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Department of Otolaryngology [M. G., K. F., T. E., S. N., K. N.] and Department of Molecular Genetics, Institute of Cellular and Molecular Biology [M. O., H. H., K. S.], Okayama University Medical School, Okayama 700-8558, Japan
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Materials and Methods |
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DNA and RNA Extraction.
Genomic DNAs were isolated from frozen tissues by SDS/proteinase
K treatment, phenol-chloroform extraction, and ethanol precipitation.
Total RNAs were prepared by using a modified acid guanidinium
phenol chloroform method (ISOGEN; Nippon Gene Co., Tokyo, Japan).
Microsatellite Analysis.
Primers for amplification of microsatellite markers D13S285,
D13S796, D13S278, D13S158, and D13S779 are available through the
internet genome
database.4
PCR was carried out in 20 µl of reaction mixture with 10 pmol of each
primer, 100 ng of genomic DNA, 1x PCR buffer, 200 µM of
each deoxynucleotide triphosphate, and 0.5 unit of Taq DNA polymerase
(Takara, Kyoto, Japan). Initial denaturation at 94°C for 3 min was
followed by 25 cycles of a denaturation step at 94°C for 30 s,
an annealing step at 52°C (D13S278 and D13S158) or 56°C
(D13S285, D13S796, and D13S779) for 30 s, and an extension step at
72°C for 1 min. A final extension step at 72°C for 7 min was added.
After amplification, 2 µl of the reaction mixture were mixed with 8
µl of loading dye (95% formamide, 20 mM EDTA, 0.05%
bromphenol blue, and 0.05% xylene cyanol), heat denatured, chilled on
ice, and then electrophoresed through an 8% polyacrylamide gel
containing 8 M urea. The DNA bands were visualized by
silver staining (10)
. LOH was scored if one of the
heterozygous alleles showed at least 50% reduced intensity in tumor
DNA as compared with the corresponding normal DNA.
Analysis of the Genomic Structure of the Human
ING1 Gene.
PCR analysis with ING1-specific primers was used to
screen a BAC library, and a positive BAC clone (118N21) was purchased
from Genome Systems, Inc. (St. Louis, MO). The BAC DNA was digested
with appropriate restriction enzymes, and regions containing
ING1 exons were mapped by Southern blotting with
ING1 cDNA probes. Some restriction fragments containing
ING1 exons were subcloned into pBluescript KS(-)
(Stratagene, La Jolla, CA) and sequenced.
Luciferase Assay.
Each putative promoter region around exon 1a and exon 1b was subcloned,
and sequential 5' truncation was introduced by restriction digestion.
These DNA fragments were inserted into the promoterless luciferase
plasmid pOA-luciferase (11)
to produce reporter plasmids
for the luciferase assay (see Fig. 2
). Each reporter plasmid (0.5 µg)
was cotransfected with 0.25 µg of an internal control Renilla
luciferase plasmid, pRLTK (Promega, Madison, WI), into 293 human
embryonic kidney cells or Saos2 osteosarcoma cells using the calcium
phosphate coprecipitation method. The cells were harvested 48 h
after transfection. The activities of firefly and Renilla luciferase
were measured simultaneously using the Dual-Luciferase Reporter Assay
kit (Promega) and normalized for the variation in transfection
efficiency. These assays were repeated at least twice in duplicate.
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Results and Discussion |
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shows the summary and representative examples. Twenty-three of 34
informative tumors (68%) showed LOH of at least one microsatellite
marker (Fig. 1A
). This prevalence may be underestimated
because most of the specimens were not microdissected, and some
apparently normal tissues might have loss by field cancerization. Five
tumors (samples 27, 28, 36, 37, and 44) displayed a large deletion that
included most polymorphic markers tested. In the other 18 cases, a
partial deletion was found, providing information about the areas of
preferential loss. The most frequent loss (49%) was seen at marker
D13S278. In particular, two cases (samples 14 and 15) exhibited LOH
with marker D13S278 but retained heterozygosity with flanking markers
D13S796 and D13S158. This deleted area was estimated to be, at most, 6
cM. It has been reported that a candidate tumor suppressor gene,
ING1, is located in this region (5)
. By
using the Stanford G3 radiation hybrid panel, ING1 was
linked to marker SHGC-5819, with a LOD score of 10.35 (5)
.
The markers SHGC-5819 and D13S278 are colocalized within less than 3
cM.4
We therefore determined the genomic
structure of the ING1 gene and searched for its mutation in
HNSCCs.
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. Recently, three alternatively spliced forms of ING1 mRNA
have been described (12)
. Our results showed that the
ING1b form (GenBank accession number AF181850), which
encodes a Mr 33,000 protein,
was composed of exons 1a and 2 and that the ING1a form
(GenBank accession number AF181849), which is predicted to encode a
Mr 47,000 protein, was composed of
exons 1b and 2 in our map (Fig. 2
). The third spliced form,
ING1c (GenBank accession number AB031269), which is
predicted to encode a Mr 24,000
protein, was composed of a truncated ING1a message including
the first ATG codon in exon 2 on our map. The size of the coding region
in exons 1a, 1b, and 2 is 136, 565, and 701 bp, respectively. Our sequence results showed some differences from previously published cDNAs (3) : (a) GTG to GCG at codon 123; (b) GTT to GCT at codon 129; (c) AAT to AAA at codon 135; (d) GAT to GAG at codon 137; (e) GTA to GCA at codon 139; and (f) TCT to GCT at codon 142, according to numbering from exon 1a. These new sequences were reconfirmed by our genomic DNA and cDNA analyses and by the database corrections made recently by Ma et al. (12) .
When our study was almost complete, the genomic organization of the mouse ING1 gene and the functional diversity of alternatively spliced variants of the gene product were reported (13) . In the mouse ING1 gene, there are three alternative exons corresponding to human exons 1a and 1b, but only two final protein products were predicted [p37 (279 aa) and p31 (185 aa), corresponding to human p33ING1b (279 aa) and p24ING1c (210 aa), respectively]. The largest form, p47ING1a, appears to be unique to the human. Three putative promoters in the mouse gene were also inferred by sequencing (13) .
Promoter Analysis of the Human ING1 Gene.
To analyze the promoter for the p33ING1b form, the 5'-flanking
3.5-kb region containing exon 1a was digested at six restriction sites
described in Fig. 2
, (1), and six luciferase reporter
plasmids were constructed and assayed. Four luciferase constructs with
the promoter region between the XbaI and NotI
sites showed about a 39–59-fold increase in luciferase activity as
compared with the promoterless luciferase plasmid. A promoter region of
about 1.7 kb from the ApaLI site to the translational
initiation site was sequenced and searched for binding sites of
transcription factors. There were some motifs such as the GC box, the
GATA box, and the TATA box-like and CCAAT box-like motifs
(6)
. The CCAAT box-like and TATA box-like motifs were
detected at almost the same position as shown in the putative promoter
for exons 1a and 1b of mouse ING1 (13)
. The
promoter analysis for the p47ING1a form was performed by using the
1.1-kb fragment between the NotI site in intron 1 and the
PflMI site located 12 nucleotides upstream from the
initiation codon for p47ING1a as shown in Fig. 2
, (2). The
NotI-PflMI luciferase construct and the four
derivative constructs showed a 2–12-fold increase in luciferase
activity as compared with the promoterless luciferase plasmid. To
analyze the promoter for the p24ING1c form, the 1.6-kb fragment between
the NotI site in intron 1 and the SmaI site in
exon 1b was used in the luciferase assay as shown in Fig. 2
,
(3). This region showed a 262-fold increase in luciferase
activity, despite the lower luciferase activity on the
NotI-PflMI region described above, suggesting
that this strong promoter existed in the coding exon between the
PflMI and SmaI sites. To confirm these results,
we analyzed each spliced form by PCR with mRNA from head and neck
tissues or cDNA libraries from human fetal lung cells and HeLa cells.
The ING1c form was readily detected with a sense primer
locating at a 150-nucleotide downstream region from the initiation
codon for p47ING1a and an antisense primer on exon 2, whereas no
ING1a form was detected with another sense primer set on the
initiation codon for p47ING1a and an antisense primer on exon 2.
Instead, we detected a weakly expressed form composed of the first 29
codons of exon 1b and exon 2 in frame that contained a limited region
amplified with the primer pair
used6
(summarized in Fig. 2
), but the importance of this form is not yet
known. The promoter for p47ING1a was relatively weak, suggesting
that it may be activated in a tissue-specific manner. We could not
identify the transcriptional start site for the ING1a form
as reported (12)
by 5' extension of cDNA ends as
long as it was reported (12)
. In the promoter regions for
the human ING1a and ING1c forms, we found three
motifs for transcriptional binding factors: (a) the GC box;
(b) the CCAAT box-like sequence; and (c) the TATA
box-like sequence6
(summarized in Fig. 2
). RT-PCR
analysis also showed that the ING1b form was expressed as a
major transcript in human normal head and neck tissues and in cDNA
libraries from human fetal lung and HeLa cells.6
Mouse exon 1c is included within human exon 1b with a limited homology, whereas the putative promoter sequence for mouse exon 1c was conserved with a higher homology (about 70%) within the intraexonic promoter in human exon 1b. In contrast, coding and noncoding regions in human exon 1a as well as the upstream promoter region conserved a high homology (about 80%) with mouse ING1 exons 1b and 1a and promoter sequences (13) . It is noteworthy that human sequences similar to mouse exon 1a or 1c did not show any proper structure as a functional exon.
Mutation Analysis.
To investigate whether the ING1 gene is the target of
functional loss in tumors, we searched for mutations in the coding
regions of the gene in 23 samples of HNSCC with LOH at chromosome
13q34. Six possible mutations were identified by SSCP analysis. Three
samples showed missense mutations with aa change (13%) (Fig. 3
). The other three samples displayed a common alteration from TCG to TCA
at codon 173 of p33ING1b without an aa change. All of these point
mutations were confirmed by repeated, independent PCR amplification and
SSCP analyses. The ING1 gene encodes nuclear proteins that
contain a zinc finger motif in their COOH-terminal half
(3)
. Zinc finger motifs are classified into several
subfamilies, and most of them have a role in the recognition of
macromolecules such as DNA, RNA, and protein (14)
. The PHD
finger, a C4HC3-type zinc finger spanning 50–80 aa residues, has been
found in a number of chromatin-mediated transcriptional regulators from
a variety of sources (15)
. One of the mutations in this
study showed an aa substitution from cysteine (TGC) to serine (TCC) at
codon 215, which is one of the seven cysteine residues composing the
C4HC3 motif in ING1. This change may affect the PHD finger and break
the three-dimensional structure of ING1 protein, leading to loss of the
function. The second mutation at codon 216 is just next to the
above-mentioned cysteine residue. This missense mutation showed
an aa change from asparagine (AAC) to serine (AGC) and may also affect
the conformation of the zinc finger domain and the ING1 protein. The
third mutation we detected caused an aa change from alanine (GCC) to
aspartic acid (GAC) at codon 192. This aa was next to a lysine within
the possible nuclear localization signal of ING1. This substitution
from a nonpolar to an acidic aa may affect the nuclear localization
signal that is abundant in positively charged aa and may ultimately
interfere in the accumulation of ING1 protein in the nucleus. All three
mutations were located within the area of exon 2 encoding the common
COOH-terminal portion of all ING1 protein species.
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As compared with the LOH value (49%), the frequency of missense mutation of the ING1 gene (13%) is still low in HNSCC. Several possible explanations may explain this result.
First, the ING1 gene and flanking regions are highly GC rich, and methylation of the promoter region may cause a loss of gene products. A well known tumor suppressor gene, p16, has rarely demonstrated homozygous loss or point mutations because the gene is usually inactivated by aberrant methylation in head and neck cancers (17) . Research aimed at examining methylation in the promoter region of the ING1 gene may uncover another mechanism for inactivation of this gene. Although we have just started to look for methylation of the ING1 gene, one sample with LOH showed a tumor-specific complete loss of the ING1 transcripts by RT-PCR.6 This suggests that methylation of the ING1 promoter region does occur. Our data on the structure of the promoters of ING1 may be useful for these analyses. Second, it has been shown that ING1 protein interacts physically with p53 and that the growth-inhibitory effect of ING1 requires the activity of wild-type p53 (18 , 19) . Because mutations of p53 have been reported frequently in various human tumors including HNSCCs (20) , ING1 may not exhibit its growth-inhibitory activity in cells in which wild-type p53 is lost, and alterations in ING1 may not be necessary in these cases. With regard to this issue, it was recently reported that the p33 equivalent of the mouse ING1 gene acts as a p53 suppressor and not as an activator, as p24ING1c does (13) . Thus, the situation may be complicated. Because the control mechanism(s) of the ING1 gene in the human and mouse forms appears to have both common and different pathways, the exact nature of each protein variant must be analyzed separately, including the p53-dependent and -independent pathways as suggested previously (13) . No matter what ING1 protein variant is responsible for tumor suppression, we suppose that the observed mutations in the COOH-terminal portion of the protein influence the tumor-suppressing ability of ING1 proteins. The high prevalence of LOH at 13q33–34 in HNSCCs suggests the importance of tumor suppressor ability rather than oncogenic ability for the gene(s) located here.
Thirdly, Knudson’s definition (16) of a tumor suppressor gene requires the demonstration of inactivation of both alleles of a candidate gene in tumors. However, a new class of tumor suppressor gene with haploid insufficiency, in which one allele is lost and the remaining allele is haploinsufficient, has been described recently, and these hemizygous tumor suppressor genes show a tumor-prone phenotype when challenged with carcinogens (21 , 22) . Most of the head and neck cancer patients continued smoking for a long time, and inactivation of only one allele of the ING1 gene or a moderately decreased expression may be sufficient to predispose cells to tumorigenesis. Finally, it can also be thought that there may be another tumor suppressor gene in chromosomal region 13q33–34. More detailed studies will clarify this possibility in the near future.
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Acknowledgments |
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FOOTNOTES |
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1 Supported by grants-in-aids from the Ministry of
Education, Science, Sports and Culture and from the Ministry of Health
and Welfare of Japan (to K. S.). 
2 To whom requests for reprints should be
addressed, at Department of Molecular Genetics, Institute of Cellular
and Molecular Biology, Okayama University Medical School, Shikata-cho
2-5-1, Okayama 700-8558, Japan. Phone: 81-86-235-7378; Fax:
81-86-235-7383; E-mail: shimke47{at}med.okayama-u.ac.jp
3 The abbreviations used are: HNSCC, head and neck
squamous cell carcinoma; LOH, loss of heterozygosity; BAC, bacterial
artificial chromosome; RT-PCR, reverse transcription-PCR; SSCP,
single-strand conformational polymorphism; aa, amino acid(s).
5 The nucleotide sequences reported in this
study will appear in the DDBJ/EMBL/GenBank nucleotide sequence
databases with accession numbers AB037386 and AB037387.
6 M. Gunduz and M. Ouchida, unpublished results.
Received 1/31/00. Accepted 5/ 2/00.
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, coding region in exon; 
, noncoding
region in exon. Dots above the line indicate the
distribution of cis elements for transcription factors.
One alternative variant indicated by an asterisk is a
new form identified in this study by RT-PCR, and the amplified region
with the primers is shown. Only relevant recognition sites for
restriction enzymes are shown. Promoter regions used in the luciferase
assay are denoted by horizontal bars and the restriction
sites. The luciferase activity measured with 293 cells is shown to the
right of each bar as compared with that of the
promoterless reporter plasmid.
, not informative (homozygous).
B, primary LOH data from three representative HNSCCs.
N, normal DNA; T, tumor DNA. Cases 14 and
15 show LOH of marker D13S278 but retention of the flanking markers.
Case 20 also shows LOH of markers D13S796 and D13S278 but retention of
the flanking markers.