ABSTRACT. We have analyzed the stability of microsat- ellites in cell lines derived from human ovarian cancers and found that 5 out of 10 of the ovarian tumor ...
Proc. Natd. Acad. Sci. USA Vol. 91, pp. 9495-9499, September 1994 Genetics
Genetic instability in human ovarian cancer cell lines KIM ORTH*, JACLYN HUNGt, ADI GAZDARt, ANNE BOWCOCK*, J. MICHAEL MATHISt, JOSEPH SAMBROOKt§
AND
*Howard Hughes Medical Institute,
tSimmons Cancer Center, and tMcDermott Center, University of Texas Southwestern Medical School, Dallas, TX 75235
Communicated by Michael S. Brown, June 21, 1994
ABSTRACT We have analyzed the stability of microsatellites in cell lines derived from human ovarian cancers and found that 5 out of 10 of the ovarian tumor cell lines are genetically unstable at the majority of the loci analyzed. In clones and subclones derived serially from one of these cell lines (2774; serous cystadenocarcinoma), a very high proportion of microsatellites distributed in many different regions of the genome change their size in a mercurial fashion. We conclude that genomic instability in ovarian tumors is a dynamic and ongoing process whose high frequency may have been previously underestimated by PCR-based allelotyping of bulk tumor tissue. We have identified the source of the genetic instability in one ovarian tumor as a point mutation (R524P) in the human mismatch-repair gene MSH2 (Salmonella MutS homologue), which has recently been shown to be involved in hereditary nonpolyposis colorectal cancer. Patient 2774 was a 38-year-old heterozygote, and her normal tissue carried both mutant and wild-type alleles of the human MSH2 gene. However the wild-type allele was lost at some point early during tumorigenesis so that DNA isolated either from the patient's ovarian tumor or from the 2774 cell line carries only the mutant allele of the human MSH2 gene. The genetic instability observed in the tumor and cell line DNA, together with the germ-line mutation in a mismatch-repair gene, suggest that the MSH2 gene is involved in the onset and/or progression in a subset of ovarian cancer.
(13, 14). Most colon tumors in patients with Lynch syndromes manifest changes in the size of randomly chosen (CA). and other simple repeated sequences (15), and cell lines established from HNPCC patients are deficient in repair of slipped-strand mismatches (16). In addition to HNPCC, the hMSH2 gene may also be involved in the onset and/or progression of a substantial fraction of human sporadic cancers. Expansion and contraction of microsatellite sequences have been reported in a discrete subset of sporadic colon cancers (2, 4), in small cell lung carcinomas (17), and in tumors of the pancreas, bladder, stomach, breast, and endometrium (18-23). However, the incidence reported for ovarian tumors is very low (=5%) (19, 24, 25). In this study, we show that genetic instability is common in cell lines derived from ovarian cancers, that this instability is ongoing and dynamic, and, in one case, is caused by a mutant allele of the hMSH2 gene.
with other types of cancers, predominantly of the gastrointestinal, upper urologic, and female reproductive tracts (Lynch syndrome II) (8-11). The human MSH2 (hMSH2) gene maps to chromosome 2p16 (12-14), and mutations in the hMSH2 gene have been detected in small HNPCC pedigrees
MATERIALS AND METHODS Cell Lines. We obtained human ovarian tumor cell lines SK-OV-3, OVCAR3, PA-1, Caov-3, and Caov4 from American Type Culture Collection; 2774 and 2008 from P. Disaia, University of California, Irvine; UCI 101 and UCI 107 from A. Manetta, University of California, Irvine; and 222 from B. Bonavida, University of California, Los Angeles. Primers. Primer sets used for microsatellite analysis included DSS299 (26), Thra-1 (27), interferon A (28), C13-373 (29), D2S123, D3S1283, and D17S791 (30), D17S261 (31), D9S126 (32), human thyroid peroxidase (hTPO) (33), D15S169 and DJSS171 (34), D18S34 (35), and WT-1 (D. Haber, Massachusetts Institute of Technology). Analysis of Polymorphic Mirosatellites. DNA was purified from confluent cultures of cells as described (36). Microsatellites were analyzed as described (24). Clonal Analysis. Single cells of the 2774 cell line were isolated by limiting dilution as follows. Two hundred microliters of a suspension containing approximately two viable 2774 cells per ml was dispensed into each well of a 96-well microtiter dish and incubated until the colonies were large enough to seed T-25 tissue culture flasks (=z7 weeks). Confluent monolayers from the T-25 flasks were expanded into three 90-mm Petri dishes. At confluency (=w5 x 106 cells per plate), DNA was isolated (36) from two 90-mm plates (zero time point). The third 100-mm plate was split into three 90-mm plates. Cells were passed for 10 weeks (80-90 cell doublings), with DNA isolated at the 3rd, 6th, and 10th weeks. Cells from four of the original clonal lines at 10 weeks were subcloned at 10 weeks and expanded into 90-mm Petri dishes by the method described above. DNA was then isolated from these expanded subclonal lines. Alelotyping of Mcrodissected DNA. Minute samples of normal and tumor cells were precisely dissected from ar-
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Abbreviations: MSH2, Salmonella MutS homologue; hMSH2, human MSH2; HNPCC, hereditary nonpolyposis colorectal cancer. §To whom reprint requests should be addressed.
Genes of the MSH2 (Salmonella MutS homologue) superfamily encode proteins that bind to mismatched nucleotides in DNA and coordinate the activities of a group of other proteins involved in "long-patch" excision repair (1). Mutations in MSH2 genes cause a strong mutator phenotype in their host organisms-from bacteria to man. In eukaryotes, an outward sign of this genetic instability is the increased rate of expansion and contraction of microsatellite sequences that are widely distributed over the entire genome (2-4). This fluctuation in size of simple repeated sequences occurs because errors resulting from strand slippage during DNA replication remain uncorrected (for review, see ref. 5). In humans, mutations of the MSH2 gene also are responsible for hereditary predisposition to nonpolyposis colorectal cancer (HNPCC). This condition, which is inherited in an autosomally dominant fashion, is defined clinically by the occurrence of early onset cancer of the colon and other organs in first-degree relatives spanning at least two generations (6, 7). Families affected by HNPCC can be divided into two groups-those with site-specific hereditary colon cancer (Lynch syndrome I) and those with colon cancer associated
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chived 22-year-old hematoxylin/eosin-stained slides as described (37), with slight modifications. Briefly, microdissection was performed under direct observation with an inverted microscope using a microcapillary tube that had been pulled to a fine tip by a micropipette puller. The tip was introduced into the field of view at an angle of -30°, and the movements in the x, y, and z axes were controlled by ajoystick-operated hydraulic manipulator (Nikon-Narishige, New York). The scraped cells (about 30 in each manipulation) were allowed to adhere to the microcapillary tip, collected in a microcentrifuge tube by breaking off the glass tip, followed by extraction of the DNA with proteinase K. The DNAs were stored at -200C and allelotyped using the PCR-based assays as described (24) with the following modifications. Reactions contained template DNA derived from '100 microdissected cells. After initial denaturation at 940C for 2 min, 25 cycles of PCR consisting of 10 sec at 94TC, 10 sec at the temperature appropriate for primer annealing, and 30 sec at 72TC, then 15 cycles consisting of 10 sec at 90TC, 10 sec at the annealing temperature, 30 sec at 720C, with a final extension of 2 min at 720C. Sequence Analysis. Direct cycle DNA sequencing was performed on PCR products amplified from cDNA copied from the hMSH2 transcripts expressed in the 2774 cells as described (14). Primers for PCR analysis and sequencing of the mutated region in genomic DNA included 51OF (5'GATTAAACTGGATTCCAGTGCAC-3'), 540R (5'CTAAAGTTTTTATTGTTACGAAGG-3'), and 548R (5'-
GCTGTTGGTAAATTTAACACC-3').
RESULTS Microsatellites in Ovarian Cancer Cell Lines Are Unstable. Cell lines established from 10 ovarian tumors were analyzed for genetic instability at 13 different polymorphic loci, each of which contained a (CA), microsatellite. In 5 of the 10 ovarian cell lines examined, most of the PCR-amplified allelic markers no longer appeared as typical single bands with faster running shadows (38). Instead, they have radiated into tightly bunched ladders of bands (Fig. 1) that are similar to those originally described by Aaltonen et al. (15) in tumors
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FIG. 1. Genetic instability atD17S791 in ovarian tumor cell lines. DNA from 10 ovarian tumor cell lines was tested for genetic instability using the PCR-based assay. The human histiocytic lymphoma cell line U-937, used as a control, is heterozygous at this locus and exhibits two distinct alleles (lane 1). The PCR-based assay produces a shadow band under each distinct allele. The ovarian cell lines 222 (lane 5), 2008 (lane 7), OVCAR3 (lane 8), Caov-3 (lane 10), and Caov-4 (lane 11) appear genetically stable at D17S791. The other five ovarian tumor cell lines appear genetically unstable at this locus. The upper allele of 2774 is genetically unstable, whereas the lower allele exhibits only a distinct band (lane 2); SK-OV-3 (lane 3), UCI 101 (lane 6), and PA-1 (lane 9) are heterozygous at this locus, and both alleles appear genetically unstable; UCI 107 (lane 4) appears genetically unstable at this locus, displaying one long ladder.
arising in affected members of HNPCC families. These laddering patterns were not artifacts of the PCR, nor did they result from contamination since (i) they were consistently observed in independent PCRs and separate gel loadings, (ii) clonal and subclonal derivatives of the original cell lines displayed different patterns of laddering (see below), and (iii) the pattern of laddering for any one marker or allele varied from cell line to cell line. It is therefore very likely that laddering patterns were the consequence of addition or deletion of DNA within the (CA), repeat embedded within each marker. These multiallelic loci therefore reflect the accumulation of replication errors and strand misalignment during development of the original tumor and/or passage of the cell line. The percentage of altered loci in cell lines displaying laddered alleles varied from 36% to 86%, and instability was not restricted to any particular marker or region of the genome (Fig. 2). Nevertheless some markers were more stable than others. For example, neither allele of the marker 9p22-interferon A displayed evidence of laddering in any of the cell lines. In addition, at several loci, one allele appeared to be more stable than the other. For example, the larger allele of marker DI 7S791 in cell line 2774 displayed laddering while the smaller allele appeared to be constant in size (Fig. 1, lane 2). The alleles of the same marker in cell line 107 displayed a single broad ladder (Fig. 1, lane 3). Cell lines SK-OV-3, 101, and PA-1 were unstable at both alleles and displayed two distinct ladders (Fig. 1, lanes 2, 5, and 8, respectively). Genetic Instability in Ovarian Tumor 2774. The 2774 ovarian tumor cell line was derived from ascitic fluid of a patient with serous cystadenocarcinoma (39). Genetic instability in this cell line was analyzed by comparing the profiles of polymorphic (CA)n microsatellites in normal, tumor, and cell line 2774 DNA (Fig. 3). Normal epithelial and tumor cells were precisely microdissected from 22-year-old archival hematoxylin/eosin-stained slides, and DNA was extracted. In Fig. 3A, D17S261 appeared to be homozygous and stable in both the normal (lane 1) and tumor (lane 2) DNA, but, by the presence of a ladder, was unstable in DNA extracted from the 2774 cell line (lane 3). In Fig. 3B, D3S1283 appeared to be homozygous and stable in microdissected normal DNA (lane 1), but was unstable in both tumor DNA (lane 2) and cell line DNA (lane 3). In Fig. 3C, D2S123 appeared to be heterozygous and stable in the normal DNA (lane 1) and unstable in the tumor DNA (lane 2) as demonstrated by a downward shift in the size of the PCR products. The simple profile of the allelotype in the tumor DNA also suggested a loss of heterozygosity at this locus. In DNA from the 2774 cell line (lane 3), this locus displayed a high degree of heterogeneity. In Fig. 3D, D17S791 appeared to be homozygous and stable in the z marker CO) OD CM 4 C# cc0 CD 4 1)%1 cJ1 cell C;' 7 a V' u lines r U-937 J]- - -2774
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FIG. 2. Analysis of ovarian tumor cell lines with polymorphic microsatellites. Cell lines were assayed for genetic instability (+) or stability (-) with markers throughout the genome.
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Proc. Natl. Acad. Sci. USA 91 (1994) D1 7S791 N T CL
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normal DNA but polymorphic and unstable in the tumor and cell line DNA. Although both alleles appeared to have altered in size, one of the alleles appeared to have shifted to a smaller size and then stabilized while the other allele has expanded more
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FIG. 3. Analysis of normal, tumor, and cell line DNA atDi7S261 (A), D3S1283 (B), D2S123 (C), and D17S791 (D). Normal epithelial and tumor cells were precisely microdissected from 22-year-old archival hematoxylin/eosin-stained slides, and DNA was extracted. PCR-amplified fragments were prepared from normal (N), tumor (T), and cell line (CL) DNA, separated by polyacrylamide gel electrophoresis, followed by autoradiography.
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that expansion and contraction of microsatellite repeats was a property of the original ovarian tumor in patient 2774 and was not a consequence of the establishment of a cell line. The multiple alleles detected at many loci in tumor and cell line DNA must reflect heterogeneity in the cell populations rather than the presence of supernumerary chromosomes. Cell line 2774 was triploid (ref. 39 and data not shown) while the unstable ovarian tumor cell lines 107 and 101 were both diploid (n = 46; ref. 40 and data not shown). Genetic Instability Is a Dynamic Process. To ascertain whether genetic instability was an ongoing and dynamic process, we analyzed the allelotypes of polymorphic microsatellites in a series of clonal lines and subclonal lines derived from the parental cell line 2774. The uncloned parental cell line contained a complex mixture of alleles. However, the clonal cell lines displayed simpler patterns, consisting, in most cases, of two or three alleles at each locus analyzed (DSS299, Fig. 4A; D2S123, D1SS169, D15S171, and D17S791, data not shown). Most of the alleles in the clonal lines were easily recognized in the original uncloned population. However, in some cases, the alleles carried in the clones were barely detectable in the original cell line (Fig. 4A, lanes 6, 7, and 9). These results confirmed that the uncloned cell line consisted of a mixture of different alleles at individual loci and showed that individual sets of alleles could be isolated from the mixture by cloning the cell line. To investigate whether the genetic instability of (CA),, microsatellites was a dynamic process, 4 of the 2774 clonal cell lines were passaged for 10 weeks. Dynamic instability of microsatellites was assayed by studying changes in allelotypes after 3, 6, and 10 weeks of serial culture. Changes in allele size were detected only in one clonal line (Cl) at seven of the nine microsatellites tested (markers changed: D2S123, C13-1136, DS299, C13S373, D15S169, D15S171, and D17S791; markers unchanged: D3S1283 and D3S1293) (Fig. 4B, lanes 3-6). To eliminate the possibility that the observed changes in allele size was due to an artifact resulting either from cross-contamination of the clonal cultures or from starting with a mixed cell population rather than a clonal cell, a series of subclonal lines were generated from the C1 clonal line after 10 weeks of culture. The results shown in Fig. 4C
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FIG. 4. (A) Analysis at D5S299 in U-937 cell line (lane 1), 2774 cell line (lane 2) and 2774 clonal cell lines (lanes 3-12). (B) Analysis at D2S123 in 2774 clonal cell lines Cl (lanes 3-6) and C2 (lanes 7-10) over 10 weeks. Controls U-937 (lane 1) and 2774 (lane 2) cell lines are shown for this polymorphic microsatellite. (C) Analysis at DI7S791 in C1 subclonal cell lines (lanes 3-12) made from clonal C1 (lane 2) at the 10-week time point. The profile for the 2774 cell lines is shown in lane 1. (D) Analysis at D1SS171 in C2 subclonal cell lines (lanes 3-12) made from clonal C2 (lane 2) at the 10-week time point. The profile for the 2774 cell lines is shown in lane 1.
confirm that the C1 clonal line was genetically unstable, as demonstrated by the appearance of altered allele sizes in the subclonal lines (lanes 4-13). This conclusion was confirmed with four other microsatellite markers (D2S123, D5S299, D15S169, and D5SS71; data not shown). As demonstrated previously, the degree of instability varied from marker to marker and from allele to allele. At the locus D17S791 (Fig. 4C), the larger of the two alleles was much more unstable than the smaller. In fact, of over 50 different clonal and subclonal lines analyzed, only subclonal line Cl-10 (Fig. 4C, lane 12) showed a change at the smaller allele. During 10 weeks of serial passage, three other clonal lines (C2, C3, and C4) showed no detectable changes in allele size at the nine different loci (example for D2S123 is shown for C2; Fig. 4B). Although these clonal lines appeared to be genetically stable, isolation of subclonal lines proved otherwise. The subclonal lines (Fig. 4D, lanes 4-13) contained alleles different in size from those present in the clonal line. As was observed with the previous clonal and subclonal selection, alleles that were rare in the clonal lines could be rescued by subcloning. These results were confirmed with four other markers (D2S123, DSS299, D1SS169, and D17S791; data not shown). These studies demonstrated (i) that genetic instability was a dynamic and ongoing process and (ii) that the failure to detect laddering or other changes in allelotype in a mixed-
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cell population did not necessarily mean that the population was genetically stable. Mutation at the hMSH-2 Locus Identified in 2774 Normal, Tumor, and Cell Line DNA. Because mutations in the hMSH2 gene can cause genetic instability in (CA),, microsatellites (13, 14, 16), we investigated whether the hMSH2 gene might be involved in the genetic instability observed in ovarian tumor cell lines. Using a polymorphic tetranucleotide repeat marker (human thyroid peroxidase; 2p23; ref. 33) located near the hMSH2 gene (2pl6) we showed a loss of heterozygosity in both the tumor and cell line 2774 DNA (Fig. 5), implying that the loss of the 2p region was involved in tumorigenesis. This result was supported by the observation of a loss of heterozygosity detected at markerD2S123, also near the hMSH2 locus (Fig. 3C). We used reverse transcriptase-PCR to copy and amplify segments of hMSH2 mRNA from the 2774 cell line (14). The amplified fragments of hMSH2 cDNA were sequenced by the dideoxy-mediated chain-termination method, and the sequence of the hMSH2 cDNA in 2774 cells was assembled. The sequence was identical to the hMSH2 cDNA sequence of Leach and colleagues (14) with the exception of one codon (524) (Fig. 6B). Codon 524 was mutated from CGT to CCT, resulting in the substitution of a proline residue for a highly conserved arginine, which is also found in the yeast homologue of hMSH2 (14, 41). Intron/exon borders flanking this mutation were identified in the cell line 2774 genomic DNA using PCR and direct sequencing of the amplified fragments. The exon containing the mutation was amplified from normal, tumor, and cell line 2774 DNA and sequenced directly with a nested primer (SlOR). The hMSH2 sequence of the exon amplified from microdissected tumor DNA was identical to that of the 2774 cell line genomic DNA sequence (Fig. 6B). However, the genomic DNA extracted from microdissected normal tissue of patient 2774 was heterozygous at position two of codon 524, with one wild-type allele (CGT) and one mutant allele (CCT) (Fig. 6A). We therefore concluded that patient 2774 was heterozygous at the HNPCC locus and carried one wild-type and one mutant copy of the hMSH2 gene. At some stage in the onset or progression of the ovarian tumor, the wild-type allele was lost, which accounts for the ongoing genetic instability detected in both the original tumor and the cell lines derived from it.
DISCUSSION Our data show that genomic instability, as assayed by changes in the size of microsatellite repeats, occurs in 50o of cultured lines of ovarian tumor cells and is an ongoing and dynamic event. In one case (tumor 2774), we have been able to demonstrate that genetic instability was present in the original tumor and hence is not a consequence of the estabN
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FIG. 5. Analysis of the tetranucleotide marker human thyroid peroxidase (2p23) in normal (N), tumor (T), and cell line (CL) DNA. The normal DNA is heterozygous at this loci, while the tumor and cell line DNA display a loss of heterozygosity.
Proc. Nat. Acad. Sci. USA 91 (1994)
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