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Molecular Biology and Genetics |
Department of Experimental Pathology, Division of Laboratory Medicine, Mayo Clinic, Rochester, Minnesota 55905 [V. S., J. C., J. S., R. A., D. I. S.]; Millennium Predictive Medicine, Cambridge, Massachusetts 02139 [A. S., S. K., J. Li.]; and Corning, Acton, Massachusetts 01720 [J. Le.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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There are several PCR-based approaches to analysis of gene expression changes including mRNA DD-PCR3 (2 , 3) , RNA fingerprinting by arbitrary primed-PCR (4 , 5) , and RDA (6, 7, 8) . In RDA, several rounds of subtractions are needed. In addition, RDA does not resolve the problem of the wide differences in abundance of individual RNA species. Whereas DD-PCR and arbitrary primed-PCR are potentially faster methods of identifying expression differences between two populations, both of these methods have high levels of false positives and are biased for high-copy-number mRNAs. SSH (9, 10, 11) has the distinct advantage over other PCR-based techniques in that SSH is used to selectively amplify target cDNA fragments (differentially expressed) while simultaneously suppressing nontarget DNA amplification and generating a library of differentially expressed sequences. The normalization step equalizes the abundant cDNAs within a target population, and the subtraction step excludes the common sequences between the driver and tester populations.
In this study, we report on down-regulated genes identified from two early- and two late-stage primary ovarian tumors subtracted against normal ovarian epithelial cell brushings. Collectively our studies demonstrate that (a) several genes, identified in the SSH libraries as down-regulated genes, map to known regions of deletions in ovarian cancer; and (b) LOH analysis revealed novel regions of deletions not previously identified in early-stage tumors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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. Tumors were staged according to the criteria proposed by International Federation of Gynecology and Obstetrics.
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Suppression Subtraction Libraries.
Four down-regulated libraries were generated from individual tumors. OV 338 (stage I endometrioid), OV 402 (stage II serous), and two stage III serous tumors (OV 4 and OV 13) were all subtracted against normal ovarian epithelial cell brushings.
Tester and Driver Preparations.
Total cellular RNA from primary ovarian tumors (driver) and from 20 pooled normal ovarian epithelial cell brushings (tester) was prepared using Trizol reagent (Life Technologies, Inc., Rockville, MD) followed by purification by RNAeasy kit (Qiagen Inc, Valencia, CA). The integrity of the RNA was assessed by agarose gel electrophoresis. One µg of total RNA was then used for first- and second-strand cDNA synthesis in a 10-µl reaction volume using Smart II oligonucleotides and cDNA synthesis (CDS) primer (Clontech, Palo Alto, CA) following the manufacturer’s instructions. The concentration of reverse-transcribed cDNA was adjusted to 25 ng/µl. The resulting cDNAs were amplified, and the cycle number was optimized for each sample after amplification with PCR primer (5'-AAGCAGTGGTAACAACGCAGAGT-3'). For cycle optimization, aliquots of the PCR reactions were removed after 15, 18, 21, and 24 cycles of amplifications. The resulting products were resolved on a 1.5% agarose gel, and optimum cycle number was chosen after southern hybridization with GAPDH and transferrin receptor genes as probes. For most samples, the optimum cycle numbers were between 17 and 19 cycles of amplification. The reaction was scaled up to generate 3 µg of double stranded cDNAs. The resulting cDNA was precipitated, washed with 70% ethanol, dissolved in 40 µl of deionized water, and digested with RsaI in a 50-µl reaction mixture containing 100 units of enzyme (Boeringher Mannheim, Indianopolis, IN) for 3 h. The blunt-ended cDNAs were then purified using PCR purification columns (Promega, Madison, WI). The driver cDNAs from primary tumors were adjusted to 300 ng/µl in a final 7-µl volume. Fifty ng of digested double-stranded tester (normal ovarian epithelial cell brushings) cDNA was ligated in two separate reactions with 2 µl of adapter 1 (10 µM) and adapter 2 (10 µM; provided in the kit), respectively, and 1.0 unit of T4 DNA ligase (Life Technologies, Inc.) in a 10-µl total volume with buffer supplied by the manufacturer. After ligation, 1 µl of 0.2 M EDTA was added and the samples were heated at 75°C for 5 min to inactivate the ligase and stored at -20°C.
Subtractive Hybridization.
SSH was performed between tester and driver mRNA populations using the PCR-select cDNA subtraction kit (Clontech) according to the manufacturer’s recommendations. Two µl of driver double-stranded cDNA (150–200 ng/µl) was added to each of two tubes containing one µl of adapter-1 and adapter-2 ligated tester cDNA (10 ng) with 1x hybridization buffer in a total volume of 4 µl. The solution was overlaid with 10 µl of mineral oil and the cDNAs were denatured (1.5 min, 98°C) and allowed to anneal for 8–9 h at 68°C. After the first hybridization, the two samples were combined and an additional heat-denatured driver (300 ng) in 1x hybridization buffer was added. The sample was allowed to hybridize for another 16 h at 68°C. The final hybridization reaction was diluted with 200 µl of dilution buffer provided by the manufacturer, heated at 68°C for 7 min, and stored at -20°C.
PCR Amplification.
For each subtraction, two PCR amplifications were performed. The primary PCR reaction in 25 µl contained 1 µl of subtracted cDNA, 1 µl of PCR primer1 (10 µM, 5'-CTAATACGACTCACTATGGGC-3'), and 0.5 µl each of 50x Advantage cDNA polymerase mix (Clontech) and 10 mM dNTP mix. The cycling parameters were 75°C for 7 min, followed by 27 cycles at 94°C for 30 s, 68°C for 30 s, and 72°C for 2 min. The amplified products were diluted 10-fold with deionized water and 2 µl were used in the secondary PCR reactions with NP1 and NP2 primers (provided in the kit). The cycling conditions were the same as in the primary PCR amplification, except the reactions were in a 50-µl volume for 11 cycles only, with a final extension cycle for 7 min at 68°C. The subtraction efficiency was determined by both PCR and-Southern based methods as instructed by the manufacturer.
Cloning and Analysis of the Subtracted cDNAs.
Products from the secondary PCRs were inserted into PCR2.1-TOPO TA cloning kit (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. Prior to ligation, the subtracted cDNA mix was incubated for 1 h at 72°C with dATP and AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Foster City, CA) to ensure that most of the cDNA fragments contained "A" overhangs. Approximately 100 ng of PCR-amplified cDNA were ligated into 50 ng of vector without further purification. Two µl of ligated products (10 ng of vector and 50 ng of cDNAs ligated in 10-µl volume) were transformed into 40 µl of DH10B cells by electroporation (Bio-Rad, Hercules, CA). Routinely, 50- and 200-µl aliquots of the transformed cells (grown in 1 ml of medium) were plated onto 150-mm Luria-Bertani/agar plates containing 100 µg/ml of ampicillin, with 100 µM isopropyl-1-thio-ß-D-galactopyranoside (IPTG) and 50 µg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) to discriminate white from blue colonies. The transformation efficiency was 2–4 x 106 colonies/µg of DNA.
Hybridization and Screening for Differentially Expressed Transcripts.
The differential hybridization was performed initially on 96 randomly picked clones to determine subtraction efficiency. The inserts in the plasmid were amplified using NP-1 and NP-2 primers provided in the kit. The PCR conditions were 94°C for 4 min followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 2 min, followed by a final extension at 72°C for 5 min. The products of the PCR reactions were resolved on a 2% agarose gel run in duplicate. After Southern blotting of the amplified inserts onto Hybond N membranes (Amersham, Piscataway, NJ). The membranes were stained with methylene blue in 0.2x SSC and visualized to ensure complete transfer of all of the products. The blots were then hybridized with RsaI-digested cDNA probes (reverse Northern). Fifty ng of RsaI-digested tester and driver cDNAs were labeled using random primer labeling kit (Stratagene, LA Jolla, CA) with 50 µCi of [33P]dCTP following manufacturer’s instructions. Equal counts (1–2 x 107 cpm/µl) of the cDNA probes from normal and tumor tissues were heat-denatured and used to probe duplicate blots. Hybridization was performed at stringent conditions in 0.5 M Na2PO4 (pH 7.2), 7% SDS at 65°C. The next day, the filters were washed twice in 2x SSC, 0.5% SDS at 68°C, then once in 0.1% SSC, 0.1% SDS at 68°C, and exposed to phosphorimager screens overnight. The signal intensity of each spot in the membranes was compared between tester and driver hybridized duplicates. cDNA fragments displaying differential expression levels of >1.8-fold or higher were selected to estimate the efficiency of the differential hybridization.
Approximately 600–700 unique clones from each of the four libraries were successfully sequenced with M13 forward primer using an ABI Prism dye terminator cycle sequencing in the sequencing core at Millennium Predictive Medicine, Cambridge, MA. Sequences were compared with the National Center for Biotechnology Information sequence database using the BLAST program.
Semiquantitative RT-PCR.
Fifty to 100 ng of reverse-transcribed cDNAs were used in a multiplex reaction with a pair of gene-specific primers and GAPDH forward (5'-ACCACAGTCCATGCCATCAC-3') and reverse primers (5'-TCCACCACCCTGTTGCTTGTA-3'), which yield a 450-bp product. The PCR reaction mixes contained 50 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 400 µM gene-specific primers, 50 µM each of the GAPDH primers, and 0.5 units of Taq polymerase (Promega, Madison, WI), in a 12.5-µl reaction volume. The conditions for amplification were as follows: 94°C for three min, then 29 cycles of 94°C for 30 s, 50–62°C for 30 s depending on the gene- specific primers being tested, and 72°C for 30 s in a Perkin-Elmer-Cetus 9600 Gene-Amp PCR system. The products of the reactions were resolved on a 1.6% agarose gel. Band intensities were quantified using the Gel Doc 1000 photo-documentation system (Bio-Rad, Hercules, CA) and its associated software.
The following gene-specific primers were used: for CTSK, (forward) F-GGA GAT ACT GGA CAAC CCA CTG and (reverse) R-CCA ACT CCC TTC CAA AGT GC; for PAI1, F-AAT CGC AAG GCA CCT CTG AG and R-GAT CTG GTT TAC CAT CTT TT; for cyclin D2, F-AGC TGC TGT GCC ACG AGG T and R-ACT GGC ATC CTC ACA GGT C; for FGF7, F-TAA TGC ACA AAT GGA TAC and R-ATT GCC ATA GGA AGA AAG; for EGR1, F-GAC ACC AGC TCT CCA GCC TGC and R-GGA AGG GCT TCT GGT CTG GGG; for SPARC, F-CCA CTG AGG GTT CCC AGC AC and R-GGA AAC ACG AAG GGG AGG GT; for decorin, F-CCT GGT TGT GAA AAT ACA TGA and R-TGA CAT TAA CAA GAT TTT GCC; for THBS2, F-TGG TCA CCA GGA CAA AGA CAC and R-ATC CTG CCA GCA AGC TGA CA; for ITM2A, F-CGC AGC CCG AAG ATT CAC TAT G and R-CTT ATT ACC AAG GAC ACT CTA TCT; for PEG3, F-CGG AGA ACT GTG AGA AGC TCG TC and R-GGT GGG GCT AGG CTA GAA GG.
Northern Blot Analysis.
Fifteen µg of total RNA was fractionated on 1.2% formaldehyde agarose gels and blotted in 1x SPC buffer [10 mM sodium phosphate (pH 6.8), 1 mM CDTA (Sigma Chemical Co., St. Louis, MO)] onto Hybond N membranes (Amersham, Piscataway, NJ). The control small ribosomal protein S9, (RPS9) and gene-specific probes were labeled using the random primer labeling system (Life Technologies, Inc., Gaithersburg, MD) and purified using spin columns (TE-100) from Clontech. Filters were hybridized at 68°C with radioactive probes in a microhybridization incubator (Model 2000, Robbins Scientific, Sunnyvale, CA) for 1–3 h in Express hybridization solution (Clontech, Palo Alto, CA) and washed according to the supplier’s guidelines. The primers, F-GCA ACA TGC CAG TGG CCC GG and R-ATC CTC CTC CTC GTC GTC TC for RPS9 yield a 586-bp cDNA fragment, and the conditions for amplification are similar to the semiquantitative RT-PCR conditions described above.
LOH Analysis.
Fifteen early- and 18 late-stage tumors of differing histologies (Table 1)
were analyzed. The 15 early-stage tumors included 3 clear-cell, and 6 each of endometrioid and serous tumors. The 18 late-stage tumors included 3 clear- cell, 4 endometrioid, and 12 serous tumors. The markers (Research Genetics, Huntsville, AL) used in this study are listed in Table 1
along with their chromosomal locations. Two new microsatellite markers, one near Methylation Controlled J protein on 13q14.1 (14)
and another marker within BAC CIT-B-470f8 on 19q14.3 (AC006115) are: MCJ-NF-5'-GATTGACCACAGTCTTATCT-3' and MCJ-18–5'-TAAGAGGTCTACTCATTGCTCAC-3', and 19-F-5'-GCACCTGGCCCAACTGTAAC-3' and 19R-5'-CCAGCTGCTGGCTCACCTT-3', respectively. The individual oligonucleotides were synthesized in the Mayo Molecular Technology Core at the Mayo Clinic, Rochester, MN. The PCR reaction mix contained: 50 ng of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 200 µM concentration of each primer, 0.05 µl of [32P]dCTP (10 µCi/µl), and 0.5 units of Taq polymerase (Promega, Madison, WI) in a 10-µl reaction volume. The conditions for amplification were: 94°C for two min, then 30 cycles of 94°C for 30 s, 52–57°C for 30 s, and 72°C for 30 s in a Perkin-Elmer-Cetus 9600 Gene-Amp PCR system in a 96-well plate. PCR products were denatured and run on 6% polyacrylamide sequencing gels containing 8 M urea. The gels were dried and autoradiographed for 16–24 h and scored for LOH. Multiple exposures were used before scoring for LOH. Allelic imbalance indicative of LOH was scored when there was more than 50% loss of intensity of one allele in the tumor sample with respect to the matched allele from normal tissue. The evaluation of the intensity of the signal between the different alleles was determined by visual examination by two independent viewers (V. S. and J. S.).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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and B show the subtraction efficiency of libraries OV 402 (panel 1) and OV 4 (panel 2) by Southern- and PCR-based methods (Fig. 1B)
, respectively. We estimated a 60- to 70-fold enrichment of the differentially expressed genes in all four libraries. This was confirmed with the Southern-based analysis, in which we saw a complete subtraction of GAPDH in the subtracted cDNAs (Fig. 1A)
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. After transfer of the PCR products onto nylon membranes, we performed reverse Northern analyses to identify differentially expressed transcripts. The cDNA probes used for hybridization were restricted with RsaI to minimize background hybridization. Faint signals representing rare transcripts could easily be distinguished with this approach (Fig. 2B)
. After densitometric analysis of each of the corresponding bands hybridized with tester and driver cDNAs, the percentage of these clones that showed the expected differential hybridization was 70–80%.
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2000 randomly picked clones from each of the four libraries. After consolidating for clones that appeared more than once in the libraries, we estimated that there were 600 distinct clones sequenced from each of the four libraries. |
Analysis of SSH Library Genes. |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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. These potentially represent genes that may consistently be down-regulated in both early- and late-stage ovarian tumors. Similar comparison of genes that were isolated from any three of four libraries revealed 80 common genes. There were 130 common genes in the two early-stage tumors. Sixty of these 130 genes were also present in one of the two late-stage tumors. A similar kind of analysis comparing sequences in the two late-stage libraries revealed that there were 210 genes that were common between them. Only 55 of 210 genes were also identified in either one of the two libraries generated from early-stage tumors.
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; Table 3
). For example, HSD3B1 (15)
, which was represented by only two clones in each of the four libraries, showed complete loss of expression in all of the seven tested cell lines (Table 4)
. However, PAI1 (16)
, which appeared several times (100–140) in each of the two late-stage libraries, showed complete loss of expression in only two of seven cell lines. We also tested the differential expression of these genes in 20 early (I/II)-stage and 16 late (III/IV)-stage primary tumors of mixed histological subtypes by semiquantitative RT-PCR comparing them with normal epithelial cell brushings. The 20 early-stage tumors included 5 clear cell, 6 endometrioid, and 9 serous tumors. The late-stage tumors included 1 clear cell, 4 endometrioid, and 11 serous tumors (Table 1)
. Fig. 3B
shows the results of this analysis for PAI1 (16)
, ITM2A (17)
, FGF7 (18)
, PEG3 (19)
, and a novel gene on 8q.
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. HSD3B1 showed complete loss of expression in all of the cell lines and the primary tumors. PRSS11 showed complete loss of expression in four of seven cell lines in three of eight primary tumors. Lower levels of PRSS11 expression was also detected in four of eight primary tumors. Control probe RPS9 was hybridized to the cell line and primary tumor blots to indicate equal loading of RNA.
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Chromosomal Sorting of SSH Genes. |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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lists additional genes mapping to specific chromosomal regions of deletions in ovarian cancer. Of interest are chromosomal bands 5q31–32, 10q11, and 10q25.3–26.2, because several of the down-regulated genes, isolated from these bands, were common to three, or were in all four, of the libraries. The 5q31–32 genes are catenin (28)
, FGF1 (29)
, HDAC3 (30)
, selenoprotein P, plasma1 (SEPP1; Ref. 31
), testican (SPOCK; 32
), transcription elongation factor B (SIII) polypeptide-like (TCEB1L; Ref. 33
), transforming growth factor, ß-induced, Mr 68,000 (TGFß1); Ref. 34
), CDC23, (35)
EGR1 (36)
, and osteonectin (SPARC; Ref. 37
). Down-regulated genes from chromosomal band 10q11.2 and 10q25.3–26.2 that were identified from all four libraries were annexin A8 (ANXA8; Ref. 38
) and PRSS11 (39)
, respectively. Other genes such as nuclear receptor coactivator 4 (ELE1, 10q11.2; Ref. 40
), proteoglycan, secretory granule (PRG1, 10q22.1; Ref. 41
), vinculin (VCL, 10q22.1–23; Ref. 42
), lipase A (LIPA, 10q23.3; Ref. 43
), and protein phosphatase regulatory (inhibitor) subunit 5 (PPP1R5, 10q23–24; Ref. 44
) were identified only from the two late-stage libraries, OV 4 and OV 13. |
LOH Analysis of Chromosomal Regions 1p, 6q, 7q, 8p, 9p, 10q, 13q, 17p, and 19q in Stage I/II and Stage III/IV tumors. |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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. Fig. 5
shows the overall LOH profile obtained. Down-regulated genes mapping to chromosomal regions of loss identified from the libraries are HSD3B1, EGR1, serum glucocorticoid kinase (SGK; Ref. 45
), and forkhead (Drosophila) homologue 1 (rhabdomyosarcoma; FRKH; Ref. 46
) mapping to 1p12–13, 5q31.1–31.2, 6q23.3, and 13q14.1, respectively. The approximate positions of these genes in relation to the markers of their respective chromosomes are also shown (Fig. 5)
. The chromosome 1p11–13 and 6q 23.3 markers showed a higher frequency of loss in late stage-tumors compared with early-stage tumors. Other markers on chromosomes 8, 9, and 10 also showed more losses in high-stage tumors. However, two markers on 5q31 and 13q14.1 and a marker within the BAC CIT-B-470f8 100-kb distal to the PEG3 locus on 19q13.4 had a higher frequency of LOH in early- compared with late-stage tumors. Markers D1S440, D1S534, D6S377, and D19S572 showed no LOH in early-stage tumors. To test whether loss, and/or lower levels, of expression of a gene corresponded to a region of loss, we used a microsatellite repeat present within intron 2 of a novel gene on 8q that was identified in this study to determine the frequency of LOH in these tumors. This marker showed 50% loss both in low- and high-stage tumors (Table 5
; Fig. 5
). Fig. 6
shows the pattern of LOH of this marker in ovarian tumors with the loss of expression of this gene. For example, as shown in Fig. 6
, there was a direct correlation between lower levels of expression of this gene and loss of an allele by LOH (tumor numbers 684 and 208). In tumor with complete loss of expression and deletion of one of the alleles by LOH (tumor number 182), the remaining allele could be inactivated either by hypermethylation or by transcriptional inactivation as a result of other mechanisms. Tumors without LOH (tumors numbers 13 and 234) showed no loss of expression, as evidenced by semiquantitative RT-PCR.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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Analysis of the differentially expressed sequences from early and late tumors allowed us to compare the library sequences to one another. In the four SSH libraries, we identified several genes the function of which in carcinogenesis is known and others with no known roles in cancer. Some of the common genes, such as tissue plasminogen activator inhibitor 1, SPARC, caveolin1, and NOEY2 have been demonstrated by others (16 , 25 , 26) to be differentially regulated in tumors. The potential tumor-associated function of genes such as aldehyde oxidase, HSD3B1 and 2, ITM2A, alcohol dehydrogenase 2 (48) , PRSS11, and PEG3 have not previously been linked with ovarian cancer. In addition, the function of several novel ESTs and genes identified from these libraries remain to be determined. Some of these same genes have been identified as down-regulated genes by other techniques such as cDNA microarray analysis (47 , 49) and DD-PCR (25) .
It is a well-accepted concept that functional inactivation of both the alleles is a prerequisite for a tumor suppressor gene to be defined as such. The loss of expression of a gene could be caused by the deletion of both alleles (homozygous deletions), or deletion of one of the alleles and inactivation of the other allele either by inactivating mutations or by hypermethylation (50 , 51) and/or by altered activity of a transcriptional repressor (52) .
However, chromosomal sorting of 600 genes and ESTs from each of the libraries revealed some interesting trends. Many of the genes identified from the SSH libraries were already mapped to known regions of deletions in ovarian cancer (21, 23, 24
, 53 , 54)
. We wanted to determine whether some of the known and novel genes identified from this screen would also map to regions of loss in ovarian cancer.
As evidenced in the LOH analysis, several of these coincided with regions of deletions observed in ovarian cancer. We identified several genes mapping to 5q31–32 in the SSH libraries. Of interest is EGR1, which has recently been identified as a down-regulated gene in ovarian cancer by cDNA microarray analysis (49)
. Two markers in the region, D5S396 and D5S500, showed a high frequency of LOH in early-stage tumors not previously seen. SPARC, an acidic, cysteine-rich component of the extracellular matrix, is directly regulated by progesterone and dexamethasone and indirectly by cytokines (55)
. Mok et al. transfected the full length SPARC into SKOV3 cells and showed both a reduced growth rate in cells expressing SPARC and a reduced ability of these cells to form tumors in nude mice, which lent support to SPARC as a tumor suppressor. SPARC was identified as a down-regulated gene in all four libraries from 5q31. Serum glucocorticoid kinase on 6q23.3, another region of deletion (23)
, was recently shown by Brunet et al. (56)
to act in concert with Akt in phosphorylating forkhead transcription factor, FKHRL1. This phosphorylation event leads to the activation of the phosphatidylinositol 3-kinase cascade. Bagnoli et al. (52)
have shown a reciprocal negative regulation of 
folate receptor (FR) and caveolin 1 (Cav-1, on 7q31.1) proteins providing evidence for a new mechanism of Cav-1 silencing in ovarian cancer. As indicated above, comparison of some of the down-regulated genes identified from the libraries corresponded with chromosomal regions of loss identified from the LOH studies. A marker 100 kb distal to paternally expressed gene 3 on 19q13.4 that was identified in all four libraries showed a higher frequency of deletion in low-stage compared with high-stage tumors. This is the first report of such a high frequency of deletion (33%) in early-stage tumors in this region. Combining genomic with expression-based analysis, we were able to identify novel regions of loss in ovarian cancer. We have identified several known and novel genes, including ESTs, whose functions in cancer have yet to be discerned. These genes could potentially lead to the identification of candidate tumor suppressor genes involved in ovarian cancer.
However, not all of the down-regulated genes in these libraries could potentially represent tumor suppressor genes. This is essentially true for ribosomal genes. Other investigators also have reported the loss of expression of several ribosomal genes (Cancer Genome Anatomy Project-Digital Differential Display) in cancer and yet the functional consequence of loss of expression of these genes have not been directly linked to tumor suppression. Although some of the genes such as ARHI, caveolin 1, and SPARC that map to regions of deletions in ovarian cancer have known tumor-suppressing functions, for other genes, neither the mechanistic basis for the loss nor the functional consequence of such a loss is known. One of the novel genes identified in this screen showed 50% LOH in both early- and late-stage tumors. Using a microsatellite marker associated with this gene, we were able to correlate the loss and/or lower levels of expression of this gene with the loss of an allele by LOH.
The data from the SSH library analysis revealed that there were many genes that were differentially expressed in both early- and late-stage tumors. Genes identified in only one library could potentially indicate tumor-specific differences. We do not yet know what changes are critical in an early-stage tumor to progress to a more malignant tumor. The genes identified from the SSH libraries are all based on expression differences. This technique cannot detect gross genomic changes, including chromosomal rearrangements or the mutator phenotype (57) , unless they result in concomitant changes in transcript levels of genes. However, one very important epigenetic phenomenon, namely, methylation (58 , 59) , has been associated with changes in the levels of gene expression. Evidence seems to indicate that methylation changes are early events in carcinogenesis leading to the possibility that the majority of the genes inactivated in early-stage tumors could be hypermethylated. Some of the genes isolated in this screen have been reported by others to be inactivated by hypermethylation in ovarian cancer (60 , 61) . For example, two of the down-regulated genes identified from chromosomal band 12p13 from the library sequences such as cyclin D2 (62) and complement component 1 subcomponent (63) do not map to known regions of deletion in ovarian cancer. These genes could be inactivated by methylation. The inactivation of cyclin D2 by methylation in Burkitt’s lymphoma (62) and breast cancer (64) has previously been reported. Transcriptional inactivation can also result because of aberrant regulation of factors.
In conclusion, we have identified several known and several novel genes that are down-regulated both in early- and in late-stage tumors. Several of these genes were later mapped to the regions of loss by LOH analysis. However, we do not rule out the possibility that loss of expression of some of these genes could also be attributable to decreased transcriptional inactivation or through promoter hypermethylation. Thus, combining expression- and genomic-based analyses has provided us with novel regions of alterations in ovarian cancer not previously reported. We are currently pursuing the cloning and characterization of some of the novel genes identified from these libraries to address the functional roles of these genes in ovarian cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Department of Defense Grant DAMD 17-99-1-9504 (to V. S. and D. I. S.) and by the Mayo Foundation. 
2 To whom requests for reprints should be addressed, at Division of Experimental Pathology, Mayo Clinic/Foundation, 200 First Street SW, Rochester, MN 55905. Phone: (507) 266-2775; Fax: (507) 266-5193; E-mail: shridv{at}exrch.mayo.edu
3 The abbreviations used are: DD-PCR, differential display-PCR; RDA, representational difference analysis; SSH, suppression subtraction hybridization; LOH, loss of heterozygosity; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSD3B1, hydroxy-
-5-steroid dehydrogenase 3ß-steroid -isomerase 1; EGR1, early growth response 1; EST, expressed sequence tag; NP, nested primer; OSE, ovarian surface epithelial cells.
Received 6/26/01. Accepted 11/ 1/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSAnalysis of SSH Library...Chromosomal Sorting of SSH...LOH Analysis of Chromosomal...DISCUSSIONREFERENCES |
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5-4-isomerase close to D1S514 and the centromeric D1Z5 locus. Cytogenet. Cell Genet., 69: 59-62, 1995.[Medline]
-folate receptor overexpression. Oncogene, 19: 4754-4763, 2000.[Medline]
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 A. D. Santin, S. Cane, S. Bellone, M. Palmieri, E. R. Siegel, M. Thomas, J. J. Roman, A. Burnett, M. J. Cannon, and S. Pecorelli Treatment of Chemotherapy-Resistant Human Ovarian Cancer Xenografts in C.B-17/SCID Mice by Intraperitoneal Administration of Clostridium perfringens Enterotoxin Cancer Res., May 15, 2005; 65(10): 4334 - 4342. [Abstract] [Full Text] [PDF]
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M. J. Goodheart, J. M. Ritchie, S. L. Rose, J. P. Fruehauf, B. R. De Young, and R. E. Buller The Relationship of Molecular Markers of p53 Function and Angiogenesis to Prognosis of Stage I Epithelial Ovarian Cancer Clin. Cancer Res., May 15, 2005; 11(10): 3733 - 3742. [Abstract] [Full Text] [PDF]
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 S. Grau, A. Baldi, R. Bussani, X. Tian, R. Stefanescu, M. Przybylski, P. Richards, S. A. Jones, V. Shridhar, T. Clausen, et al. Implications of the serine protease HtrA1 in amyloid precursor protein processing PNAS, April 26, 2005; 102(17): 6021 - 6026. [Abstract] [Full Text] [PDF]
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 A. De Luca, M. De Falco, L. De Luca, R. Penta, V. Shridhar, F. Baldi, M. Campioni, M. G. Paggi, and A. Baldi Pattern of Expression of HtrA1 During Mouse Development J. Histochem. Cytochem., December 1, 2004; 52(12): 1609 - 1617. [Abstract] [Full Text] [PDF]
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A. De Luca, M. De Falco, V. Fedele, L. Cobellis, A. Mastrogiacomo, V. Laforgia, I. L. Tuduce, M. Campioni, D. Giraldi, M. G. Paggi, et al. The Serine Protease HtrA1 Is Upregulated in the Human Placenta During Pregnancy J. Histochem. Cytochem., July 1, 2004; 52(7): 885 - 892. [Abstract] [Full Text] [PDF]
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 C. Oka, R. Tsujimoto, M. Kajikawa, K. Koshiba-Takeuchi, J. Ina, M. Yano, A. Tsuchiya, Y. Ueta, A. Soma, H. Kanda, et al. HtrA1 serine protease inhibits signaling mediated by Tgf{beta} family proteins Development, March 1, 2004; 131(5): 1041 - 1053. [Abstract] [Full Text] [PDF]
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 J. M. Garcia Pedrero, M. P. Fernandez, R. O. Morgan, A. Herrero Zapatero, M. V. Gonzalez, C. Suarez Nieto, and J. P. Rodrigo Annexin A1 Down-Regulation in Head and Neck Cancer Is Associated with Epithelial Differentiation Status Am. J. Pathol., January 1, 2004; 164(1): 73 - 79. [Abstract] [Full Text] [PDF]
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A. De Luca, M. De Falco, A. Severino, M. Campioni, D. Santini, F. Baldi, M. G. Paggi, and A. Baldi Distribution of the Serine Protease HtrA1 in Normal Human Tissues J. Histochem. Cytochem., October 1, 2003; 51(10): 1279 - 1284. [Abstract] [Full Text] [PDF]
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J. Lai, J. Chien, J. Staub, R. Avula, E. L. Greene, T. A. Matthews, D. I. Smith, S. H. Kaufmann, L. R. Roberts**, and V. Shridhar** Loss of HSulf-1 Up-regulates Heparin-binding Growth Factor Signaling in Cancer J. Biol. Chem., June 13, 2003; 278(25): 23107 - 23117. [Abstract] [Full Text] [PDF]
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 P. F. Macgregor and J. A. Squire Application of Microarrays to the Analysis of Gene Expression in Cancer Clin. Chem., August 1, 2002; 48(8): 1170 - 1177. [Abstract] [Full Text] [PDF]
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, early stage; 
, late stage. % LOH (on Y axis), frequency of LOH with specific markers. Arrows, approximate positions of these genes in relation to the markers on their respective chromosomes.