Download Pol Is a Candidate for the Mouse Pulmonary

[CANCER RESEARCH 64, 1924 –1931, March 15, 2004]
Advances in Brief
Pol ␫ Is a Candidate for the Mouse Pulmonary Adenoma Resistance 2 Locus, a
Major Modifier of Chemically Induced Lung Neoplasia
Min Wang,1 Theodora R. Devereux,2 Haris G. Vikis,4 Scott D. McCulloch,3 Wanda Holliday,2 Colleen Anna,2
Yian Wang,1 Katarzyna Bebenek,3 Thomas A. Kunkel,3 Kunliang Guan,4 and Ming You1
1
Department of Surgery and The Alvin J. Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri; 2Laboratory of Molecular Carcinogenesis and
Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina; and
4
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan
3
Abstract
In this study, we performed systematic candidate gene analyses of the
Pulmonary adenoma resistance 2 locus. Differential gene expression in
lung tissues and nucleotide polymorphisms in coding regions between A/J
and BALB/cJ mice were examined using reverse transcription-PCR and
direct sequencing. Although not all genes in the interval were analyzed at
this moment due to the recent database updating, we have found that the
Pol ␫ gene, encoding the DNA polymerase ␫, contains 25 nucleotide polymorphisms in its coding region between A/J and BALB/cJ mice, resulting
in a total of ten amino acid changes. Primer extension assays with purified
BALB/cJ and A/J proteins in vitro demonstrate that both forms of Pol ␫
are active but that they may differ in substrate discrimination, which may
affect the formation of Kras2 mutations in mouse lung tumors. Altered
expression of POL ␫ protein and an amino acid-changing nucleotide
polymorphism were observed in human lung cancer cells, suggesting a
possible role in the development of lung cancer. Thus, our data support
the Pol ␫ gene as a modifier of lung tumorigenesis by altering DNA
polymerase activity.
Introduction
Lung cancer is the leading cause of cancer-related death in the
United States (1). Although cigarette smoke is implicated in ⬃90% of
male and 75– 80% of female lung cancer deaths, only ⬃15% of heavy
smokers will ultimately develop lung cancer, which suggests that
there is variation in individual susceptibility to lung cancer (2, 3).
Inbred strains of mice vary markedly in their susceptibility to spontaneous and chemically induced lung tumorigenesis, thus representing
a valuable model to study genetic susceptibility to lung cancer. On the
basis of their mean tumor multiplicities induced by a lung carcinogen,
the inbred mouse strains can be categorized into sensitive, intermediate, and resistant groups (4). The A strain is the most susceptible
strain, whereas the C57BL/6J strain is the most resistant. Other strains
such as the BALB/cJ strain belong to the intermediate group and are
less susceptible to lung tumorigenesis than the A strain. Experimental
crosses of inbred mouse strains have revealed Pulmonary adenoma
susceptibility (Pas) loci, Pulmonary adenoma resistance (Par) loci,
and Susceptibility to lung cancer (Slucs) loci (5).
A/J and BALB/cJ inbred strains of mice carry the same Pas1 allele
but show different susceptibility to urethane-induction of lung tumors.
In (A/J ⫻ BALB/c) F1 mice, the relatively resistant BALB/cJ phe-
notype was dominant over the high susceptibility of A/J mice (6).
Additional analysis on F2 hybrids and backcross mice supported the
hypothesis that a major locus named Par2 accounts for the difference
in adenoma susceptibility between A/J mice and BALB/cJ mice (7).
Par2 is mapped to the mouse chromosome 18 and accounts for 60%
phenotype variance (7–9). The resistance of BALB/cJ mice appears
due to the interaction between the Pas1 QTL and Par2 QTL in the
BALB/cJ mouse genome (10). We previously used (A/J ⫻ BALB/
cJ) ⫻ BALB/cJ congenic mice to successfully fine map the Par2
locus into a candidate region encompassed by the D18Mit103 and
D18Mit162 markers (11). In a recent study, this region has been
additionally narrowed down to a region encompassed by the marker
D18Mit103 and D18Mit188 (12), which has resulted in a ⬃2.4-Mb
candidate region based on the Celera and public mouse genome
databases. Four known genes are located in this region, i.e., StarD6,
Pol ␫, Mbd2, and Dcc. Preliminary analysis of the polymorphisms in
the Dcc and Pol ␫ genes were reported recently (11, 13).
Pol ␫ represents an interesting candidate for the Par2 locus. It is the
most error-prone DNA polymerase and preferentially incorporates G
rather than A across from template T (14 –16). Human POL ␫ has at least
two distinct catalytic activities including translesion DNA synthesis and
5⬘-deoxyribose phosphate lyase activity (17–19). Its 5⬘-deoxyribose
phosphate lyase activity and capability for filling short gaps implicate that
this polymerase may play a role in certain base excision repair (BER)
reactions (19). It may also play a role in lung tumorigenesis by affecting
DNA adduct repair and thus Kras2 mutations that are found in a high
proportion of mouse and human lung tumors (13, 20).
In the present study, we identified multiple nucleotide polymorphisms
and alternatively spliced transcript isoforms in the Pol ␫ gene between A/J
and BALB/cJ mice. We provide initial biochemical support for the
hypothesis that the amino acid differences between the two mouse isoforms may affect their substrate discrimination properties. An amino
acid-changing nucleotide polymorphism and altered expression of POL ␫
protein in human lung cancer cell lines was observed. These data support
the Pol ␫ gene as a modifier of lung tumorigenesis by altering DNA
polymerase and, possibly, repair activity.
Materials and Methods
Expression and Nucleotide Polymorphism Analyses of Genes in the
Par2 Region. Inbred mouse strains were purchased from The Jackson Laboratory (Bar Harbor, ME). DNA was isolated from tail snips. For RNA isolaReceived 9/30/03; revised 1/6/04; accepted 1/12/04.
tion, 100 mg of lung tissue were pulverized and total RNA extracted using
Grant support: NIH Grants R01CA099147 and R01CA58554.
The costs of publication of this article were defrayed in part by the payment of page
TRIzol reagent according to the manufacturer’s protocol (Life Technologies,
charges. This article must therefore be hereby marked advertisement in accordance with
Inc., Gaithersburg, MD). The quality of the isolated RNA was assessed by
18 U.S.C. Section 1734 solely to indicate this fact.
absorbance at 260 nm, the A260/A280 ratio (1.7–1.9), and electrophoresis on
Note: M. Wang, T. Devereux, and H. Vikis and their laboratories contributed equally
1% agarose/formaldehyde gels that indicated the intensity and integrity of the
to this article. Data deposition: The Pol ␫ A/J and BALB/c coding sequences reported in
this article have been deposited in the GenBank database (accession nos. AY515316 and
28S and 18S bands. Two ␮g of total RNA were used in a reverse transcription
AY515317).
reaction to synthesize the first-strand cDNA using oligo-dT primer. Primers
Requests for reprints: Ming You, Department of Surgery, The Washington Univerwere designed for each gene based on their published sequences. The followsity School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail:
ing PCR condition was used for each primer set: 95°C for 3 min, followed by
[email protected].
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Pol ␫ IS A CANDIDATE MOUSE LUNG TUMOR MODIFIER GENE
30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, and finally 72°C 0.01% xylene cyanol), heated to 94°C for 3 min, and separated by 12%
for 6 min. Annealing temperature was optimized for each primer set. Electro- denaturing PAGE. Gels were quantified by phosphorimage analysis.
phoreses on 1.5% agarose gels were performed to resolved PCR products.
Human POL ␫ Mutation and Expression Analyses. Eleven human lung
Genomic DNA- and DNase I-treated cDNA samples were used for those cancer cell lines, including A427 (carcinoma), A549 (carcinoma), CaLu-1
containing only one exon. Amplified PCR products were purified with QIA- (metastatic epidermoid carcinoma), CaLu-3 (adenocarcinoma), CaLu-6 (adequick gel extraction kit (Qiagen, Valencia, CA) and subjected to direct se- nocarcinoma), SK-LU1 (adenocarcinoma), SK-MES1 (squamous cell carciquencing.
noma), NCI-H460 (large cell carcinoma), NCI-H596 (adenosquamous carciTo detect alternative transcripts and nucleotide polymorphisms in the Pol ␫ noma), NCI-H520 (squamous cell carcinoma), and NCI-H661 (large cell
gene, five primer sets were used to cover the entire Pol ␫ coding region: primer carcinoma), and the normal fetal lung cells MRC5 [all available from the
set 1: forward, 5⬘-GAGGAAGAAGACGCTCCTC-3⬘, and reverse, 5⬘-TTC- American Type Culture Collection, Manassas, VA] were used in this study.
CTCCAACAATTCTGTGAC-3⬘; primer set 2: forward, 5⬘-GAGCCGCTA- The cells were grown in Eagle’s MEM ⫹10%FBS (MRC-5, A549, A427,
CAGAGAGATG-3⬘, and reverse, 5⬘-GTAGTAAGACCGTCTGCTG-3⬘; CaLu-1, CaLu-3, CaLu-6, SK-LU-1, and SK-MES-1) or RPMI 1640 ⫹ 10FBS
primer set 3: forward, 5⬘-GTGGCTCCTAATAAACTCTTG-3⬘, and reverse, (NCI-H460, NCI-H596, NCI-H520, and NCI-H661) and were harvested at
5⬘-AGGCCCTTTCTTAGCACTGC-3⬘; primer set 4: forward, 5⬘-ATGGT- 80 –90% confluence. DNA, RNA, and protein were isolated from these cells by
GAACGTGAAGATGCC-3⬘, and reverse, 5⬘-GCCTTGACTCGTTTGCT- standard techniques. Primers used to amplify the human POL ␫ gene (GenBank
CAT-3⬘; and primer set 5: forward, 5⬘-TCGTGCGGAAAGGACTGTTC-3⬘, accession no. NM_007195) are the following: 1F, 5⬘-GCGACG ACGAGGAAand reverse, 5⬘-ACTAGAATTTCCTTCCTGTGC-3⬘. Alternative mRNA GACG-3⬘ (33-50); 2R, 5⬘-GTATTCCCTTGCTTTTCAGAC-3⬘ (2230-2250); 3F,
splicing was detected by using primer sets 1 and 2.
5⬘-GATCTCAGATTGCAGCAGAG-3⬘ (591-610); 4R, 5⬘-AGAAGCCACTCA/J and BALB/cJ GST-Pol ␫ Plasmid Constructs. A full length of Pol ␫ CAGCACAG-3⬘ (649-667); 5F, 5⬘-GGATTTCCTACCAAGTGGAAG-3⬘ (1447coding sequence was separately amplified using two sets of oligonucleotides: 1467); 6R, 5⬘-GAAGCTGCTTGAAGACTTCTTG-3⬘ (1574-1595); 7F, 5⬘-CAAP1 set: forward, 5⬘-GAACGCGGATCCGCGGCCATGGAGCCCTTGCACGC- GAAGTCTTCAAGCAGCTTC-3⬘ (1574-1595); and 8R, 5⬘-GTCCAATG3⬘(A/J),
forward,
5⬘-GAACGCGGATCCGCGGCCATGGAGCCCTCGC- TGGAAATCTGATC-3⬘ (1284-2204).
ACGC-3⬘ (BALB/cJ), and reverse, 5⬘-ATAAGGATATCAATCAGGGGAGGCTo examine the 2147 base coding region of the human POL ␫ gene for
3⬘; and P2 set: forward, 5⬘-CCTCCCCTGATTGATATCCTTATG-3⬘, and polymorphisms or mutations, we made cDNA from the RNA and amplified
reverse, 5⬘-CTCCCTCCATCGATGGACTTATCTGTGCGCCGAGG-3⬘. P1 overlapping regions with primers 1F ⫻ 4R, 3F ⫻ 6R, and 5F ⫻ 2R. After Qiagen
and P2 PCR products were digested with BamHI/EcoRV and EcoRV/ClaI PCR purification (Qiagen), the PCR products were both sequenced directly and
restriction enzymes. Digested P1 and P2 PCR products were separately cloned cloned using a TOPO-TA PCR cloning kit (Invitrogen). DYE-ET (Amershaminto the pBluesript II SK vector, and their sequences were confirmed by direct Pharmacia, Sunnyvale, CA) and BIG DYE (Applied Biosystems, Foster City, CA)
sequencing in both directions. The Pol ␫ -pBluesript II SK plasmid containing fluorescent terminator cycle sequencing kits were used. Two to four clones were
a full length of Pol ␫ open reading frame was produced by subcloning the P2 sequenced in each direction, and direct sequencing of products without cloning in
fragment into P1 plasmid.
at least two reactions was used to confirm the polymorphisms detected. Western
The GST-Pol ␫ plasmid was generated by subcloning the ⬃2.2-kb BamHI to blot analysis was performed to examine the protein expression of POL ␫ in the
ClaI fragment from Pol ␫ -pBluesript II SK plasmid into BamHI/ClaI double- human lung cancer cell lines and normal MRC5 line. A polyclonal antibody to the
digested glutathione S-transferase (GST)-tagged expression vector pEBG-3X- full-length POL ␫ protein made in rabbits was used. Ponceau Red staining was
HV. The in-frame open reading frame of GST-Pol ␫ fusion was confirmed by used to confirm the even loading of protein.
direct sequencing.
Purification of GST-Tagged Pol ␫ Proteins. The GST-Pol ␫ expression Results
plasmids were transfected into HEK293 cells using Lipofectamine (Invitrogen,
Carlsbad, CA), and 48 h after transfection, cells were lysed in NP40 buffer [20
Expression and Nucleotide Polymorphism Analyses of Genes in
mM Tris (pH 7.5), 100 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM DTT, 0.1 mM the Par2 Region. The present minimum Par2 physical region is
phenylmethylsulfonyl fluoride, 5 ␮g/ml aprotinin, and 5 ␮g/ml leupeptin].
encompassed by the D18Mit103 and D18Mit188 markers. We obLysates were cleared by centrifugation, and NaCl was added to 1 M before the
tained the marker sequence information from the Whitehead Institute/
addition of glutathione-agarose beads and incubation for 2 h. Beads were
MIT Center for Genome Research.5 Using the marker sequences to
washed by NP40 buffer and eluted in glutathione elution buffer [50 mM Tris
blast
against the Celera and National Center for Biotechnology Infor(pH 8), 100 mM NaCl, 10 mM glutathione, and 1 mM DTT]. Eluted proteins
mation
(NCBI) mouse genome databases, we localized these two
were resolved by SDS-PAGE to determine purity and concentration. In addition, protein concentrations were estimated by Western blot analysis using markers on both maps. The physical distance between the D18Mit103
human GST-i as a standard. The antibody to full-length human POL ␫ was and D18Mit188 markers was ⬃2.4 Mb in the Celera map with both
kindly provided by Rajendra Prasad and Samuel H. Wilson.
markers located on the scaffold GA_x6K02T2NR3J. In the NCBI
Primer Extension Assays. A set of experiments were designed to compare mouse map, these two markers were localized to the contig
the incorporation of dATP by the A/J and PALB/c Pol ␫ isoforms. The DNA NT_082383 and encompassed a ⬃2.6-Mb region (D18Mit103 at the
template used was 5⬘-CTCGTCAGCATCTTCATCATACAGTCAGTG-3⬘. nucleotide position 2932731-2932845 and D18Mit188 at the nucleoThe matched primer was 5⬘-CACTGACTGTATGATGA-3⬘, and the mistide position 5505960-5506058). The chromosome position for each
matched primer was 5⬘-CACTGACTGTATGATGG-3⬘. For matched primergene in the candidate region is illustrated in Fig. 1. Generally, the gene
template extension, 10, 25, 50, 110, and 225 ng of A/J and BALB/cJ Pol ␫
proteins were used. Reactions were performed at 37°C degree for 15 min. For annotations on both maps are consistent to each other. For example,
G/T mismatched primer extension, when dATP was added, 100, 200, 300, and four known genes, namely StarD6, Pol ␫, Mbd2, and Dcc, are located
in both Par2 regions. Sequence comparison revealed that the
400 ng of A/J and BALB/cJ Pol ␫ proteins were used.
To examine the abilities of A/J and BALB/cJ Pol ␫ proteins to incorporate mCG9249 in the Celera map represents the same gene as the
dGTP compared with dATP opposite template T, the primer oligonucleotide 2310002L13Rik does in the NCBI map. Both the mCG9258 and
(5⬘-AATTTCTGCAGGTCGACTCCAAAGGCT-3⬘) was 5⬘-end labeled with Loc225699 genes encode a protein similar to 60S ribosomal protein
32
P using T4 polynucleotide kinase and annealed at a 1.5:1 ratio with the
L5 and should represent a same gene. However, there are also some
template oligonucleotide (5⬘-CCAGCTCGGTACCGGGTTAGCCTTTG- discrepancies between the Celera and NCBI mouse Par2 region. The
GAGTCGACCTGCAGAAAT-3⬘). Reactions contained 40 mM Tris-Cl (pH
Celera Par2 region has 7 annotated genes, whereas the NCBI Par2
8.0), 30 mM NaCl, 5 mM MgCl2, deoxynucleoside triphosphate (as indicated in
region has 11, which may reflect different gene prediction tools and
Fig. 3), 10 mM DTT, 1.25% glycerol, 250 ␮g/ml BSA, 2 pmols of primer:
template DNA, and 2 ng of GST-Pol ␫. Samples (5 ␮l) were removed at the prediction stringency used by annotators. Another apparent discrepindicated times and added to an equal volume of formamide loading buffer
5
(95% deionized formamide, 25 mM EDTA, 0.01% bromphenol blue, and
Internet address: http://www.broad.mit.edu/cgi-bin/mouse/sts_info?database⫽mouse.
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Pol ␫ IS A CANDIDATE MOUSE LUNG TUMOR MODIFIER GENE
Fig. 1. Annotated genes in the Par2 candidate region. D18Mit103 and
D18Mit188 were separately anchored to Celera (released on August 17,
2003) and National Center for Biotechnology Information (NCBI)
Build32 (released on February 10, 2003) mouse genome maps by performing BLASTn search. The unit of gene position is “Mb.”
ancy is the position of Mbd2 gene (Fig. 1). For the four known genes,
their order in the NCBI mouse genome map is Mbd2-Stard6-Pol
␫-Dcc, which is different from their order (Stard6-Poli-Mbd2-Dcc) in
the other three maps, i.e., the NCBI human, Celera mouse, and Celera
human genome maps. Because of the high homology between human
and mouse Par2 region, the Mbd2 gene apparently has been somehow
mistakenly placed during the NCBI sequence assembling. We used
reverse transcription-PCR (RT-PCR) and direct sequencing to examine differential expression in lung tissues and coding-region nucleotide polymorphisms for the annotated genes. The result is presented in
Table 1. Because the 4930503Rik, Loc383391, Loc383392, and
Loc381176 genes were annotated in the Par2 region after this work
was submitted, relevant work is still ongoing in our laboratory. A
previous study using Northern blot suggested that the Stard6 gene is
expressed exclusively in the mouse testis, and its function has been
suggested to be specific for fertility (21). However, our RT-PCR
results indicate that Stard6 is expressed in mouse lung tissues but has
no differential expression or nucleotide polymorphism between A/J
and BALB/cJ inbred strains. Our previous study has revealed neither
expression difference nor nucleotide polymorphism between A/J and
BALB/cJ mice for the Dcc gene (11). Real-time RT-PCR analysis for
the Mbd2 gene only revealed a slight expression difference (A/J:
BALB/cJ, 0.84 ⫾ 0.03; data not shown), and no nucleotide polymorphism has been found for this gene either. Thus, Stard6, Mbd2, and
Dcc are less likely to be the Par2 gene. Two other genes, i.e.,
A430085C19 and Loc225699, only have one exon and were not
detected by DNase I-treated RT-PCR in lung. We could not detect
2310002L13Rik/mCG9249 in either A/J or BALB/cJ lung tissues.
Nucleotide Polymorphisms and Alternative mRNA Splicing in
the Pol ␫ Gene. The remaining known gene in the Par2 region, Pol ␫,
has a 2154-bp open reading frame and encodes a 717-amino acid protein.
By sequencing its entire coding region, we found a total of 25 nucleotide
polymorphisms in the Pol ␫ coding region between A/J and BALB/cJ
mice. As shown in Fig. 2A, we have identified 10 amino acid-changing
polymorphisms and two alternatively spliced exons in Pol ␫ between A/J
and BALB/cJ mice. Among them, 9 amino acid polymorphisms and 1
alternative splicing variant were consistent with those reported previously
(13). The newly observed amino acid changing polymorphism at codon
606 encodes a positively charged arginine (CGA) in BALB/cJ but a
neutral Glutamine (CAA) in A/J (Fig. 2B).
Pol ␫ exon 4a transcript isoform was reported as a product of
alternative splicing on exon 4 (13). In our study, we have found that
in addition to this isoform (detected by our primer set 2; data not
shown), another isoform also exists in mouse lung tissues. The new
alternative transcript was detected by primer set 1 and has a shorter
nucleotide sequence compared with the regular one (Fig. 2C, a).
Directly sequencing this shorter PCR product has revealed that the
exon 2 in the full-length Pol ␫ mRNA is spliced out without changing
open reading frame (Fig. 2C, b). We present this isoform as exon 2d
(“d” means “deleted”). In C57BL/6J mice, the exon 2d isoform seems
to be more abundant than the regular isoform that contains exon 2,
whereas A/J, BALB/cJ, 129/SvJ, and C3H/HeJ lung tissues have more
of the regular-length isoform transcript than the exon 2d isoform. We
did not observe differential expression of the new exon 2d transcript
isoform between A/J and BALB/cJ mice.
Initial Biochemical Characterization of Pol ␫ Protein. To investigate the properties of the Pol ␫ isoforms encoded in A/J and BALB/cJ
mice, we expressed and purified both enzymes as full-length GST fusion
proteins and measured their polymerization activity. Both enzymes were
active in extending a correctly paired primer-template through the addition of dAMP opposite template T (Fig. 3A, a). Thus, the amino acid
differences between the two polymerases do not result in loss of polymerization activity by either protein. At concentrations of Pol ␫ estimated to
be equivalent, more extension of the correctly paired primer was ob-
Table 1 Expression and nucleotide polymorphism analyses for genes in the Par2 candidate region
Gene ID
mCG9249/2310002L13
4930503L19
Stard6
Pol ␫
A430085C19
Mbd2
Loc225699/mCG9258
Dcc
Loc383391
Loc382292
Loc381176
a
Lung
expression
Description
Riken transcript
Riken transcript
START domain-containing protein 6
DNA polymerase ␫
Methyl-CPG binding domain protein
Similar to 60S ribosomal protein L5
Tumor suppressor protein
Similar to DCC tumor suppressor protein
No
NDa
Yes
Yes
No
Yes
No
Yes
NDa
NDa
NDa
Nucleotide polymorphisms
between A/J and BALB/c
No
Ten amino acid polymorphisms
No
No
No
ND, not determined because of recent database update.
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Pol ␫ IS A CANDIDATE MOUSE LUNG TUMOR MODIFIER GENE
Fig. 2. Amino acid alterations and transcript isoforms of the mouse Pol ␫ gene. A, schematic illustration of amino acid-changing nucleotide polymorphisms and alternative splicing. The full
length of Pol ␫ mRNA consists of 10 exons (f, E1-E10) with a total of 2497 bp (GenBank accession no. NM_011972). Exon 2d isoform is produced by alternatively splicing out exon 2 (s)
without changing the entire open reading frame. The asterisk sign (ⴱ) indicates that the exon 4a isoform has extra 32 bp (䡺) on regular exon 4, which produces a truncated protein caused by
an early termination codon (tga) in exon 5. Ten amino acid changing codons are located in exon 1 (2), exon 8 (1), exon 9 (1), and exon 10 (6). The start codon (atg) is at 95 bp position, and
the stop codon in the regular isoform is at 2246 bp position. B, amino acid-changing nucleotide polymorphism on codon 606 of the Pol ␫ regular transcript. Direct sequencing results revealed
that the codon 606 of the Pol ␫ full-length transcript encodes a positive-charged arginine (CGA) in BALB/cJ but a neutral glutamine (CAA) in A/J mice. C, alternative mRNA splicing on exon
2. a, reverse transcription-PCR result using primer set 1 (see “Materials and Method” for primer sequences). Two transcript isoforms were detected in A/J and BALB/cJ mouse lung. The longer
one is regular Pol ␫ transcript with exon 2. The shorter one is a new isoform without exon 2. b, sequencing result revealed that the new Pol ␫ isoform is caused by in-frame exon 2 skip.
served for the BALB/cJ enzyme than for the A/J enzyme. However, in
parallel reactions using somewhat higher enzyme concentrations, a difference between the two isoforms was not apparent for extension of a
primer containing a terminal T-G mismatch (Fig. 3A, b). These data
suggested that the two isoforms of Pol ␫ may differ in their ability to use
aberrant substrates. To further test this hypothesis, we examined a property that distinguishes Pol ␫ from all other DNA polymerases studied to
date—the preferential incorporation of incorrect dGMP over correct
dAMP opposite template thymine (14 –17). The results show that both
mouse isoforms of Pol ␫ do exhibit this noncanonical behavior (Fig. 3B).
Thus, when examined at two different concentrations of dGTP or dATP
(0.1 and 1.0 mM), incorporation of dGTP is preferred by both enzymes
(compare squares to circles). However, at the higher deoxynucleoside
triphosphate concentration (black symbols), the A/J Pol ␫ prefers dGTP
by a factor of 3– 4-fold, whereas the BALB/cJ Pol ␫ prefers dGTP by a
factor of ⬍2-fold. This observation additionally supports the hypothesis
that the amino acid differences between the two mouse isoforms may
alter their substrate discrimination properties.
Human POL ␫ Mutation and Expression in Lung Cancer Cell
Lines. To explore the possible role of POL ␫ in human lung cancer
development, we examined 11 human lung cancer cell lines for
mutations in the coding region of the human POL ␫ gene. Two silent
polymorphisms at codon 12 (TCG to TCT, Ser) and codon 293 (GTG
to GTC, Val) were identified in the CaLu-1 cells, and a common
polymorphism at position 2180, which changes the amino acid at
codon 706 (ACA to GCA, Thr to Ala), was identified in A427, A549,
CaLu-3, CaLu-6, NCI-H460, and NCI-H596 cells. These polymorphisms in the lung cancer cell lines all appeared to be heterozygous
with the published sequence. Fig. 4A shows a sample without and one
with the polymorphism at codon 706.
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Pol ␫ IS A CANDIDATE MOUSE LUNG TUMOR MODIFIER GENE
Fig. 3. Primer extension assays. A, differential activities in incorporation of dATP opposite template T at high enzyme concentration. Incorporations of dATP extend matched (a)
and G/T mismatched (b) primer-template T. Pol ␫ enzymatic activity is reflected by percentage of primers being converted. B, preferential incorporation of dGTP versus dATP opposite
template T. Two ng of A/J and BALB/cJ full-length Pol ␫ proteins were used in each reaction. At 0.1 and 1 mM deoxynucleoside triphosphate (dNTP) concentrations, both proteins
exhibit classical Pol ␫ enzymatic property, preferring incorporation of dGTP opposite template T. At 1 mM dNTP concentration, the BALB/cJ Pol ␫ protein is less efficient (⬃2-fold)
at incorporating dGTP opposite template T than the A/J protein.
In addition to the sequence analyses, an assessment of POL ␫
protein expression in the human lung cancer cell lines was made.
Western analysis demonstrated that POL ␫ expression varied among
the cell lines from low expression in the normal MRC-5 and the A427,
SK-LU-1, and NCI-H460 cancer cells to very high in the CaLu-1 and
NCI-H520 cell lines with the other cells in between (Fig. 4B). These
expression differences did not correlate with the amino acid change at
codon 706 observed in certain cell lines or with the tumor morpho-
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Fig. 4. Alteration of POL ␫ in human cancer cell lines. A, codon 706 polymorphism in
human POL ␫ gene. Sequencing has been done with reverse primer 2R. ACA to GCA
(THR to ALA) was identified in A427, A549, CaLu-3, CaLu-6, NCI-H460, and NCIH596 cells. The polymorphism appeared to be heterozygous with the published sequence
as indicated by arrow in Calu-3 sequence. B, Western Blot of human POL ␫ protein in lung
normal and cancer cell lines. A polyclonal antibody was used to examine protein
expression. Ponceau Red staining has been performed to make sure that a similar amount
of protein was loaded to each lane.
logical subtypes (e.g., adenocarcinoma or squamous cell carcinoma)
from which the cells were derived.
The protein similarities between the human POL ␫, mouse Pol ␫, and
other species such as Arabidopsis thaliana, Caenorhabditis elegans,
Escherichia coli, as well as Saccharomyces cerevisiae Pol ␫-like
proteins are 76, 29, 29, 32, and 20%, respectively (UniGene).6 Most
of conserved amino acids are clustered within the NH2-terminal
region, which contains five conserved motifs (22). The evolutionary
conservation of the Pol ␫ codons was examined using the human,
mouse, and fruit fly’s Pol ␫ sequences. The fruit fly Pol ␫ sequence was
derived from the Drosophila melanogaster transcript CG7602-RA
(GenBank accession no. AAF54198). We aligned these three sequences using the Clustalw software.7 As shown in Fig. 5, the conserved amino acids are mainly clustered in NH2-terminal region
consistent with the previous study (22). The codon 706 shows weak
conservation among these three species. At this codon, the fruit fly Pol
␫ has a proline, whereas mouse Pol ␫ has an alanine. Both are
hydrophobic and neutral amino acids.
Discussion
In this study, we demonstrated the candidacy of Pol ␫ for the Par2
locus. Linkage analysis and fine mapping in various mouse crosses and
congenic mice have significantly narrowed the Par2 region (7–12).
6
Internet address: http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG⫽Hs&CID
⫽438533.
7
Internet address: http://www.ebi.ac.uk/clustalw/.
Among all of the annotated genes in the Par2 region, Pol ␫ was identified
as a candidate mouse lung resistance gene based on two lines of evidence:
(a) genetic variants were identified between susceptible A/J and resistant
BALB/cJ mice, and changes including genetic polymorphisms and altered expression of POL ␫ were also observed in human lung cancer cells;
(b) primer extension assays with purified BALB/cJ and A/J proteins in
vitro found that both forms of Pol ␫ are active but that they may differ in
substrate discrimination. This result strongly supports the hypothesis that
the amino acid differences between the two mouse isoforms may alter
their substrate discrimination properties, which may affect the formation
of Kras2 mutations in mouse lung tumors. A major characteristic of the
highly error-prone human POL ␫ is its preference for inserting wobble
base G rather than A opposite template T. Primer extension assays
demonstrated that the full-length BALB/cJ Pol ␫ protein is less efficient
than the A/J protein in incorporating G compared with A opposite a
template T. At higher concentrations, the full-length BALB/cJ Pol ␫
protein incorporates Watson-Crick base A opposite template T more
efficiently than the A/J protein. These results are consistent with a genetic
modifier role for the Pol ␫ variant in mouse and, possibly, human lung
cancer susceptibility.
Human chromosome 18q21.1 locus is deleted in many human cancers,
including squamous cell carcinoma, osteosarcoma, colon cancer, and
breast cancer (23–26). Recent studies have indicated that deleted segments of 18q21 contained a lung cancer tumor suppressor gene (27, 28).
Because the mouse Par2 region is highly homologous to human chromosome 18p21.1 region, positional cloning of the Par2 gene may facilitate the identification of the human lung cancer suppressor gene. To date,
there are three known tumor suppressors (DCC, SMAD2, and SMAD4)
located in the human chromosome18q21.1 region, although there is no
evidence that these genes play a role in lung cancer. Because the mouse
Smad2 and Smad4 genes are located outside of the refined Par2 candidate
region, they can be excluded as Par2 candidates. The status of the Dcc
gene as a Par2 candidate has been challenged by the fact that neither
nucleotide polymorphism nor significant lung expression difference has
been found between A/J and BALB/c mice (11). The MBD2 gene
encodes methyl-CpG binding proteins that suppress transcription from
methylated promoters and is a candidate tumor suppressor. However, one
recent mutation study suggests that the MBD2 gene may only have a
limited role, if any, in lung cancer tumorigenesis (29).
Multiple nucleotide polymorphisms and functional testing results (i.e.,
different enzymatic activities) provide evidence that the Pol ␫ gene is a
strong candidate for the mouse Par2 gene. As with tumor suppressors
DCC, SMAD2, and SMAD4, the human POL ␫ was also mapped to the
chromosome 18q21.1 (22). The gene belongs to the Rad30 branch of the
recently described UmuC/DinB/Rev1/Rad30 family of DNA polymerases
and encodes a 715-amino acid DNA-dependent polymerase POL ␫ in
human and a 717-amino acid Pol ␫ protein in mouse (30). On the basis of
in vitro studies, human POL ␫ has the lowest fidelity of any eukaryotic
polymerase studied to date, which suggests that its activities must be
highly specialized. The fact that POL ␫ orthologues are evolutionarily
conserved in higher eukaryotes from Drosophila to humans also suggests
that it provides some selective advantage. Several studies show that the
POL ␫ gene may play a role in somatic hypermutation (31–33) by which
specific mutations occur as part of antibody diversity. Human POL ␫
protein has also been shown carrying an intrinsic 5⬘-deoxyribose phosphate lysase activity and can substitute for POL ␤ during BER reactions
in vitro (19).
Twenty-five nucleotide polymorphisms in the Pol ␫ coding region
were observed between A/J and BALB/cJ strains that resulted in 10
amino acid alterations. Although none of these amino acid polymorphisms has been located in the conserved enzymatic regions, recent
studies have shown that the COOH-terminal region of human POL ␫
protein may also have additional critical functions (34 –36). For ex-
1929
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Pol ␫ IS A CANDIDATE MOUSE LUNG TUMOR MODIFIER GENE
Fig. 5. Clustalw alignment of human, mouse, and fruit fly Pol ␫
proteins. Mouse Pol ␫ protein is represented by the A/J sequence. “ⴱ”
indicates the identical residues in that column in the alignment.“:”
indicates the conserved substitutions. “.” indicates semiconserved
substitutions. The codon 706 is highlighted.
ample, the motif SRGVLSFF (in which the conserved residues are
indicated in bold) is present in residues 540 –547 of human POL ␫ and
may contribute to PCNA binding (34). Another study has shown that
the localization of POL ␫ protein in the nucleus requires sequences
within amino acids 219 – 451, and sequences within amino acids
492–539 and 636 –715 are critical for interaction between POL ␩ and
POL ␫ proteins (35, 36). The results in Fig. 3 are nonetheless consistent with the possibility that the BALB/cJ and A/J isoforms of Pol ␫
differ in substrate discrimination properties. The higher apparent
activity for incorporation of dAMP (Fig. 3A) and the lower ratio of
dGTP to dATP incorporation (Fig. 3B, 1.0 mM) by the BALB/cJ
enzyme are both consistent with the possibility that this enzyme may
be more accurate than the A/J isoform, at least for T-dGMP mismatches that would lead to T-A to C-G transition mutations. A more
comprehensive test of this hypothesis for this and other mismatches is
currently underway.
An amino acid polymorphism at the codon 706 of human POL ␫
gene was identified in human lung cancer and normal cell lines. It
is unclear at this time if this polymorphism will alter its function.
The Environmental Genome Project sampled 178 chromosomes
during its resequencing of POL ␫ and identified A in position 2180
(codon 706) at a proportion of 0.758 and G at 0.242. The estimated
heterozygosity was 0.366 for this SNP.8 This frequency is not
statistically different from our finding. We also found that the
human lung cancer cell lines exhibit varied expression of POL ␫
protein, which may suggest a role in the development of lung
cancers. Although these expression differences did not correlate
with the amino acid change at codon 706 observed in certain cell
lines or with the tumor morphological subtypes, it is noteworthy
8
Internet address: http://www.genome.utah.edu/genesnps/cgi-bin/frame.cgi_gene_id⫽350.
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Pol ␫ IS A CANDIDATE MOUSE LUNG TUMOR MODIFIER GENE
that there are a number of polymorphisms in the 5⬘-end flanking
region of the gene identified by Environmental Genome Project
that may affect expression or function. Thus, additional analysis of
the POL ␫ promoter region should be pursued.
The Kras2 gene plays an important role in mouse lung tumor development, with most adenomas and adenocarcinomas containing Kras2
mutations (5, 20). After treatment with urethane, nearly all Kras2 mutations arise in codon 61. Kras2 codon 61 mutations were very frequent
(90.9% for CAA3 CGA and 6.1% for CAA3 CTA) in lung tumors
following urethane treatment from BALB.B6-Par2 congenic mice and
slightly lower (86% for CAA3 CGA) in tumors from BALB/cJ mice
(13). After treatment with another carcinogen, diethylnitrosamine, the
CAA3 CGA mutation rates in tumors were 46% for parental BALB/cJ
mice and 79% for BALB.B6-Par2 congenic mice, respectively (13). It
was unclear whether these differences were because of differential protein expression in lungs of BALB/cJ and A/J mice (e.g., parental
BALB/cJ mice producing less than half amount of full-length Pol ␫
protein as congenic mice) or qualitative (i.e., enzymatic activity) factors.
Our primer extension assays present, for the first time, the evidence for
the hypothesis that enzymatic activity differences between the BALB/cJ
and A/J (or C57BL/6J) Pol ␫ proteins may contribute to differences in
Kras2 mutation frequency. Indeed, preferential DNA damage and poor
repair have been demonstrated to be responsible for the mutation hotspot
at codon 12 of Kras gene in human lung cancer (20). The urethaneinduced DNA adduct 1, N6-ethenodeoxyadenosine, has been shown to be
a strong mutagenic DNA adduct when replicated in mammalian cells
(37). A recent study has revealed that the level of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine were ⬃70% higher in A/J mice
than in C57BL/6J mice (38). Because of the conservation between mouse
Pol ␫ and human POL ␫ proteins, we speculate that mouse Pol ␫ proteins
are also involved in BER and may affect Kras2 mutation during BER
reactions. On the basis of our results, the BALB/cJ Pol ␫ protein would
less efficiently incorporate dGTP and might more efficiently incorporate
correct dATP base into the damaged DNA strand than the A/J Pol ␫
protein during BER. Thus, the probability of forming a CAA3 CGA
mutation at codon 61 in BALB/cJ Kras2 gene will be less than in A/J
Kras2 gene. Further in vitro enzymatic studies, e.g., BER test using
Kras2 gene sequence as template, are needed to confirm this hypothesis.
Additionally, it has been revealed recently that the functional Pol ␫ protein
is deficient in 129 strains of mice, which is consistent with its susceptibility to chemically induced lung tumorigenesis (39, 40). Thus, 129
strains of mice provide an ideal model to evaluate the relationship
between Kras2 mutation and Pol ␫ proteins in vivo.
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Pol ι Is a Candidate for the Mouse Pulmonary Adenoma
Resistance 2 Locus, a Major Modifier of Chemically Induced
Lung Neoplasia
Min Wang, Theodora R. Devereux, Haris G. Vikis, et al.
Cancer Res 2004;64:1924-1931.
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