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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1767-1772
NEOPLASIA
MSH2-deficient murine lymphomas harbor insertion/deletion
mutations in the transforming growth factor beta receptor type 2 gene
and display low not high frequency microsatellite instability
Robert Lowsky,
Anthony Magliocco,
Ryo Ichinohasama,
Armin Reitmair,
Stuart Scott,
Michele Henry,
Marshall E. Kadin, and
John F. DeCoteau
Saskatoon Cancer Centre and Department of Pathology,
Royal University Hospital and University of Saskatchewan, Saskatoon,
Saskatchewan, Canada; Department of Pathology, Tohoku University
Hospital, Aoba-Ku, Sendai 980, Japan; Ontario Cancer Institute and
Department of Pathology, Princess Margaret Hospital and the University
of Toronto, Toronto, Ontario, Canada; Department of Pathology, Beth
Israel Hospital and Harvard Medical School, Boston, MA.
 |
Abstract |
High-frequency microsatellite instability (MSI), defined as more
than 20% unstable loci, is an inconsistent finding in hematologic malignancies; consequently, the significance of deficient DNA mismatch
repair (MMR) to their pathogenesis has been questioned. To further
investigate the relationship between MMR deficiency and genomic
instability in hematologic malignancies, this study evaluated
MSH2 / murine lymphomas for insertion/deletion (ID) mutations within the transforming growth factor (TGF)-beta receptor type II (T R-II) gene and MSI at 10 neutral microsatellites.
The lymphomas displayed ID mutations within short mononucleotide runs of TR-II at a high frequency, whereas nonmalignant tissue
from corresponding animals lacked mutations. Loss of
TR-II transcripts and protein was seen in 6 of 7 murine
lymphomas harboring acquired TR-II mutations. In the
analysis of paired nonmalignant and tumor DNA samples, low-frequency
but not high-frequency MSI was found. Low-frequency MSI occurred in 8 of 20 lymphomas and 12 displayed microsatellite stability. MSI was even
less frequent in nonmalignant tissue as only 3 of 20 samples displayed
low-frequency MSI and 17 displayed stability. Evaluation of 20 single
cell clones from the MSH2/ lymphoma cell lines R25 and
L15 identified high-frequency MSI in 4 and 2 clones,
respectively. The remaining clones showed low-frequency MSI or
stability. These findings suggest that acquired TR-II
mutations represent important inactivating events in tumor pathogenesis following MSH2 deficiency. Furthermore, for
some hematolymphoid malignancies, the evaluation of cancer-associated genes for ID mutations may represent a more sensitive marker of MMR
deficiency than evaluation of neutral microsatellites for high-frequency MSI.
(Blood. 2000;95:1767-1772)
© 2000 by The American Society of Hematology.
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Introduction |
Cancers displaying high-frequency microsatellite
instability (MSI), defined as more than 20% unstable loci, are
considered to have a defective DNA mismatch repair (MMR)
system.1,2 This relationship has been conclusively
demonstrated in hereditary non-polyposis colorectal carcinoma (HNPCC)
and in some sporadic endometrial, colon, gastric, and pancreatic
cancers not associated with HNPCC.2-5 In contrast, tumors
displaying low-frequency MSI often appear to have intact MMR gene
systems.1-3 High-frequency MSI has not been a consistent
finding in hematolymphoid malignancies and thus the significance of MMR
deficiency to the molecular pathogenesis of these tumors has been
questioned.6-9
Despite the inconsistent finding of high-frequency MSI in
hematolymphoid malignancies, strong evidence implicates defects in MMR
genes with their development. We reported that MSH2 knockout mice (MSH2/) uniformly develop aggressive
lymphomas of thymic origin at an early age that recapitulate the human
entity precursor T-cell lymphoblastic lymphoma (LBL).10
Others have shown that PMS2 and MLH1 knockout mice are
prone to develop lymphomas.11,12 In humans, coding region
mutations in hMSH2 and hMLH1 have been identified in
primary tumor samples from patients with LBL and in acute
lymphoblastic leukemia (ALL) cell lines, respectively.10,13 Zhu et al found 14 of 43 (32.6%) primary acute myelogenous
leukemia (AML) samples lack hMSH2 protein expression.14 Additionally, ID mutations within the BAX
(G)8 mononucleotide run were reported in 4 of 29 (14%) human
hematolymphoid tumor cell lines not selected for MMR
deficiency.15
Nonmalignant tissue harvested from mice lacking MSH2 exhibit
hypermutability. MSH2/ mice carrying the
lacI reporter transgene show an increase in spontaneous
transition, transversion, and insertion/deletion (ID) mutations and
hypermutability on exposure to DNA methylating agents when compared
with MSH2+/+ or MSH2+/ mice.16,17
Furthermore, the finding of identical ID mutations within short
mononucleotide runs (3-5 base pairs) in multiple tissues and in
different animals extends the spectrum of sequences that are altered as
a consequence of MMR deficiency beyond microsatellites.16
In humans, the TR-II gene contains a mononucleotide (A)10
repeat within the 5' end of the coding region that appears to be a consistent target for inactivating ID mutations in gastrointestinal cancers deficient in MMR.18,19 A (GT)3 repeat within the
3' end of the coding region of TR-II is also a
reported target of mutational inactivation in a minority of
MMR-deficient human colon cancers.18 To further investigate
the relationship between MMR deficiency and genomic instability we
evaluated the thymic lymphomas arising in MSH2/
mice for ID mutations within the TR-II gene and the
frequencies of MSI at 10 neutral microsatellites. Compared with
nonmalignant tissues, the murine lymphomas displayed a high frequency
of ID mutations within short mononucleotide runs in the
TR-II gene. A high level of concordance was found between TR-II ID mutations and loss of TR-II mRNA
transcripts and protein. In addition, the murine lymphomas and
nonmalignant tissues showed low-frequency but not high-frequency MSI at
neutral microsatellites. These findings indicate that disruption of the
TR-II gene is likely an important inactivating molecular
event in lymphomagenesis following MMR deficiency. Furthermore, for
some hematolymphoid malignancies, the evaluation of genes implicated in
tumor pathogenesis for ID mutations may represent a more sensitive
marker of MMR deficiency than evaluation of neutral microsatellites for
high-frequency MSI.
| |
Materials and methods |
Specimens
Details for the generation of MSH2/ mice and
characterization of the lymphomas they develop have been previously
reported.10,20 Briefly, the median time to tumor
development was 3.8 months and all tumors represented a single
histopathologic entity closely resembling human precursor T-cell LBL.
By histology, the tumors were comprised of more than 95% malignant
cells. Tumor specimens and histologically nonmalignant tail (germline)
tissue from each animal were snap frozen in liquid nitrogen and stored
at 70°C before DNA and RNA extraction. A portion of each
tumor specimen and nonmalignant small intestine tissue was fixed in
formalin and embedded in paraffin blocks. The cell lines L15 and R25
were established from thymic LBLs from 2 different
MSH2/ mice. Twenty single cell clones from each
cell line were generated by limiting dilution and expanded into
5 × 105 cells (approximately 19 cell divisions) in
tissue culture wells. Cells were harvested by centrifugation and stored
as a cell pellet at 70°C before DNA extraction.
Detection of ID mutations in the TR-II gene by denaturing
polyacrylamide gel analysis
High-fidelity polymerase chain reaction (PCR) amplification was
performed with PfU polymerase (Invitrogen, San Diego, CA) using
genomic DNA isolated from tumor samples and corresponding nonmalignant
tissue. The primers JR-1 5'-GAAGATGCCGCTTCTCCCAA-3', nucleotides 377 to 396, and JR-2
5'-GCTGGTGGTGTATTCTTCCG-3', nucleotides 486 to 505, were
used to amplify a region of the murine TR-II gene (GenBank
Accession # S69 114) containing the mononucleotide (A) repeat
corresponding to the (A)10 repeat of the human TR-II gene.
The primers JR-3 5'-GAGACTTTGACCGAGTGCTG-3', nucleotides 1580 to1599, and JR-4 5'-CCATCTTCTGGAATCTTCTC-3',
nucleotides 1700 to1719, amplified the area of the gene containing the
3' (GT)3 repeat tract. The blunt-ended PCR products generated by PfU polymerase were resolved on 6% denaturing polyacrylamide, 8.3 mol/L urea gels, transferred to nylon membranes and UV
cross-linked. The PCR products were hybridized to internal
oligonucleotide probes, JR-14 5'-GGCGAGACTTTCTTCATGTG-3',
nucleotides 425 to 444, or JR-15 5'-GCAGAGCGCTTCAGTGAGCT-3', nucleotides 1640 to 1659, end-labeled using T4 polynucleotide kinase and  32P-dATP.
Sequence analysis
The PCR products were cloned using the Zero-Blunt cloning system
(Invitrogen). Ten clones from 2 separate PCR experiments were sequenced
using the ABI Prism Dye Terminator Cycle Sequencing kit with AmpliTaq
FS (Perkin Elmer ABI, Foster City, CA) and analyzed on a 310 Genetic
Analyzer (Perkin Elmer ABI).
Reverse transcriptase PCR (RT-PCR)
Total cellular RNA was isolated from tumor samples by the RNAzol B
method according to the manufacturer's instructions (Cinna/Biotecx, Friendswood, TX). Single-stranded complementary DNA (cDNA) was prepared from total cellular RNA by reverse transcription according to
the manufacturer's recommendations (Perkin-Elmer, Branchburg, NJ). To
evaluate expression of TR-II messenger RNA (mRNA), cDNA was
amplified using the primers JR-5
5'-CTCACCTACCACGGCTTCAC-3', nucleotides 353 to 372, and
JR-6.5'-CTCAGCTTCTGCTGCCGGTG-3', nucleotides 611 to 630. The cycling conditions were 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute for 30 cycles. The primers JR-7
5'-GCTGCTTTCAGGTTTATGAG-3', nucleotides 701 to 720, and
JR-8 5'-GATGCAAGCTAAAAGACATA-3', nucleotides 899 to 918, were used to evaluate the expression of TR-I mRNA as a
control (GenBank Accession # L15 436). Cycling conditions for these
sets of experiments were 95°C for 1 minute, 55°C for 1 minute,
and 72°C for 1 minute for 30 cycles. PCR products were visualized
by electrophoresis in a 1.8% agarose gel containing ethidium bromide.
All experiments were performed in duplicate.
Immunohistochemistry
Immunohistochemical detection of TR-II was performed using an
affinity purified rabbit polyclonal antibody to human TR-II prepared
against a synthetic peptide corresponding to amino acid residues 246 to
266 (L-21; Santa Cruz Biotechnology, Santa Cruz, CA) that cross-reacts
to mouse TR-II. Staining was done on formalin-fixed paraffin
embedded tissue sections using the ImmunoCruz Staining System (Santa
Cruz Biotechnology). In brief, sections were de-paraffinized and heated
at 95°C in 10 mM sodium citrate for 5 minutes. Quenching of
endogenous peroxidase activity and blocking of nonspecific protein
binding was accomplished using reagents supplied by the manufacturer.
Sections were sequentially incubated with prediluted L-21 primary
antibody for 2 hours, a biotinylated secondary antibody for 30 minutes,
and horseradish peroxidase-strepavidin complex for 30 minutes with
phosphate-buffered saline (PBS) washing steps between incubations.
After a final wash step, the immunostaining reaction was revealed by
incubation with a substrate prepared from substrate buffer, DAB
chromogen and peroxidase substrate supplied by the manufacturer. Slides
were then counterstained in Gill's hematoxylin.
Western blot analysis
Protein samples were separated by electrophoresis on a 10% sodium
dodecyl sulfate (SDS)-polyacrylamide gel, transferred to nitrocellulose
membranes (Bio-Rad, Hercules, CA), and blocked in 5% nonfat milk
powder and 0.1% Tween 20 in buffered saline (PBS-T). The membranes
were then incubated with a diluted (1:500 in 1% milk powder, PBS-T)
rabbit polyclonal antibody, corresponding to amino acids 1 to 567 of
human TR-II that cross-reacts with mouse TR-II (H-567; Santa Cruz
Biotechnology) overnight at 4°C. Following incubation with the
primary antibody, the membranes were incubated with a horseradish
peroxidase conjugated antirabbit antibody (1:2000 in 1% milk powder
PBS-T) for 2 hours at room temperature. The membranes were then
developed using a chemiluminescence detection system (NEN Life Science
Products, Boston, MA) and exposure to Hyperfilm MP (Amersham Pharmacia
Biotech, Piscataway, NJ). The membranes were then incubated in
stripping buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 100 mM
2-mercaptoethanol) for 30 minutes at 50°C, washed several times in
PBS-T and re-probed with a rabbit polyclonal antibody corresponding to
an internal domain of TR-I of human origin that cross-reacts with
mouse TR-I (V-22; Santa Cruz Biotechnology) with detection as for
TR-II.
Microsatellite instability analysis
DNA was extracted from tumor and corresponding nonmalignant tissue
from 20 different MSH2/ mice and from 20 single
cell clones from each cell line using DNAzol (Gibco-BRL, Bethesda, MD)
according to the manufacturer's instructions. To assay for MSI, DNA
was genotyped at 10 loci: D1Mit4, D2Mit16, D3Mit11, D4Mit11, D5Mit10,
D6Mit8, D7Mit12, D8Mit4, D9Mit17, and D10Mit2. PCR was performed using
approximately 50 ng genomic DNA, with primers and conditions as
recommended by the protocol supplied by Research Genetics for
MouseMapPairs (Huntsville, AL). Equal volumes of PCR reaction products
and STOP solution (Sequenase kit Version 2.0, USB, Cleveland, OH) were
mixed, boiled, and placed on ice. Then 2 µL of the mixture was loaded
onto 6% polyacrylamide gels containing 8.3 mol/L urea and
electrophoresed at 70 W. Products were transferred to prewetted nylon
membranes and the DNA was fixed by UV cross-linking. Microsatellite
alleles were detected by hybridization to end-labeled sense primers
using T4 polynucleotide kinase and 32P-dATP and exposure
to x-ray film. High-frequency MSI was defined by the presence of
unambiguous band shifts in more than 20% of loci evaluated.
Low-frequency MSI was defined as 20% or less unstable loci and
microsatellite stability as 0% unstable loci.2
| |
Results |
ID mutations in TR-II MSH2/ murine
lymphomas
Frameshift ID mutations within the (A)10 repeat of the
TR-II gene occur frequently in MMR-deficient human colon and
gastric cancers.18,19 The sequence of the (A) repeat of the
murine TR-II gene 5'-AAAAGAAAAG-3', nucleotides
411 to 420, differs from the human (A)10 repeat because it contains
guanine nucleotides at positions corresponding to the 5th and 10th
adenine nucleotides of the human repeat. We analyzed 10 of the
MSH2/ murine lymphomas for ID mutations within a
129 bp DNA fragment containing the (A) repeat by denaturing
polyacrylamide gel electrophoresis. Comparison of tumor DNA with
corresponding nonmalignant DNA showed mobility shifts consistent with
acquired insertion mutations in 6 of 10 (60%) murine lymphomas (Figure
1). Sequence analysis confirmed the
presence of acquired mutations within the (A) repeat in all 6 lymphomas. In 4 of the 6 lymphomas, the second half of the repeat (nucleotides 416-419) was expanded by 1 adenine nucleotide. The wild-type TR-II protein consists of 567 amino acids and these frameshift mutations result in the generation of a premature STOP codon
predicted to encode a truncated protein of 137 amino acids. In the
other 2 lymphomas, acquired insertion mutations occurred in both the
first and second halves of the repeat (Figure
2). Sequence analysis of the 129 bp DNA
fragment from the 4 lymphomas lacking mobility shifts and from
nonmalignant tissue from the 6 mice with ID mutations in the tumor DNA
showed the wild-type sequence in all cases.
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| Fig 1.
Denaturing polyacrylamide gel analysis of the
TR-II gene in MSH2/ murine lymphomas.
Examples of the analysis of DNA containing the mononucleotide (A)
repeat from paired nonmalignant (N) and whole lymphoma tumor specimens
(T). Slow migrating bands consistent with acquired frameshift insertion
mutations are seen in the tumors in 3 of the 4 paired specimens.
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| Fig 2.
Sequence analysis of the TR-II gene in
MSH2/ murine lymphomas.
Examples of the genomic sequence profile of clones obtained from tumor
DNA specimens demonstrating denaturing polyacrylamide gel band shifts
in the region containing the mononucleotide (A) repeat (A and B) and in
the region containing the (GT)3 repeat (C and D). (A) Mutant sequence
with adenine insertion mutations (arrows), (B) corresponding wild type
sequence, (C) mutant sequence with cytosine deletion mutation (arrow),
(D) corresponding wild type sequence.
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The (GT)3 repeat within the 3' end of TR-II coding
region is also a reported target of mutational inactivation in a
minority of MMR-deficient human colon cancers.18 We
analyzed the same 10 MSH2/ murine lymphomas for
acquired ID mutations within a 140 bp DNA fragment of the
TR-II gene containing the (GT)3 repeat. One lymphoma showed
a mobility shift by denaturing polyacrylamide gel electrophoresis
consistent with an acquired deletion mutation. Sequence analysis
confirmed an acquired deletion mutation as 1 of 3 cytosines in
nucleotides 1617 to 1619 located just upstream of the (GT)3 repeat was
deleted (see Figure 2). This deletion results in a premature STOP codon
predicted to code for a truncated protein of 528 amino acids.
Sequencing of the 9 lymphomas whose PCR products lacked mobility shifts
and nonmalignant tissue from 6 mice showed the wild-type sequence in
all cases.
The ID mutations located in the (A)10 repeat of the human
TR-II gene are associated with reduced levels of
TR-II mRNA.18 Therefore, we evaluated all 10 murine lymphomas for TR-II transcripts by RT-PCR. All 6 of
the lymphomas harboring ID mutations within the (A) repeat showed an
absence of TR-II mRNA (Figure
3). In contrast, TR-II mRNA was
detectable in the lymphoma containing the deletion mutation within the
region of the (GT)3 repeat and in lymphomas lacking TR-II
gene mutations. Immunohistochemical and Western blot analysis confirmed
the RT-PCR results. Murine lymphomas without detectable RNA transcripts
by RT-PCR lacked detectable protein by either of these methods, whereas
those with positive RT-PCR results showed detectable protein. In
contrast, all of the murine lymphomas had TR-I protein by Western
blot analysis (Figure 4).
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| Fig 3.
TR-II mRNA expression in MSH2/
murine lymphomas.
RT-PCR was used to assess the expression of TR-II (lanes
1-10) and TR-I (lanes 11-20) mRNA in 10 whole lymphoma tumor
specimens. Tumor specimens with frameshift mutations in the
mononucleotide (A) repeat of TR-II (lanes 1, 3-6, 9) show
absence of TR-II mRNA transcripts, whereas all other tumor
specimens (lanes 2, 7, 8, 10) express TR-II. All tumor
specimens, including those with TR-II frameshift mutations,
show TR-I mRNA transcripts.
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| Fig 4.
Immunohistochemical and Western Blot detection of
TR-II protein.
(A) Example of a thymic lymphoma with wild-type TR-II (A)
repeat and detectable mRNA by RT-PCR. All tumor cells show strong
immunohistochemical staining for TR-II. (B) Example of a thymic
lymphoma harboring an (A) repeat ID mutation, and showing no detectable
mRNA by RT-PCR, demonstrates the absence of immunohistochemical
staining for protein. (C) Nonmalignant tissue from the small intestine
of the animal from panel B shows strong immunohistochemical staining
for TR-II. (D) Representative Western analysis of 2 murine
lymphomas. Lane 1, lymphoma harboring TR-II (A) repeat ID mutation;
lane 2, lymphoma showing wild-type TR-II (A) repeat. Top
panel demonstrates absence of TR-II protein in lane 1. Bottom panel
is the same blot re-probed with an antibody to TR-I as control.
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Microsatellite instability in MSH2/ murine lymphomas
A common strategy to detect underlying MMR deficiency is the
comparison of germline DNA with tumor DNA for MSI. We evaluated the
thymic lymphomas arising in 20 MSH2/ mice for
instability at 10 neutral microsatellites. In the analysis of 200 paired nonmalignant and tumor DNA samples high-frequency MSI was not
observed. Low-frequency MSI occurred in 8 of 20 lymphomas with none
showing alterations at more than one microsatellite. Twelve of 20 lymphomas displayed microsatellite stability (Figure
5). MSI was even less frequent in
nonmalignant tissue because none of the 20 tail tissue samples displayed high-frequency MSI, 3 of 20 displayed low-frequency MSI, and
17 of 20 displayed microsatellite stability.
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| Fig 5.
Microsatellite analysis of MSH2/ murine
lymphomas.
(A) Examples of the analysis of DNA from paired nonmalignant (N) and
whole lymphoma tumor specimens (T) at the D5Mit10 marker. Novel
microsatellite alleles are present in 1 tumor specimen. (B)
Examples of the analysis of DNA from paired normal tissue (N) and whole
tumor tissue (T) at the D7Mit12 marker. No microsatellite instability
is seen.
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A more sensitive strategy to detect MSI uses DNA from single cell
clones.21 We evaluated DNA from 20 single cell clones from
the MSH2/ murine thymic lymphoma cell lines R25
and L15 for instability at the same 10 neutral microsatellites used to detect MSI in whole tumor DNA (Figure 6).
For the R25 cell line, high-frequency MSI was found in 4 of 20 clones,
low-frequency MSI in 14 of 20 clones, and stability in 2 of the 20 clones. In the 4 high-frequency MSI clones, instability was seen in 6 of the microsatellite loci in 1 clone, 5 of the loci in 2 clones, and 4 of the loci in 1 clone. For the L15 cell line, high-frequency MSI was
less common than in the R25 cell line and was observed in 2 of 20 clones. Low-frequency MSI was seen in 16 of 20 clones and stability in
2 clones.
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| Fig 6.
Microsatellite analysis of MSH2/ murine
lymphoma single cell clones.
(A) Examples of analysis of DNA from single cell clones derived from
the R25 cell line at D6Mit8 marker. No microsatellite instability is
seen. (B) Examples of analysis of DNA from single cell clones derived
from the L15 cell line at D8Mit14 marker. Novel microsatellite alleles
are present in 2 of the clones (lanes 2 and 6).
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Discussion |
We previously reported that MSH2/ mice
uniformly develop aggressive thymic lymphomas of a single
histopathologic subtype at an early age and demonstrate aberrant
activation of T-cell associated oncogenes, RBTN-2 and
TAL-1.10 In this study, we used the
MSH2/ murine lymphomas as a model to investigate
the spontaneous mutations arising in these tumors in vivo as a
consequence of MMR deficiency. A high frequency of ID mutations within
the TR-II gene was observed in the tumors of different
animals, whereas the corresponding nonmalignant tissue lacked
mutations. These findings suggest that in addition to the molecular
activation of RBTN-2 and TAL-1, acquired
TR-II mutations represent important molecular inactivating
events in tumor pathogenesis following MSH2 deficiency. In
contrast, when tumor and nonmalignant MSH2/ tissues were compared for the frequency of MSI at neutral
microsatellites no appreciable difference was observed because both
showed low-frequency but not high-frequency MSI. This lack of
difference implies that analysis of cancer-associated genes for ID
mutations may be a more sensitive indicator of underlying MMR
deficiency than analysis of neutral microsatellites for MSI.
It has been previously established that MSH2 and PMS2
knockout mice exhibit hypermutability consistent with deficient
MMR.16,17,22 Nonmalignant tissues harvested from
MSH2/ and PMS2/ mice
bearing the neutral reporter transgenes lacI, supF, or
supFG1 demonstrate a high frequency of base transitions,
transversions, and ID mutations in short mononucleotide runs. In the
present study, we evaluated a known tumor suppressor gene and found
that the TR-II ID mutations occurred in short mononucleotide
runs. Our findings corroborate that short mononucleotide runs may
represent potential mutational hotspots in the setting of MMR
deficiency. The high frequency of ID mutations within the (A)10 repeat
of TR-II in human MMR-deficient cancers further supports
this contention.
Escape from TGF--mediated growth inhibition, due to mutations in
TR-II or loss of TR-II surface protein expression (or both), is a defined pathogenetic mechanism in many cancers, including some hematolymphoid malignancies.23,24 In MMR-deficient
human cancers, ID mutations within the TR-II (A)10 repeat
are predominantly deletions and are associated with reduced levels of
TR-II mRNA transcripts that are thought to be secondary to
decreased mRNA stability.18 In contrast to ID mutations in
the TR-II (A)10 repeat in human cancers, all of the ID
mutations within the murine TR-II mononucleotide A repeat
were insertions. The reason for this difference is not entirely clear
but may reflect sequence differences because the murine repeat contains
guanine nucleotides at positions corresponding to the 5th and 10th
adenine nucleotides of the human repeat. A high frequency of insertion
mutations has been reported in other mononucleotide runs in the setting
of MMR deficiency. For example, in human MMR-deficient gastrointestinal cancers, the BAX (G)8 and hMSH6 (C)8 mononucleotide
runs show a high frequency of insertion mutations.19,25
Similar to studies of MMR-deficient human cancers, the
MSH2/ murine lymphomas showed concordance between
the presence of ID mutations within the mononucleotide A repeat of
TR-II and loss of TR-II mRNA transcripts and
protein as detected by RT-PCR, immunohistochemistry, and Western blot analysis. These ID mutations were detected on 1 allele. Thus, other
genetic or epigenetic changes may be present to account for the loss of
transcripts and protein. For example, our study analyzed 269 of the
1704 bp in the TR-II coding region and it is possible other
mutations may be present. Furthermore, epigenetic changes such as gene
inactivation through promoter hypermethylation remain to be
investigated. Our findings strengthen the association between acquired abnormalities in TR-II following MSH2
deficiency and tumorigenesis and suggest that some hematolymphoid
malignancies may share similar pathogenetic mechanisms as some solid cancers.
The lack of TR-II ID mutations in the nonmalignant tail
tissue of MSH2/ mice is not entirely clear but
may in part reflect tissue-specific differences in mutational
frequencies. In studies of MSH2/ mice bearing the
lacI transgene, ID mutations were greatest in tissues
characterized by having a comparatively high cell turnover rate such as
the small intestine and thymus.16,17
In contrast to the high frequency of acquired TR-II ID
mutations in MSH2/ murine lymphomas, only
low-frequency MSI was detected in the analysis of neutral
microsatellites. Others have also found a lack of high-frequency MSI in
some tumors arising in the setting of bona fide MMR
deficiency.1,26-28 Tumor growth kinetics and tumor age are
believed to be important determinants for frequency of MSI. Shibata et
al used xenografts of single cell clones of the human mutator phenotype
colorectal cell line HCT 116 to simulate mutator phenotype tumors with
rapid growth rates and found a low frequency of altered microsatellite
alleles despite a high intrinsic mutation rate.26,27 In
comparison, slow-growing adenomas displayed greater microsatellite
allele diversity.26,27 In MSH2/,
APC +/ mice, intestinal adenomas develop at a young age
that lack MSI.28 The short mitotic history of these
adenomas has been suggested as a possible explanation for the lack of
MSI.1,28 Thus, in rapidly growing tumors, absence of
high-frequency MSI may not preclude underlying MMR deficiency. Taken
together, we reason that the lack of high-frequency MSI in the
MSH2/ murine lymphomas may have its basis in the rapid growth characteristics of these aggressive high-grade lymphomas and their short mitotic history, because the median time to tumor development is 3.8 months.10
Analysis of single cell clones is a more sensitive method to detect MSI
compared with analysis of whole tumor samples.21 In keeping
with the increased sensitivity, we observed high-frequency MSI in a
minority of single cell clones obtained from the R25 and L15 cell
lines, 20% and 10%, respectively. Although analysis of single cell
clones increases the ability to detect MSI, it remains an impractical
method to screen human cancers for underlying MMR deficiency.
Our finding that links MMR deficiency with frameshift mutations within
the TR-II gene may not be generalized to all subtypes of
hematolymphoid malignancies. Childhood ALL represents a subtype of
hematolymphoid malignancy with a distinct biologic behavior associated
with a generally favorable outcome. Molenaar et al found mutations
within the repeat tracts of the BAX and TR-II genes
in 3 of 6 adult ALL cell lines but not in 55 cases of childhood ALL.29 Thus, in humans, MMR deficiency and TR-II
frameshift mutations may represent pathogenetic events more important
to the development of adult leukemia/lymphoma.
High-frequency MSI has been an inconsistent feature in human
hematolymphoid malignancies and consequently MMR gene defects have been
thought not to significantly contribute to their molecular pathogenesis. Acquired alterations within neutral microsatellites likely do not confer a selective growth advantage and may be difficult to detect amid the large background of normal alleles, even when using
a more sensitive method such as the analysis of single cell clones.1,21,26-28 The lack of high-frequency MSI in
MSH2/ lymphomas implies that MSI at neutral
microsatellites may not always reflect MMR status. Rather, we observed
a high frequency of ID mutations within a cancer-associated gene,
TR-II, in the MSH2/ lymphomas. Whether
these ID mutations represent commonly occurring early events in tumor
pathogenesis or events that contribute to clonal expansion and
dominance remains to be determined.1 Irrespective of the
potential role of these mutations in cancer development or progression,
we submit that for some rapidly growing hematolymphoid malignancies,
the evaluation of cancer-associated genes for ID mutations may
represent a more sensitive marker of MMR deficiency than
evaluation of neutral microsatellites for high-frequency MSI.
| |
Acknowledgments |
The authors would like to thank Mr Bob van den Beuken and Mr Todd
Reichert for assistance in preparing the figures, and Dr T Al-Tweigeri
for his support.
| |
Footnotes |
Submitted June 7, 1999; accepted October 28, 1999.
Supported by grants from the Medical Research Council of Canada, the
Health Service Utilization and Research Commission of Saskatchewan,
Saskatchewan Cancer Agency, and Saskatchewan Health. R.L., A.M., and
J.F.D. are recipients of Medical Research Council of Canada Awards.
Reprints: John F. DeCoteau, Department of Pathology, Royal
University Hospital, University of Saskatchewan, 103 Hospital Drive,
Saskatoon, Saskatchewan, Canada, S7N 0W8; e-mail: decoteauj{at}sdh.sk.ca.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
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Abstract
Introduction