(Received for publication, April 24, 1995; and in revised form, June 7, 1995)
From the
Human cells contain several homologs of the bacterial mutL gene required for mismatch repair, including a gene on chromosome
7 designated hPMS2. We have identified an endometrial
carcinoma cell line, HEC-1-A, that has a C
Instability of simple repetitive sequences is common in tumor
DNAs from hereditary nonpolyposis colorectal carcinoma (HNPCC) ( Supporting a role for the hMLH1 and hMSH2 gene
products in mismatch repair is the finding that extracts of colon and
endometrial tumor cell lines with a mutation in hMSH2 or hMLH1 are defective in mismatch
repair(16, 17) . These cells also exhibit
microsatellite instability, and several such lines have elevated
mutation rates in endogenous genes ((16, 17, 18, 19, 20, 21) and
references therein). Cell lines defective in mismatch binding and/or
repair have been shown to be resistant to killing by treatment with
otherwise lethal doses of DNA alkylating agents ( (20, 21, 22) and reviewed in (23) ).
Moreover, the introduction of chromosome 3 containing a wild-type copy
of the hMLH1 gene to a hMLH1-defective line restores
DNA mismatch repair activity, reduces microsatellite instability, and
increases sensitivity to MNNG (21) . These data strongly imply
that both hMSH2 and hMLH1 function in mismatch
repair. Eukaryotic cells contain several genes that share sequence
homology with the bacterial mutL gene. In yeast, these are
designated yMLH1(24) and yPMS1(25, 26) . Interaction of the yMLH1 and
yPMS1 proteins has been demonstrated by protein affinity
chromatography(27) . Moreover, ypms1 mutants have
elevated mutation rates and are epistatic for this phenotype with yeast ymlh1 mutants(24) , supporting a role for yPMS1 in mismatch repair. Additional genes exist in yeast that, when
mutated, yield phenotypes similar to ypms1 mutants ( (28) and references therein). Human cells likewise contain
several mutL homologs. In addition to the hMLH1 gene
on chromosome 3(2, 8) , there are mutL homologs designated hPMS1 located on chromosome 2 (10) and hPMS2 located on chromosome
7p22(10, 29) . The latter is closely related by
sequence homology to the yPMS1 gene. The hPMS2 has
been described as a member of a subfamily that includes at least two
related genes located on chromosome 7q(29) . Recently, Horii et al.(30) have isolated additional PMS-like
sequences and have suggested that hPMS genes constitute a
family containing at least 11 members. Given the large number of hPMS sequences, what is the evidence that any of these
function in mismatch repair? A role for both hPMS1 and hPMS2 in cancer, and by extrapolation, in mismatch repair, has
been inferred from a mutation found in a single HNPCC family for each
gene(29) . However, since HNPCC individuals are at risk for
developing alterations in many genes, it is possible that these single
examples of hPMS1 and hPMS2 mutations are not
causally related to disease progression or to the presumed mismatch
repair defect. A functional role for hPMS2 in mismatch repair is also
suggested by the observation that a mismatch repair-defective extract
made from a cell line containing a mutant hMLH1 gene is
complemented by a purified fraction containing hMLH1 and a second
protein that is either hPMS2 or a closely related gene
product(31) . As noted by the authors, the identity of this
second protein is uncertain because the complementing protein is 110
kDa while the deduced amino acid sequence of the hPMS2 gene
product predicts a 95,808-kDa protein and because one putative peptide
sequence derived from the 110-kDa protein was not found in the
predicted hPMS2 polypeptide. Thus, the role of hPMS2 in
mismatch repair and in cancer is not yet as clearly established as for hMSH2 and hMLH1, where a wealth of information is
available on many mutations in HNPCC families, in tumor DNA, and in
tumor cell lines. Beyond the report by Nicolaides et
al.(29) , no additional HNPCC kindreds, tumor DNAs, or
tumor cell lines have been reported to contain mutations of any hPMS gene. Establishing a role in mismatch repair (or a lack
thereof) for hPMS genes would be facilitated by identifying
and characterizing tumor cell lines containing mutations in these
homologs. Toward this end, we describe here a mutation in the hPMS2 gene in a human endometrial carcinoma cell line (HEC-1-A) that
yields a truncated gene product but no detectable wild-type hPMS2
protein. We then show that it has the properties predicted of a cell
line defective in mismatch repair.
Figure 1:
Mutation
detection in HEC-1-A cells. a, protein truncation test of
RT-PCR products of codons 415-862 of hPMS2. The proteins
produced by in vitro transcription and translation were
separated on a 15% SDS-polyacrylamide gel. Sizes of prestained
molecular weight standards are indicated to the left. Lane1 is the protein produced from normal uterine tissue. Lane2 is the protein from HEC-1-A cells. b,
sequence of RT-PCR products. Sequence 1 is from HEC-1-A cells,
demonstrating the C to T change in the first position of codon 802 of hPMS2 (marked by an asterisk). Sequence 2 is from the
normal human uterus RT-PCR product and is the wild-type hPMS2 sequence.
To determine the exact nature of the mutation in hPMS2, the sequence of the C-terminal end of the gene was
determined from the PCR product that was used to generate the truncated
protein. A single C to T mutation was found at nucleotide 2428 in codon
802 (Fig. 1b), creating a TGA termination codon that
predicts a protein truncated by 61 amino acids. This result is
consistent with the mobility shift seen in Fig. 1. No band
indicative of the wild-type nucleotide was observed at this position on
the sequencing gel. To confirm the absence of a wild-type gene
sequence, an independent RT-PCR amplification was performed, and the
resulting fragment was cloned and sequenced. Ten of 10 clones contained
the expected C to T transition, and no wild-type clone was observed. To
establish that the observed mutation was in hPMS2 and not in a
closely related PMS gene, we sequenced 325 nucleotides of the
gene, corresponding to amino acid residues 753-861. Except for
the substitution at codon 802, the sequence was identical to the
published hPMS2 gene sequence(29) .
Figure 2:
Microsatellite analysis at a CTT repeat. a, analysis of single cell HEC-1-A clones. The lane marked C represents the clonal population of cells from
which the single cell clones were generated. b, analysis of
single cell KLE clones.
As previously reported(17) , repair of substrates containing
single base mismatches and one or more unpaired nucleotides is readily
detected in a TK6 cell extract used as a positive control (Fig. 3). An extract of KLE cells also repairs these same
mismatches. In contrast, an extract of HEC-1-A cells is defective in
repair of all substrates examined. This deficiency is observed for
substrates containing the nick on either the 3` or 5` side of the
mismatch (Fig. 3).
Figure 3:
Mismatch repair activity in three cell
lines. The analysis was performed as described under ``Experimental Procedures'' using the substrates
indicated (described in (17) ) and reactions incubated for 15
min. Substrates designated A contain a nick 3` to the listed
mismatch, while the one designated B contains a nick 5` to the
G
Figure 4:
MNNG cytotoxicity in endometrial cancer
cell lines. Relative survival of HEC-1-A and KLE cells after a 45-min
exposure to an increasing concentration of MNNG is shown. Each point is the average of three independent
experiments.
A defective hPMS2 gene has been implicated in the
development of colon cancer by the observation of a mutation in one
HNPCC kindred(29) . A functional role for hPMS2 in mismatch
repair has previously been suggested by the close gene sequence
homology to the yeast mutator gene yPMS1(29) and by
complementation of a mismatch repair-defective extract by two proteins,
one of which is either hPMS2 or a closely related gene
product(31) . The present study directly supports a role for
hPMS2 in mismatch repair by evaluating predicted phenotypes in an
endometrial carcinoma cell line containing a nonsense codon in the hPMS2 gene. Although we cannot exclude the presence of
mutations in other genes in the HEC-1-A cell line, the protein
truncation and DNA sequencing data (Fig. 1) establish that
HEC-1-A cells do lack wild-type hPMS2. The protein that is
observed is shorter and presumably missing 61 C-terminal amino acids.
These amino acids are highly conserved among yPMS1, hPMS1, and hPMS2 genes(29) , suggesting that
they are functionally important. The evident lack of a wild-type
transcript also suggests that both copies of this gene must be
inactivated in order for the defect to be manifest. This reduction to
homozygosity is consistent with similar allelic inactivation observed
with hMLH1 and hMSH2 mutations in cell lines and
primary
tumors(8, 9, 10, 12, 16, 17) .
In the previously reported hPMS2 mutation, deletions were
identified in both alleles of an unspecified tumor arising in this
kindred, one of which was germline and the other somatic(29) . Supporting a functional role for hPMS2 in mismatch repair is the
observation that HEC-1-A cell extracts are deficient in mismatch repair (Fig. 3). The substrates used here already contain an incision
to direct repair to the minus strand, and repair is defective
regardless of whether the nick is 3` or 5` to the mismatch. Thus the
defect is not in incision activity, but it could be prior to incision
or it may involve the excision step. Both possibilities are consistent
with the recent study suggesting that hPMS2 or a related protein forms
a heterodimer with hMLH1(31) . This complex has been designated
hMutL HEC-1-A cells also
exhibit the microsatellite instability characteristic of mismatch
repair-deficient cell lines. Instability was noted using five different
repeat sequences. The greatest instability was seen with a
(CTT) As has been noted
previously, mismatch repair in bacteria and humans shares a number of
common mechanistic features (reviewed in (36) ). Nonetheless, E. coli contains one mutL gene while yeast and humans
clearly contain two or more genes sharing close sequence homology to mutL. These homologs could be involved in substrate- or
tissue-specific mismatch repair processes. The results presented in
this study directly implicate a biochemical role for one of these
homologs, hPMS2, in the repair of mispaired and unpaired nucleotides.
They also substantiate a role for hPMS2 inactivation in the development
of tumors of the endometrium, colon, and other tissues associated with
HNPCC kindreds.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
T mutation in hPMS2 that generates a nonsense codon and yields a protein
truncated at the C terminus. No wild-type gene or gene product was
detected. The missing amino acids in hPMS2 are highly conserved among PMS homologs, suggesting that they may be critical for
function. In support of this, extracts of HEC-1-A cells are defective
in repairing a variety of mismatched substrates. Moreover, di-, tri-,
and tetranucleotide repeated sequences are highly unstable in single
cell clones of HEC-1-A cells, and HEC-1-A cells are resistant to
killing by N-methyl-N`-nitro-N-nitrosoguanidine. The
results provide strong experimental support for the involvement of the
hPMS2 gene product in mismatch repair in human cells and support the
concept that a defective hPMS2 gene may lead to predisposition
to certain forms of cancer.
)kindreds and from individuals with sporadic cancers that
are commonly associated with
HNPCC(1, 2, 3, 4, 5, 6) .
The majority of HNPCC kindreds have germline alterations in human
homologs of the bacterial mismatch repair genes mutS and mutL(7, 8, 9, 10, 11, 12, 13) .
Inactivation of the human hMSH2 and hMLH1 genes in
cells of these individuals is thought to result in a mutator phenotype
that is required for subsequent tumor progression (for review, see
Refs. 14 and 15).
Cell Lines
Human endometrial carcinoma cell
lines HEC-1-A and KLE were from the American Type Culture Collection.
TK6 cells were from P. Modrich (Duke University).Single Cell Clone Analysis of Microsatellite
Instability
Single cell clones of HEC-1-A and KLE cells were
made from a limiting dilution of a clonal population of cells. DNA
suitable for PCR was prepared from cells representing 18-25
doublings by a 1-h digestion at 56 °C in 100 µl of 1
PCR buffer (see below) without MgCl
and containing 100
µg/ml proteinase K. The DNA preparation was heated at 95 °C to
inactivate the proteinase K before amplification. DNAs were amplified
by the polymerase chain reaction using oligonucleotides specific for
several repetitive sequences. Primers were from Research Genetics
(Huntsville, AL), and references can be obtained from the Johns Hopkins
University genome data base (or, for vWFa, see (32) ). Each PCR
(10 µl) consisted of 50 mM KCl, 10 mM Tris-HCl
(pH 8.3), 1.5 mM MgCl
, 200 µM of each
deoxynucleotide triphosphate, 1 µM of each primer, 1
µl of DNA, and 1 unit of Amplitaq polymerase (Roche Molecular
Systems, Alameda, CA). One primer was end-labeled with
[
-
P]ATP and T4 polynucleotide kinase prior
to addition to the reaction. PCR was performed for 35 cycles consisting
of 20 s each at 94, 55, and 72 °C. Following amplification, the
reaction was diluted 1:1 in a denaturing loading buffer of 98%
formamide, 10 mM EDTA (pH 8.0), 0.02% bromphenol blue, 0.02%
xylene cyanol FF and denatured at 95 °C for 3 min. One-fifth of the
denatured product was subjected to electrophoresis for 3 h at 90 watts
in a 6% polyacrylamide gel containing 8.3 M urea and 30%
formamide. Following electrophoresis gels were covered in a single
layer of mylar (Ambis Systems, San Diego, CA), dried, and exposed to
x-ray film.
Analysis of hPMS2 by Protein Truncation Test and DNA
Sequencing
Total RNA was prepared from cell lines using the
single step procedure described by Chomczynski and Sacchi(33) .
First strand cDNA was prepared as described previously (34) and
used as template for amplification of hPMS2 in two fragments
spanning codons 1-472 and codons 415-863, using
oligonucleotides containing T7 RNA polymerase sites as
described(12) . A portion of each amplified product (5 µl)
served as a template for coupled transcription and translation in
10-µl reactions using the TNT system (Promega,
Madison, WI) that incorporates [
S]methionine to
label the protein product. The reactions were diluted 1:5 in SDS
loading buffer and denatured at 97 °C for 5 min, and 4 µl was
loaded onto 15% SDS gels. The remainder of the PCR product DNA was
gel-purified and sequenced on both strands, using Sequenase v2.0. The
primers used were PMS2-2282 (TTGTTATCGATGAAAATGCTC) and
PMS2-2463 (GTCCCAATCATCACCGACTTC). Upon identification of the
mutation (see below), a second amplification was performed, the
fragment was cloned, and clones were sequenced to confirm the mutation.
The hPMS2 gene sequence was determined for codons
753-860.
Mismatch Repair Assay
Cell-free extracts and
mismatched substrates were prepared as described(35) .
Substrates contained the indicated mismatch and a nick in
the(-)-strand at position -264, where position +1 is
the first transcribed nucleotide of the LacZ gene. Repair
reactions (25 µl) contained 30 mM Hepes; 7 mM
MgCl
; 200 µM each CTP, GTP, UTP; 4 mM ATP; 100 µM each dCTP, dATP, dGTP, dTTP; 40 mM creatine phosphate; 100 mg/ml creatine phosphokinase; 15 mM sodium phosphate (pH 7.5); 1 fmol of substrate DNA; and 50 µg
of extract proteins. Reactions were incubated for 15 min at 37 °C.
The substrate DNA was recovered and introduced into Escherichia
coli NR9162 (mutS) via electroporation and plated to
score plaques as described(34) .
MNNG Cytotoxicity
Non-confluent cell monolayers
were harvested and rinsed in several changes of serum-free medium, and
10 cells were treated with either 0, 1, 5, or 10 µM MNNG at 37 °C in serum-free medium for 45 min. Cells were
rinsed twice in growth medium and then plated on 100 mM dishes
at concentrations of 10
, 10
, and 10
cells/plate. After 10 days, cell colonies were fixed with
methanol and stained with Giemsa, and colonies with 50 or more cells
were counted. The relative surviving fraction was expressed as a ratio
of the plating efficiency in treated cultures to the survivors with no
MNNG exposure.
HEC-1-A Cells Contain a hPMS2 Gene Mutation
We
examined a variety of cell lines derived from endometrial
tumors(5) , the most common HNPCC-associated non-colon tumor
type, for mutations in the four genes reported to have alterations in
HNPCC kindreds. Among the approaches we use to screen for mutations is
a protein truncation assay (12) , in which tumor cell line RNA
for a specific gene sequence is converted by RT-PCR to cDNA. This DNA
is transcribed and translated in vitro with
[S]methionine to label the protein products,
which are then resolved by SDS-polyacrylamide gel electrophoresis and
detected by autoradiography. The procedure rapidly detects shorter
protein products resulting from mutations such as premature termination
codons, frameshifts, deletions, or aberrant splicing. We screened a
panel of endometrial tumors and tumor cell lines for mutations using
primers designed to yield PCR products spanning either codons
1-472 or codons 415-862, respectively, of the hPMS2 gene(29) . The product of the latter fragment that was
amplified from normal endometrial tissue generated the expected protein
of
50 kDa (Fig. 1a, lane1).
Although this same result was obtained with several tumor cell lines
(including KLE cells, see below), we found one cell line, designated
HEC-1-A, that yielded a hPMS2 polypeptide that was slightly shorter
than wild type (Fig. 1a, lane2). No
product of wild-type size was observed, suggesting a reduction to
homozygosity for this alteration at the level of RNA expression.
Parallel analyses of the other half of the gene and of the hMSH2, hMLH1, and hPMS1 genes revealed no
detectable truncations of any of these genes in HEC-1-A cells (data not
shown).
Microsatellite Instability in HEC-1-A Cells
We
next examined HEC-1-A cells for the microsatellite instability
characteristic of tumor cell lines having mutations in the hMSH2 and hMLH1 genes(16, 17) . Single cell
clones were generated from clonal populations of HEC-1-A cells and from
KLE, an endometrial carcinoma cell line that had no detectable hPMS2
mutation (see above) and was proficient in mismatch repair (see below).
When the clones of each line reached 18-25 population doublings,
DNA was prepared and analyzed for variations at several microsatellite
sequences using the PCR. Microsatellites were highly unstable in
HEC-1-A clones. An example of (CTT) repeat
(D7s1794) instability is shown in Fig. 2a. Eighteen of
40 single cell clones exhibited new bands, representing both expansions
and contractions. Instability was noted in 3 of 40 clones for D2s147
(CA)
, 3 of 40 clones for D14s76
(CA)
, 17 of 40 clones for D14s73
(CA)
, and 2 of 40 for vWFa (TCTA)
(data not shown). Thus, although the degree of fluctuation
varied depending on the marker used, all microsatellites examined had
at least one example of an altered allele. As expected of a mismatch
repair-proficient cell line, no variations in microsatellite sequences
were present in 29 KLE clones examined (see Fig. 2b for
D7s1794, data not shown for D2s147, D14s76, and D14s73, vWA not
examined).
Analysis of Mismatch Repair
To determine if
HEC-1-A cells are deficient in mismatch repair, cell-free extracts were
prepared and tested for their ability to repair a variety of mispaired
substrates in vitro. A circular M13mp2 DNA substrate was used,
containing a covalently closed (+)-strand and a(-)-strand
with a nick (to direct repair to this strand) located several hundred
base pairs away from the mispair located in the lacZ -complementation coding sequence. The (+)-strand encodes
one plaque phenotype (either colorless or blue) while
the(-)-strand encodes the other plaque phenotype. If the
unrepaired heteroduplex is introduced into an E. coli strain
deficient in methyl-directed heteroduplex repair, plaques will have a
mixed plaque phenotype on selective plates, due to expression of both
strands of the heteroduplex. However, repair occurring during
incubation of the substrate in a repair-proficient human cell extract
will reduce the percentage of mixed plaques and increase the ratio of
the (+)-strand phenotype relative to that of the(-)-strand
phenotype, because the nick directs repair to the(-)-strand.
G mismatch at position 89. Results are expressed as percent
repair determined from counting several hundred plaques per variable.
Measurements with two of the substrates were performed multiple times
with similar results. Repair values of
1% are represented as 1%.
Repair of the (A
-) substrate in a KLE extract was not examined.
All three extracts were competent for SV40 origin-dependent DNA
replication activity (e.g. see (17) ), demonstrating
that the repair-defective extracts are not defective in all DNA
transactions.
MNNG Cytotoxicity
Several mismatch
repair-deficient cell lines have been shown to be resistant to the
cytotoxic effect of treatment with MNNG ( (20, 21, 22) and reviewed in (23) ).
In one instance, sensitivity to MNNG has been restored to a mismatch
repair-defective, alkylation-resistant hMLH1 mutant cell line
via transfer of a wild-type chromosome 3 that restores mismatch repair
activity(21) . To determine if the correlation between
defective mismatch repair and MNNG resistance holds for HEC-1-A cells
containing a hPMS2 mutation, we treated these cells with
increasing concentrations of MNNG (Fig. 4). MNNG concentrations
up to 10 µM were without significant effect, while
mismatch repair-proficient KLE cells were more sensitive.
, by analogy to the homodimeric MutL complex of E.
coli, whose function is suggested to be formation of a complex
with MutS that activates incision by MutH and then initiates excision
of nucleotides (for review, see (36) ).
repeat (D7s1794, Fig. 3) and one of
three (CA)
repeats examined (D14s73). Instability
was also detected with a (TCTA)
repeat (vWFa) and
two other (CA)
repeats. The degree of instability
was less with these latter markers, just as has been seen in other
mismatch repair-defective cell lines. (
)The instability
reported here is in accord with previous reports of microsatellite
instability in mismatch repair-defective hMSH2 and hMLH1 mutant cell lines(16, 17) .
We thank John D. Roberts and Kenneth R. Tindall for
evaluating the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.