deficient cells and the rates reported for the human colorectal carcinoma cell lines deficient in hMLHJ or hMSH6. Mutation rates at the HPRT locus in the human tumor cell lines are elevated 100—700-fold (16, 18), whereas the rates of mutation to ouabain resistance are elevated 24—30-fold.5 The Hprt mutation rate reported here for the 5 B. Richards
and
M.
Meuth,
unpublished
data.
(pM)
Msh2—/—lines is very similar to the rates measured for the human tumor cell lines. However, the rate of Hprt mutation in the mouse Msh2+I+ lines appears to be about 10-fold higher than the rate for mismatch repair-proficient human cell lines. Mismatch repair-deficient mouse fibroblasts were resistant to 6-tg, much like the repair-deficient colon cancer cell lines (43). However, the E7/Ras-transformed mouse fibroblast lines derived from Msh2—I-- mice showed no change in sensitivity to cis-platinum, UV light, or ionizing radiation. Spontaneously immortalized mouse cells derived from the Msh2—/—mice were significantly more resistant to cis-platinum and UV light but not ionizing radiation. Presumably, cells deficient in Msh2 were unable to recognize the platinum adducts and initiate the futile repair cycle. The resistance to UV light is unexpected, given the increased sensitivity of human mismatch repair deficient cell lines reported previously (26). Nevertheless, the differ ences in the responses in the two sets of mouse embryo fibroblast lines suggest that the altered sensitivity to some of these DNA-damaging agents may be a product of immortalization or alterations of growth controls, as well as mismatch repair status. For example, previous studies indicate that mutations ofp53 may induce resistance to DNA damaging agents, such as UV light, by eliminating p53-dependent apoptosis (44). Thus, some of the phenotypic alterations found in the
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MUTATOR PHENOTYPE IN MSH2-DEFICIENTMOUSE FIBROBLASTS
mismatch repair-deficient human tumor cell lines may not be solely the result of the repair deficiency. Because of the high penetrance of HNPCC (34), another question that we addressed in these studies was whether the Msh2 heterozy gotes displayed any changes in mutation rate or sensitivity to DNA damaging agents that might predispose these cells to loss of the remaining Msh2 wild-type allele. No increase in mutation rate was seen in these heterozygous cells; in fact, they had consistently lower mutation
rates
than
the
Msh2+/+
cells.
It is also
notable
that
the
Msh2 heterozygous mouse cells showed no alteration in sensitivity to
Uv relativeto theMsh2+/+ cells.Theseresultsshouldbe considered in light of the recent demonstration of a deficiency in transcription coupled repair in a human lymphoblastoid cell strain heterozygous for hMSH2 and proficient in mismatch repair (26). Thus, except for the increased resistance to cis-platinum seen in the spontaneously trans formed mouse cells, our studies fail to reveal any phenotype in the Msh2-heterozygous mouse cells that might provide a selective growth advantage or predispose to the acquisition of further mutations in mismatch
repair
genes
or genes
that control
cell growth.
These studies support the idea that the loss of mismatch repair results
in an increase
in spontaneous
mutation
rate. Secondary
alter
ations of replication fidelity, other repair genes, or genes regulating the cell cycle may further enhance this mutator phenotype. Presum ably, this mutator phenotype increases the likelihood of mutations in tumor suppressor or proto-oncogenes that are necessary for carcino genesis. The detection of mutations in tumor suppressor genes at sites similar to hot spots of mutation in the HPRT gene in mismatch repair-deficient colon cancer cell lines further supports this proposal (16, 45—47).The questions of why tumor development is restricted to only a few tissue types despite the expression of Msh2 in a wide range of different tissues (40, 48, 49) and why only limited numbers of tumors were seen throughout the intestines of individual Msh2-defi cient animals,
although
every
crypt stem cell is homozygous
gene defect (unlike human HNPCC heterozygotes), resolved.
for the
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