From the Laboratory of Molecular Pathology, Division
of Oral Pathology, Harvard School of Dental Medicine, Boston,
Massachusetts 02115, ¶ Intramural Research Support Program, SAIC
Frederick and
Laboratory of Immunobiology, Division of Basic
Sciences, NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, ** Laboratory of Experimental Carcinogenesis,
Division of Basic Sciences, NCI, National Institutes of Health,
Bethesda, Maryland 20892, and the
Department of Oral and Maxillofacial
Surgery II, Okayama University Dental School, Okayama 700, Japan
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ABSTRACT |
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doc-1 is a putative tumor suppressor
gene isolated and identified from the hamster oral cancer model. Here,
we report the molecular cloning and the functional characterization of
the human ortholog of the hamster doc-1 gene. Human
doc-1 cDNA is 1.6 kilobase pairs in length and encodes
for a 115-amino acid polypeptide (12.4 kDa, pI 9.53). Sequence analysis
showed 98% identity between human and hamster doc-1
protein sequences. DOC-1 is expressed in all normal human tissues
examined. In oral keratinocytes, expression of DOC-1 is restricted to
normal oral keratinocytes. By immunostaining of normal human mucosa,
DOC-1 is detected in both the cytoplasm and nuclei of basal oral
keratinocytes; while in suprabasilar cells, it is primarily found in
the nuclei. Human oral cancers in vivo did not exhibit
immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates
with DNA polymerase /primase and mediates the phosphorylation of the
large p180 catalytic subunit, suggesting it may be a potential
regulator of DNA replication in the S phase of the cell cycle. Using a
human doc-1 cosmid as a probe, human doc-1 is
mapped to chromosome 12q24. We identified four exons in the entire
human doc-1 gene and determined the intron-exon boundaries.
By polymerase chain reaction and direct sequencing, we examined
premalignant oral lesion and oral cancer cell lines and found no
intragenic mutations.
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INTRODUCTION |
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Squamous cell carcinoma (SCC)1 of the oral cavity is newly diagnosed in 38,000 Americans each year and in 350,000 people worldwide (1, 2). Approximately half of the patients afflicted die within 5 years of diagnosis, while surviving patients may be left with severe cosmetic and/or functional compromise (1-3). Survival curves of oral cancer patients have plateaued over the past 2 decades and remained among the worst of all cancer sites.
The hamster oral cancer model is an excellent model to study the
molecular event during oral carcinogenesis (4-7). doc-1 is
a putative tumor suppressor gene identified and isolated from the
carcinogen-induced hamster oral cancer model (8). DOC-1 is predicted to
be a 114-amino acid peptide with a molecular mass of 12.4 kDa.
Transfection of doc-1 into malignant oral keratinocytes led
to the reversion of transformation phenotypes including anchorage independence, doubling time, and morphology. The genetic sequence of
doc-1 matched to a tumor necrosis factor--induced
early-response murine transcript, TU-166 (9), suggesting that
doc-1 may be a downstream event in the tumor necrosis
factor-
signaling pathway. We have recently cloned the full-length
mouse doc-1 cDNA (GenBankTM number
AF011644); its DNA sequence in the open reading frame is 94% identical
to that of the hamster. The predicted amino acid peptides encoded by
the mouse and hamster doc-1 open reading frames are
identical. Each open reading frame encodes for a 114-amino acid peptide
that has a predicted molecular mass of 12.4 kDa and a pI of 9.53.
The highly conserved nature of the rodent doc-1 genes
prompted us to clone and examine the role of doc-1 in human
oral and other cancers. This paper describes the cDNA and genomic
DNA cloning of the human doc-1 gene, its chromosome
localization, and its expression in normal and transformed human
tissues. In addition, we examined for intragenic mutation of the human
doc-1 gene. To obtain an insight into the biology of DOC-1,
we made use of our recent finding that murine DOC-1 associates with DNA
polymerase /primase (DNA-PP) and regulates DNA replication in the S
phase of the cell cycle. The potential interaction of human DOC-1 with DNA-PP was therefore examined.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Culture Media-- Normal, diploid human oral keratinocyte cell strains, having a limited replicative lifespan, have been cultured from floor of mouth (OKF4 and OKF6) and cryopreserved within their first two serial passages in culture. Oral squamous cell carcinoma-derived, immortal cell lines (SCC-9, SCC-15, and SCC-25) were cryopreserved within their first five serial passages in culture (10, 11). POE-1 and POE-9 are dysplastic "premalignant" oral lesions from two newly diagnosed, previously untreated oral biopsies.
The human oral keratinocyte cultures were grown in Life Technologies, Inc., keratinocyte serum-free medium supplemented with hydrocortisone, insulin, transferrin, 0.1 ng/ml epidermal growth factor, and 50 mg/ml bovine pituitary extract. We added additional CaCl2 to bring the total [Ca2+] to 0.4 mM (12), a concentration that is sufficient to permit cadherin- and desmosome-mediated cell-cell junction formation. This results in closer association of sister cells in growing colonies and permits stratification of terminally differentiated cells in the central regions of larger colonies, thereby aiding the identification of individual colonies and the evaluation for growth rate and relative proportions of proliferative and differentiated cells. This calcium concentration does not inhibit proliferation of human keratinocytes. Media were supplemented with penicillin and streptomycin. Cells were cryopreserved in medium containing 10% Me2SO and 10% serum and stored in liquid nitrogen freezers.Cloning of doc-1 cDNA and Genomic DNA-- Using the plaque hybridization method (13), cDNA clones were obtained by screening a human testes cDNA library (Stratagene, La Jolla, CA) with the hamster doc-1 cDNA (8). Genomic clones were isolated from a total human genomic placental cosmid library (Stratagene, La Jolla, CA) by colony hybridization using a human doc-1 cDNA clone as a probe (14).
DNA Sequencing and Sequence Analyses-- Plasmid cDNA clones and genomic cosmid clone were sequenced by automated sequencing as was described by Duh et al. (15). Sequence editing, sequence analyses, and homology search were performed following previously published methods (15).
Fluorescent in Situ Hybridization-- Chromosomes obtained from human peripheral lymphocyte cultures after methotrexate/thymidine release treatments and normal mouse spleen cultures, were used for fluorescence in situ hybridization (FISH). The probes were labeled with biotin or digoxigenin using a random-prime DNA labeling kit (Boehringer Mannheim). The FISH protocol as described in detail elsewhere was followed (16, 17). The slides were pretreated with RNase, denatured in 2× SSC, 70% (v/v) formamide for 2 min at 70 °C. Human cosmid DNA probes (200 ng) together with human Cot-1 DNA (Life Technologies) in 2× SSC, 50% (v/v) formamide, 10% (w/v) dextran sulfate, 2× Denhardt's solution, 1% Tween 20 (v/v) were denatured for 5 min at 70 °C, reannealed for 2 h at 37 °C, and hybridized in a humid environment for 18 h at 37 °C. Posthybridization final wash was in 0.1× SSC at 60 °C. Biotin and digoxigenin-labeled DNA was detected by fluorescein isothiocyanate-conjugated avidin DCS (Vector Laboratories) and rhodamine-conjugated antidigoxigenin (Boehringer Mannheim), respectively.
Chromosomes were counterstained with propidium iodide (PI) or 4,6-diamino-2-phenylindole (DAPI) and examined with a Zeiss Axiophot epifluorescent microscope with a 100-watt mercury lamp. Digital images of selected metaphase spreads were recorded using a cold charge-coupled device camera CH250 (Photometrics, Tuscon, AZ) and a filter system consisting of a triple band pass beam splitter and emission filters. Excitation of each of fluorochromes used was accomplished by single band pass excitation filters in a computer-controlled motorized filter wheel. This made it possible to acquire sequential, registration shift-free gray scale images of two or three fluorochromes (DAPI, fluorescein isothiocyanate, and/or rhodamine). Images were processed and analyzed on an Apple Power Macintosh 8100/100 computer using Oncor recording and analytic program Image, and well as NIH Image and Yale University's Gene Join. To identify individual chromosomes and to assign the location of signal to specific chromosome regions, the method for direct visualization of fluorescent spots on LUT-inverted digital images of DAPI-banded chromosomes was used (18). To confirm the identity of chromosomes, preparations were rehybridized with human chromosome 12-specific painting probes (Oncor, Gaithersburg, MD), and previously observed labeled metaphases were recorded.Northern Blot Analysis-- Total RNA was isolated from human oral keratinocytes using the guanidine isothiocyanate method described by Davis et al. (19). Details of Northern blot analysis using the Zetabind membrane were described previously (20, 21). Random priming was used to label the cDNA inserts.
Immunohistochemical Staining for DOC-1 in Normal and Tumor Oral
Epithelia--
Three normal oral mucosal tissues (two tongues and one
gingival tissue sample) and five primary oral squamous cell carcinoma were subjected to DOC-1 immunohistochemical study. These tissues were
obtained from biopsy and surgical operations at the Department of Oral
and Maxillofacial Surgery II, Okayama University Dental School Hospital
(Okayama, Japan). All samples were embedded in OCT compound without
fixation, snap-frozen, and stored at 80 °C until used. Frozen
sections (4-6 µm) were air-dried after sectioning and then
rehydrated in phosphate-buffered saline (pH 7.4). Immunohistochemical staining was performed using a rabbit polyclonal anti-hamster DOC-1
antibody at a dilution of 1:200 to 1:500 for overnight at 4 °C.
DOC-1 immunoreactivity was detected by the indirect immunoperoxidase method with an Envision kit (DAKO, Tokyo, Japan).
Interaction of DOC-1 with DNA Polymerase /Primase--
The
human doc-1 cDNA was cloned into the GST fusion protein
vector pGEX-4T-1 (Pharmacia Biotech, Inc.) at the SalI (5')
and NotI (3') sites. GST-DOC-1 fusion protein was produced
by growing pGST-doc-1 transformed DH5-
cells to
A600 between 1.0 and 2.0, and induced with 0.1 mM isopropyl-
-thiogalactopyranoside (Stratagene, La
Jolla, CA). GST-DOC-1 fusion proteins were purified with use of
glutathione-Sepharose 4B (Pharmacia). The production of the GST-DOC-1
fusion protein was confirmed by SDS-polyacrylamide gel electrophoresis
and Western blot with a DOC-1-specific rabbit polyclonal antibody (8).
For use in experiments, the GST-DOC-1 fusion protein bound to
glutathione-Sepharose 4B was either eluted with the elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH
8.0) or cleaved by thrombin (Sigma) to separate from the GST moiety.
GST protein produced by parental pGEX-4T-1 vector was also purified in
the same fashion and used as a control.
Detection of Intragenic Mutations in the doc-1 Gene in Human Oral Keratinocyte Cell Lines-- Following the strategy that was employed by Duh et al. (15), we have determined the intron-exon boundaries of the human doc-1 gene. Primer sets (Table I) designed from the UTR sequences and intronic sequences were used to screen intragenic mutations in the four doc-1 coding exons. DNAs isolated from the premalignant and malignant human oral keratinocyte cell lines were polymerase chain reaction-amplified, and the polymerase chain reaction products were directly sequenced to examine for any intragenic mutations.
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RESULTS |
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doc-1 Is a Highly Conserved Gene-- doc-1 was initially identified and cloned from normal hamster oral keratinocytes by a subtractive hybridization procedure (8). To determine whether the doc-1 gene is present in the human genome as well as genomes of other species, a multiple-species genomic DNA blot (CLONTECH) containing EcoRI-digested genomic DNA from human, monkey, rat, mouse, dog, cow, rabbit, chicken, and yeast was hybridized with 32P-labeled hamster doc-1 cDNA (Fig. 1). doc-1 hybridizable fragments were clearly detected in the genomes of humans, rats, mice, dogs, cows, and rabbits. Faint doc-1 hybridization bands were observed in monkey and chicken genomes, and no signals were detectable in yeast.
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doc-1 cDNA and Genomic DNA Cloning--
We isolated two
full-length cDNA clones, 1.6 and 1.3 kb, and each was completely
sequenced. Both contained an open reading frame of 115 amino acids and
polyadenylation signal AATAAA. The 1.6-kb clone (clone 15) contained
522 bp of 5'-untranslated region, and the 1.3-kb clone (clone 8)
contained 219 bp of 5'-untranslated region. cDNA sequence of the
1.6-kb clone has been deposited in GenBankTM (accession
number AF006484). Results of sequence analysis showed that the homology
between the human and the hamster doc-1 genes was very high:
81% identity in cDNA sequences, and 98% identity in peptide
sequences. Fig. 2 shows that except for
the additional alanine at residue 19, the human DOC-1 protein differs
from the hamster DOC-1 only at two amino acid residues (Ala Thr at
residue 8 and Gly
Ser at residue 100). The 5'-untranslated region
sequence of the 1.6 kb was analyzed with the Web Promoter Scan
Program.3 A putative promoter
region containing AP-2, Sp-1, GCF, UCE.2, and T-Ag motifs was located
at nucleotides 27-277.
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Chromosome Mapping of the Human doc-1 Gene-- FISH was used to map the chromosome location of the human doc-1 gene. In duplicate experiments, a high efficiency of FISH was observed when biotin- and digoxigenin-labeled genomic doc-1 probe was hybridized to normal human chromosomes. The majority of 200 metaphases had fluorescent label on the chromosomes. The majority of 200 metaphases had fluorescent label on the distal region of the short arm of both chromosomes 12 regardless of detection protocol (fluorescein isothiocyanate or rhodamine), and symmetrical fluorescent spots were detected on the long arm of chromosome 12. The signal was localized at region 12q24 in 50 nonoverlapped and variably contracted chromosomes 12 showing a G-like banding pattern generated by contrast enhancement and LUT inversion of digital images of DAPI-counterstained chromosomes (Fig. 4). Within the region 12q24, where we assign the locus of the doc-1 gene, is a common fragile site where several genes are located (22).
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doc-1 Expression in Normal Human Tissues and Oral
Keratinocytes--
Expression of doc-1 in normal human
tissues was examined using a multiple tissue Northern blot
(CLONTECH) containing mRNA isolated from
various normal human tissues. All tissues examined (heart, brain,
placenta, lung, liver, skeletal muscle, kidney, and pancreas)
demonstrated high cellular levels of the 1.5-kb doc-1
transcript (Fig. 5A). The
bottom of panels A and B shows the
rehybridization of the same blot with the chicken -actin gene to
demonstrate RNA loading.
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Human DOC-1 Associates with the p180 Catalytic Subunit of DNA-PP-- Our laboratory has recently demonstrated that murine DOC-1 associates and mediates the phosphorylation of the p180 catalytic subunit of DNA-PP.2 The interaction of DOC-1 with DNA-PP is associated with suppression of DNA replication, shown by the SV40 in vitro DNA replication assay. These findings provide an insight into the mechanism whereby DOC-1 can mediate its growth and/or tumor suppressor function, by regulating DNA replication in the S phase of the cell cycle.
To determine whether human DOC-1 similarly associates with DNA-PP and mediates phosphorylation of the p180 subunit, the human doc-1 cDNA was cloned in frame into the pGEX plasmid vector (pGEX-4T-1) at the SalI and NotI sites (see "Experimental Procedures"). Human DOC-1 was expressed as a GST-DOC-1 fusion protein. SDS-polyacrylamide gel electrophoresis analysis showed that the human DOC-1 peptide was correctly expressed (data not shown). Digestion with thrombin released the DOC-1 peptide at the expected size. Immunoblotting with a DOC-1-specific rabbit polyclonal antibody detected the GST-DOC-1 and DOC-1 peptides (data not shown). 500 µg of lysate prepared from the normal human oral keratinocytes OKF4 was mixed with 5 µg of GST-DOC-1 fusion protein. The DOC-1·OKF4 lysate binding was carried out at 4 °C for 18 h, followed by centrifugation to separate from unbound proteins. A standard phosphorylation assay was set up using the DOC-1·OKF4 pull-down lysate complex in the presence of [
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Intragenic Mutation Analysis of the Coding Exons of Human doc-1 Gene in Human Normal and Malignant Oral Keratinocyte Cell Lines-- The availability of the intron-exon boundaries and the flanking sequences allow the design of primers to amplify the exon sequences to compare the coding regions of doc-1 gene between normal and tumor oral keratinocytes. We performed mutation analysis on two premalignant (POE-1 and POE-9) and three oral cancer cell lines (SCC-9, SCC-15, and SCC-25) using the four primer sets in Table I and did not detect any intragenic mutation.
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DISCUSSION |
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This paper presents the cDNA and genomic cloning as well as partial characterizations of the human doc-1 gene. doc-1 is a highly conserved gene present in the genomes of all mammalian species examined. doc-1 mapped to human chromosome 12q24. Expression of DOC-1 can be detected in all normal human tissues examined. Interestingly, expression of doc-1 is not detectable in malignant oral keratinocytes, in cell lines, and in primary tumor tissues. Intragenic mutation analysis of the doc-1 in premalignant and malignant oral keratinocytes did not reveal any mutations. Like the hamster DOC-1, human DOC-1 associates with DNA-PP and mediates the phosphorylation of the p180 subunit.
The existence of a human homolog and the highly conserved nature of the doc-1 gene prompted us to examine the role of the doc-1 gene in human oral and other forms of cancers. The doc-1 gene was initially identified and cloned from the hamster oral cancer model. In this chemically induced oral cancer model, expression of doc-1 is consistently reduced and/or lost. Loss of heterozygosity was observed in two out of three malignant oral keratinocyte cell lines (8). Transfection of doc-1 into malignant hamster oral keratinocytes altered a number of phenotypes in culture including anchorage dependent growth, doubling time, and morphology. 83% of the doc-1 transfectants lost the ability to grow in soft agar (p < 0.05).
The human doc-1 gene was mapped to chromosome 12q24. While this site has not been noted to be altered in oral/head and neck cancers, 12q24 is a recurrent break point in high grade B cell non-Hodgkin lymphoma (23). The cytogenetic abnormalities included both translocations and interstitial deletions (22).
Expression of doc-1 is consistently reduced/lost in transformed oral keratinocytes. Neither doc-1 mRNA nor protein could be detected in the malignant human keratinocyte cell lines or in freshly resected oral cancers. While no intragenic mutations could be detected in the three tumor cell lines examined, a much larger sample size of human oral and nonoral tumor cell lines needs to be examined to evaluate the potential involvement of the doc-1 gene in carcinogenesis. In addition, cis-regulatory modifications of the doc-1 promoter such as CpG methylation can alter expression of the gene, which has been demonstrated to be of importance in the inactivation of p16ink4A/CDKN2A (24).
The association of human DOC-1 with DNA-PP is of importance and sheds some light on the biochemical function of the doc-1 gene. DNA-PP is the only eukaryotic enzyme that can initiate DNA replication de novo. Based on data from our murine DOC-1 studies, we propose that DOC-1 is a negative regulator of DNA replication through the phosphorylation of the p180 subunit of DNA-PP.2 In tumor cells, where DOC-1 expression is absent or reduced, these DOC-1-mediated DNA-PP regulatory mechanisms might be compromised.
We are beginning to understand the biochemical function of
doc-1. DOC-1 is likely to be a regulator of DNA replication
in the S phase of the cell cycle, of importance in normal cells as well
as in carcinogenesis. Gordon et al. (9) have shown that doc-1 is a tumor necrosis factor--inducible gene,
suggesting that doc-1 is a downstream event in the tumor
necrosis factor-
signaling pathway. Recently, Hatakeyama et
al. (25) have shown that DOC-1 is constitutively ubiquinated
in vivo and is a substrate of the mammalian ubiquitin ligase
Nedd-4 (neural precursor cells expressed developmentally
down-regulated) (25), thus providing a mechanism whereby intracellular
DOC-1 activity is tightly regulated by the ubiquitin-mediated
proteosomal degradation pathway (26). The interconnections of DOC-1 and
these various signaling pathways are of importance in carcinogenesis
and are now being addressed in our ongoing investigations.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Wang (Stanford University) for advice on this project and for providing the polyclonal antibody to human DNA-PP and the baculovirus containing the p180 cDNA. We thank Dr. Ellen Fanning (Vanderbilt University) for advice and for providing the polyclonal antibody to mouse DNA-PP and the baculovirus containing the p68, p55, and p49 subunits to human DNA-PP. We also thank Dr. James G. Rheinwald of the Cell Culture Core at the Harvard Skin Disease Research Center, Brigham and Women's Hospital for providing the human oral keratinocyte cultures. We acknowledge the National Cancer Institute for allocation of computing time and staff support at the Frederick Biomedical Supercomputer Center.
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FOOTNOTES |
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* This work was supported by NIDR, National Institutes of Health, Grants 1 PO1 DE12467, 2 RO1 DE08680-08 (to D. T. W. W.), and 1 R29 DE11983-01 (to R. T.), and Grant 97A024 from the American Institute for Cancer Research (to D. T. W. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
§§ To whom correspondence should be addressed.
1
The abbreviations used are: SCC, squamous cell
carcinoma; DNA-PP, DNA polymerase /primase; FISH, fluorescence
in situ hybridization; DAPI, 4,6-diamino-2-phenylindole;
LUT, look-up table; bp, base pair(s); kb, kilobase pair(s); GST,
glutathione S-transferase.
2 K. Matsuo, E. Nagata, T. Tsuji, M. Lerman, J. McBride, S. Shintani, R. B. Donoff, R. Todd, and D. T. W. Wong, submitted for publication.
3 Available on the World Wide Web at http://ch.nus.sg/bio/proscan/proscan.html.
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REFERENCES |
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