10.1245/ASO.2004.03.056
Annals of Surgical Oncology 11:192-196 (2004)
© 2004 Society of Surgical Oncology
Deleted in Oral Cancer-1 Expression Upregulates Proapoptosis Elements in Microsatellite-Unstable Human Colorectal Cancer
Tara Sotsky Kent, MD,
Ziqiang Yuan, MD,
Agnes Miller, BA and
Thomas K. Weber, MD, FACS
From the Departments of Surgery (TSK, TKW) and Molecular Genetics (TKW), Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, New York; and Departments of Molecular Genetics (ZY) and Surgery (AM), Albert Einstein College of Medicine, Bronx, New York.
Correspondence: Address correspondence and reprint requests to: Thomas K. Weber, MD, FACS, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Ullmann 1219, Bronx, NY 10467; Fax: 718-430-2773; E-mail: tweber{at}aecom.yu.edu
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ABSTRACT
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Background: We previously reported differential expression of the growth suppressor, deleted in oral cancer-1 (DOC-1), in microsatellite-unstable (MSI+) versus microsatellite-stable colorectal cancer (CRC) cell lines. MSI+ CRC cell lines demonstrated decreased DOC-1 expression and decreased apoptosis. Transfection of wild-type DOC-1 into an MSI+ cell line (SW48) resulted in increased apoptosis. We undertook our current experiment to identify specific elements modulated by DOC-1 expression that result in increased apoptosis.
Methods: SW48 is an MSI+ CRC cell line that does not constitutively express DOC-1. SW48 was suspended in culture medium and incubated to 60% confluence. Half the plates were transfected with cytomegalovirus (CMV)-DOC-1. At 30 hours, RNA and protein were isolated with Trizol. Complementary DNA microarray was performed to compare SW48CMV-DOC-1 with SW48, which lacks DOC-1. Signal intensity was analyzed by GenePix Pro 3.0 software. Expression ratios 
.67 and 
1.5 were considered significant. Poor-quality spots were flagged and excluded from analysis. Real-time polymerase chain reaction was performed to determine DOC-1 levels in both cell lines.
Results: Successful transfection of DOC-1 was confirmed by real-time polymerase chain reaction and by Western blot. Microarray revealed significant differential expression of DOC-1, as expected. Increased DOC-1 expression in SW48CMV-DOC-1 was associated with significantly increased expression of proapoptosis components of the caspase cascade (CASP7, CASP9) and bcl2/bax pathway (BNIP3, BNIP3L, BID).
Conclusions: DOC-1 expression promotes apoptosis by upregulation of specific elements of the caspase cascade and bcl2/bax pathways. DOC-1 therefore deserves further study as a candidate for the therapeutic modulation of apoptosis in MSI+ CRC.
Key Words: DOC-1 • Colorectal carcinoma • Apoptosis • Microsatellite unstable
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INTRODUCTION
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The development of colorectal cancer via the Vogelstein adenoma-carcinoma sequence represents one pathway to malignancy. However, variations on this pathway continue to be elucidated.1 More than 80% of colorectal adenocarcinomas (CRC) in carriers of mismatch repair gene germline mutations1 and 20% of sporadic CRCs2,3 are characterized by microsatellite instability,4 or alterations in the length of widespread DNA repeat sequences. The large number of widely distributed altered microsatellites renders the DNA unstable and ultimately facilitates malignant transformation.5,6 These microsatellite-unstable (MSI+) tumors are thought to arise via a pathway of malignant transformation and progression distinct from that associated with microsatellite-stable (MSS) CRC.2 This distinction is supported by reports, at the genomic level, of mutations in growth-regulatory genes, such as transforming growth factor (TGF)-ßRII, insulin-like growth factor, BAX, hMSH3, and hMSH6, that are typical of MSI+ CRC.5,7 Furthermore, MSI+ and MSS tumors can often be distinguished by differing ploidy and the degree of loss of heterozygosity (LOH): MSI+ tumors tend to be diploid and lack LOH, whereas MSS tumors are often aneuploid with significant LOH.2 Identification of those elements unique to the MSI+ pathway will facilitate an understanding of the means by which tumors of this sort develop and progress and ultimately will facilitate the development of appropriate targeted therapy.
Through our prior work using a complementary DNA (cDNA) microarray to compare MSI+ and MSS human CRC cell lines, we have identified deleted in oral cancer-1 (DOC-1) as one such element with differential expression in MSI+ compared with MSS tumors.8 DOC-1 is a highly conserved gene mapped to chromosome 12q24 that was initially identified and characterized by LOH and decreased expression of its translation product, the growth suppressor p12DOC-1, in oral squamous cell carcinoma.9,10 It is constitutively expressed in normal human tissues, in which it is has been shown to interact with the growth-regulatory elements DNA polymerase 
/primase, cdk2, and pRB (retinoblastoma) as a growth suppressor. Via its interaction with DNA polymerase /primase, p12DOC-1 negatively regulates the rate of initiation of DNA replication.10,11 Its association with the nonphosphorylated form of cdk2 negatively modulates cell-cycle progression.12 Most recently, p12DOC-1 has been found to suppress cdk2-mediated RB phosphorylation, thereby mediating TGF-ß1 antiproliferative activity.13 Decreased p12DOC-1 expression, demonstrated in oral squamous cell carcinoma, has been found to correlate with tumor invasion, lymph node metastases, and decreased survival.14
We reported differential DOC-1 expression, with decreased or absent expression in MSI+ CRC cell lines,8 suggesting that loss of p12DOC-1 expression is a characteristic of malignant transformation in MSI+ CRC. Cell-cycle progression and apoptosis also differed significantly according to p12DOC-1 expression, such that MSI+/DOC-1- cell lines had increased S-phase cell populations and a lower percentage of apoptotic cells compared with MSS/DOC-1+ cell lines by flow cytometry and terminal transferase-mediated dUTP nick end labeling assay.8 We further demonstrated that p12DOC-1 expression contributes to the cell proliferation and apoptosis profiles by inducing its expression in SW48, an MSI+/DOC-1–negative cell line. Repeated flow cytometry analysis revealed cell-cycle and apoptosis profiles similar to those of the MSS cell lines that constitutively express p12DOC-1, with an increased apoptotic cell percentage and decreased S-phase population compared with native SW48 and SW48 transfected with empty vector.8
The increased S-phase percentage in MSI+/DOC-1–negative cells is consistent with previously established knowledge about the DOC-1/cdk2 interaction, in which the association of p12DOC-1 with the nonphosphorylated form of cdk2 prevents the conformational changes that allow cyclin binding, cdk2 activation, and cell-cycle progression.11,15 Increased apoptosis after DOC-1 transfection was reported in malignant hamster oral keratinocytes.16 A more recent study conducted with the squamous epithelium of the Syrian hamster cheek pouch model, with the goal of establishing it as an appropriate model for the study of oral carcinogenesis, reported changes in p12DOC-1 expression profiles associated with changes in the ratio of apoptotic to proliferating cells.17 However, no association between DOC-1 expression and apoptosis in colon cancer has previously been reported. Although our prior work supported a role for DOC-1 in modulation of apoptosis by demonstrating an association between DOC-1 status and apoptosis, it did not explain the mechanism by which the modulation may occur. Our early work, based on differential expression studies of MSI+ and MSS CRC cell lines, suggested the involvement of the caspase cascade, the bcl2/bax pathway, or both. However, those initial studies were conducted by comparing an MSI+ cell line with an MSS cell line; thus, we were unable to attribute differential gene expression to DOC-1 specifically. We therefore undertook this study with the goal of identifying the specific elements modulated by isolated changes in DOC-1 expression that result in increased apoptosis.
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METHODS AND MATERIALS
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Cell Lines
The MSI+8 CRC cell line SW48 was obtained from the American Type Culture Collection (Manassas, VA). It was maintained in Dulbecco’s modified Eagle’s medium with L-glutamine supplemented with 10% fetal bovine serum, 50 U/mL of streptomycin/penicillin, 1% nonessential amino acids, and C4-C2-hydroxyethyl piper. Cultures were incubated at 37°C with 5% CO2 to 60% confluence. RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) according to kit instructions, and protein was isolated with trypsin and lysis buffer. RNA quality was confirmed by gel electrophoresis.
Transient Transfection With p12DOC-1
SW48 culture plates were transfected with 5 µg of p12DOC-1-CMV expression plasmid (from D. T. Wong, Boston, MA). Control plates were transfected with empty vector (pcDNA3) or left untransfected. Cells were incubated as described previously and harvested at 30 hours for RNA and DNA isolation.
Complementary DNA Microarray Analysis
Total RNA was extracted from SW48 and from SW48p12DOC-1-CMV by using Trizol (Invitrogen) and was used to produce cDNA by anchored oligo(dT)12-18 primer (Invitrogen, Carlsbad, CA) reverse transcription with SuperScript II reverse transcription (Life Technologies, Inc., Carlsbad, CA). SW48 and SW48p12DOC-1-CMV cDNAs were labeled with the fluorescent dyes Cy3-dUTP and Cy5-dUTP (Amersham, Piscataway, NJ), respectively. The cDNA probes were then hybridized to silane glass slides containing 9568 cDNA human gene spots according to Albert Einstein College of Medicine standard protocol: each slide was probed with the native and the transfected SW48 probes. Slides were scanned in our microarray facility, and the images were exported to Gene Pix Pro 3.0 software (Axon Instruments, Inc., Union City, CA) for signal intensity analysis. Poor-quality spots were flagged and excluded from analysis. Signal intensity information was exported to Excel (Microsoft Corp., Redmond, WA), where the data were normalized before statistical analysis. Student’s t-test was used to evaluate the significance of the difference in signal intensity of the spot compared with the background. Data were sorted by -fold change, and genes with at least a 1.5-fold change were accepted as significant on the basis of the t-test criterion and subjected to further investigation.
Real-Time Polymerase Chain Reaction
Total RNA extracted from SW48, SW48p12DOC-1-CMV, and SW48pcDNA3 was used to produce cDNA as described previously, by using SuperScript II reverse transcription. A semiquantitative real-time reverse transcription-polymerase chain reaction (PCR) method was used to measure the messenger RNA level in the selected targets (ß-actin, DOC-1, CASP7, CASP9, BID, BNIP3, and BNIP3L) according to the manufacturer’s protocol (Ciphergen, Fremont, CA). The semiquantitative real-time reverse transcription-PCR assay was performed on the SmartCycler PCR machine (Ciphergen, CA) by using the SYBR-Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA). The matching primers were designed to cross at least one exon for each gene. For each sample, the amounts of the target and the housekeeping gene (ß-actin) were determined by real-time PCR. The ratio of the target to ß-actin was calculated as the normalized value.
Western Blot Assay
Whole cell protein extracted from the cultured SW48 cell lines (native, transfected with empty vector, and transfected with p12DOC-1-CMV) was subjected to Western blot analysis with the FLAG-p12DOC-1 polyclonal antibody (D. T. Wong, Boston, MA) as previously described.14 The membrane was incubated with anti-p12DOC-1 antibody (1/8000 dilution) and hybridized for 1 hour at room temperature with the secondary antibody, horseradish peroxidase–conjugated rabbit anti-mouse antibody. Protein was detected by a Super Signal West Pico kit (Pierce, Rockford, IL) and exposed to x-ray film. Western blot analysis was also conducted, as described by Shintani et al.14 and previously, to confirm microarray and real-time PCR findings by using antibodies to CASP7, CASP9, BID, and BNIP3.
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RESULTS
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SW48 was previously confirmed to be MSI+ and to express BAX protein.8 Successful transfection of DOC-1 was confirmed by Western blot (Fig. 1) and by real-time PCR. p12DOC-1 was present in the transfected cell line but not in the native SW48. A cDNA microarray, performed as described previously, revealed significant differential expression of DOC-1, as expected (expression ratio, 3.96; P < .05). Increased DOC-1 expression in SW48CMV-DOC-1 was associated with significantly increased expression of proapoptosis components of the caspase cascade and bcl2/bax pathways. These included CASP7 and CASP9 (caspase cascade) and BNIP3, BNIP3L, and BID (bcl2/bax pathways; Table 1). Additional genes in these pathways with significant differential expression are listed in Table 2.
Received for publication March 30, 2003.
Accepted for publication September 29, 2003.
TOP
ABSTRACT
INTRODUCTION
