Chromosome 3p14 homozygous deletions and sequence analysis of FRA3B
Chromosome 3p14 homozygous deletions and sequence analysis of FRA3BFerenc Boldog1,[Dagger], Robert M. Gemmill1,[Dagger], James West1, Misi Robinson1,Linda Robinson1, Efang Li1, Joelle Roche1,+, Sean Todd1, Barbara Waggoner1, Ron Lundstrom2, Jan Jacobson1, Michael R. Mullokandov3, Harold Klinger3 and Harry A. Drabkin1,*
1Division of Medical Oncology, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262, USA, 2Eleanor Roosevelt Institute, 1899 Gaylord, Denver, CO 80206, USA and 3Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461, USA
Received September 16, 1996;Revised and Accepted November 27, 1996
Loss of heterozygosity (LOH) involving 3p occurs in many carcinomas but is complicated by the identification of four distinct homozygous deletion regions. One putative target, 3p14.2, contains the common fragile site, FRA3B, a hereditary renal carcinoma-associated 3;8 translocation and the candidate tumor suppressor gene, FHIT. Using a ~300 kb cosmid/[lambda] contig, we identified homozygous deletions in cervix, breast, lung and colorectal carcinoma cell lines. The smallest deletion (CC19) was shown not to involve FHIT coding exons and no DNA sequence alterations were present in the transcript. We also detected discontinuous deletions as well as deletions in non-tumor DNAs, suggesting that FHIT is not a selective target. Further, we demonstrate that some reported FHIT aberrations represent normal splicing variation. DNA sequence analysis of 110 kb demonstrated that the region is high in A-T content, LINEs and MER repeats, whereas Alu elements are reduced. We note an intriguing similarity in repeat sequence composition between FRA3B and a 152 kb segment from the Fragile-X region. We also identified similarity between a FRA3B segment and a small polydispersed circular DNA. In contrast to the selective loss of a tumor suppressor gene, we propose an alternative hypothesis, that some putative targets including FRA3B may undergo loss as a consequence of genomic instability. This instability is not due to DNA mismatch repair deficiency, but may correlate in part with p53 inactivation.
LOH involving 3p occurs frequently in carcinomas of the lung, kidney, cervix, breast and other epithelial neoplasms (1 -7 ). However, 3p loss is complex, involving at least four distinct homozygously deleted regions (8 -12 ). One of the most frequently lost regions is 3p14, especially in cervical carcinomas (13 ). This region is also of interest since it contains the site of a hereditary renal carcinoma-associated translocation, t(3;8)(p14.2;q24.1) (14 ), and is the location of the most inducible common fragile site in the genome, FRA3B (15 ). We previously reported cloning of the 3;8 translocation breakpoint (16 ) and demonstrated by fluorescence in situ hybridization that FRA3B was further telomeric (17 ). These studies also suggested that breaks in FRA3B occur over a region instead of at a single site. While searching for genes in this region, we identified a homozygous deletion in the cervical carcinoma cell line, HeLa, involving marker D3S1300.
We developed a ~300 kb cosmid/[lambda] contig within FRA3B containing D3S1300. Probes from the region detected frequent homozygous deletions in cervical, lung, colorectal and breast carcinoma cell lines. Cervical carcinomas, which are associated with papilloma virus infection and p53 inactivation (18 ), were most frequently deleted and the smallest deletion occurred in cell line CC19. During our investigations, Ohta et al. reported identification of a candidate tumor suppressor gene, FHIT, which spanned the t(3;8) breakpoint and was deleted in various carcinoma cell lines (19 ). However, our analysis indicates that FHIT is not the target of these deletions. We also observed that 3p14 deletions tend not to occur in tumors with deficiencies in DNA mismatch repair.
aDefective refers to a reported deficiency in mismatch repair or microsatellite instability.bOverall, deletions (5) or rearrangements (1) were identified in 6/12 colorectal carcinoma DNAs. In addition to those listed, deletions occurred in COLO-205 and COLO-320. No deletions were identified in SKCO-1, CaCo-2, SW-1417 and T-84. cRepresents all cervical carcinoma samples examined.dPersonal communication from Dr Garret M. Hampton.NT = not tested.
Following identification of the HeLa deletion, additional cervical carcinoma lines were examined using probes spanning a ~300 kb cosmid/phage contig from the FRA3B region (Fig. 1 A and B). Figure 2 A shows an example using cosmid c136C3. Deletions were evident in DNAs from MS751, SIHA and Caski (lanes 2, 5 and 7) whereas CC19 (lane 6) shows missing and altered bands (arrow). Altogether, homozygous deletions were detected in seven out of eight lines (87.5%). Similar hybridizations were performed using DNAs from 12 colon tumor lines, and deletions or rearrangements were seen in 50% (Fig. 2 B). Other homozygous deletions (Fig. 1 C) were detected in three lung (NCI-H1648, NCI-H460 and NCI-H892) and one breast carcinoma (MDA-231) cell lines. The smallest (~40 kb) was in the cervical carcinoma line CC19. This line was characterized further by DNA sequence analysis and FHIT gene expression (see below). Two (HeLa and MDA-231) contained discontinuous deletions, which was surprising since a single deletion should have been sufficient to inactivate a tumor suppressor gene. No deletions were detected in five renal carcinoma lines (KRC/Y, CAKI-1, CAKI-2, ACHN and KV6).
Analysis with Pythia (32 ) showed that 20.2% of the sequenced region is comprised of known repeats, a level comparable with other regions chosen for comparison (Table 2 ). However, in this 110.4 kb, LINE and MER elements make up the bulk of repeats. Intriguingly, the repeat composition is very similar to a 152 kb sequence from the Fragile-X region (33 ) and differs from arbitrarily selected segments in 3p21.3 and 4p16.3 (Table 2 ). In particular, [lambda]33 (from position 96 to 110.4 kb) within the CC19 deletion is nearly identical in LINE element composition to the Fragile-X region. Similarly, both Fragile-X and FRA3B have a low level of predicted coding regions. Speculatively, these similarities may influence the observed tendency of both regions to undergo breakage. However, there are obvious differences between the two sites, notably the presence of a triplet repeat and methylated CpG island in Fragile-X (34 ) and the common versus rare nature of FRA3B.
Position 38 kb contains a cluster of terminal deletion breakpoints induced by aphidicolin treatment (GenBank U46001) further linking our sequence to FRA3B. Flanking these aphidicolin breakpoints is a long polypurine tract and two extended variable dinucleotide repeats. The telomeric breakpoint of the CC19 deletion lies within a 1.0 kb region at position 76 kb between L1 and MER/LTR elements. Within the CC19 deletion region, near position 86 kb, is an HPV16 integration site from a cervical carcinoma (35 ). It is of note that this integration was associated with an interstitial deletion (35 ,36 ) which we now know would not affect FHIT coding sequences. Interestingly, a very significant similarity (1e-77) to a small polydispersed circular (spc) DNA was observed at 106 kb (GenBank X96885). Figure 6 shows a FASTA alignment over 585 bp. The region is 72.1% identical and contains interspersed blocks having up to 89% identity (e.g., spcDNA bp 121-207). This spcDNA, which appears non-repetitive, was isolated from a tuberous sclerosis-associated angiofibroma (I. Hinkel-Schreiner, Ph.D. Thesis). Characteristics of spcDNAs include derivation from chromosomal sequences (37 ), association with clustered repeats [such as [beta]-satellites and other clustered elements although a single family member may be predominantly involved (38 )] and elevation in conditions associated with genomic instability such as Fanconi's anemia (39 ). spcDNAs are also increased by DNA-damaging agents (37 ) and inhibitors of DNA and protein synthesis including the fragile site inducer aphidicolin (40 ). Given the limited number of spcDNAs that have been sequenced, this similarity may be biologically important.
Using a ~300 kb cosmid/[lambda] contig, located ~150 kb telomeric to the 3;8 translocation breakpoint, we have identified homozygous deletions in various carcinoma cell lines that overlap the most inducible common fragile site in the genome, FRA3B. From various aphidicolin-induced breakpoints and plasmid or viral integration sites (Fig. 1 ), FRA3B represents a region rather than a single site. Studies by Wilke et al. (36 ) and Smith et al. (41 ) indicate that some clustering of breakpoints may occur. However, their rearrangements were induced by aphidicolin in a single chromosome 3-containing hybrid and, unlike the interstitial deletions we observed in tumor and non-tumor samples, appear to represent terminal breaks. Where we have defined the boundaries for the carcinoma-associated deletions accurately, one or both are contained within the FRA3B region.
Our initial hypothesis was that the smallest deletion (CC19) would contain elements of a tumor suppressor gene. During our studies, Ohta et al. (19 ) identified the FHIT gene with reported abnormalities in RT-PCR products. However, FHIT has similarity to a yeast di-adenosine hydrolase which would represent an unexpected function for a tumor suppressor gene. Our results indicate that FHIT is not the target of these deletions. First, the CC19 deletion does not involve FHIT coding sequences (Fig. 1 B and C) and, based on RT-PCR and cDNA sequence analysis, the coding portion of the FHIT transcript is normal. Second, we have observed deletions in genomic DNAs from non-tumor sources (Fig. 3 A). The somatic cell hybrid 2A3CT was formed by spontaneous terminal deletion at 3p21.3 from a non-tumor-derived chromosome 3 hybrid, UCTP2A3 (11 ). However, 2A3CT also acquired a 3p14 interstitial deletion overlapping an aphidicolin-induced breakpoint, a pSV2neo integration into FRA3B (20 ) and the telomeric borders of several carcinoma-associated deletions. We also identified an overlapping deletion in an unselected subclone from YAC 74B2. While neither the hybrid nor YAC 3p14 DNA segments are in a `normal' background, they clearly are unselected from a tumorigenic standpoint. We note that `hotspots' of recombination in human DNA can be maintained in a yeast background (42 ), thus it is not unreasonable that unstable regions may behave similarly or more so. Third, discontinuous deletions appear common in this region (i.e. HeLa, MDA231), both from our analysis and from that reported by Ohta et al. (19 ). Multiple deletions might be expected if there were no common target gene and if the region was unstable. Fourth, we have observed that FHIT undergoes alternative splicing in normal tissues (Fig. 4 B) which explains some previously reported abnormal PCR products (19 ,30 ,43 ). Thiagalingam et al. (44 ) recently reported lack of FHIT involvement in colorectal carcinomas and suggested that PCR artifacts might be responsible for some previously observed alterations (19 ,43 ). It also seems likely that many deletions would have been missed in their study since only a few markers were tested. One of the possible features that suggested FHIT could be a tumor suppressor gene was that it crossed the hereditary renal carcinoma-associated 3;8 breakpoint (19 ). However, we found no alterations in RT-PCR products from five renal carcinoma cell lines. Moreover, Bugert et al. (manuscript submitted) have observed normal FHIT transcripts and no point mutations in a large series of renal cancers. Thus, FHIT does not appear to be involved in renal carcinoma. With respect to other possible target genes, from our sequencing studies we identified a 100% identity to two expressed sequence tags from a liver/spleen library. However, we determined that both clones were identical, were not expressed using a commercial Northern blot (Clontech), were co-linear with genomic DNA containing a poly(A) tract corresponding to their 3' end, encoded no significant open reading frame and overlapped a partial LINE element.
While other tumor suppressor genes may exist within FRA3B, an alternative possibility is that the deletions are due to primary genomic instability affecting a particularly susceptible region. This hypothesis is consistent with several of the observations reported here including their discontinuity and occurrence in non-tumor cell lines. By using numerous probes, we were able to identify a high frequency of homozygous deletions, especially in cervical carcinomas where p53 inactivation is very common (18 ). In this regard, it is interesting that we observed an inverse correlation between 3p14 deletions and microsatellite instability (RER+). Importantly, p53 mutations, which have been shown to destabilize the genome (45 ,46 ), appear infrequent in RER+ colorectal carcinomas and gastric tumors [(47 ,48 ) and P. Cottu, presented at Cancer and the Cell Cycle, Lausanne, Switzerland 1996]. Thus, these findings would be consistent, at least in part, with 3p14 deletions resulting from the genomic instability which accompanies p53 inactivation. We note that although CC19 is HPV negative, it expresses a mutant p53 protein (53 ).
The sequences used for comparison were derived from Xq27.3 (contains the Fragile-X region, GenBank L29074) and 4p16.3 (in the region of Huntington's disease, GenBank Z69837). The sequence from 3p21.3 was downloaded from ftp://genome.wustl.edu/pub/gsc1/sequence/st.louis/human/shotgun/3/H_LUCA14.seq.
Cell lines. Breast, colon and cervical carcinoma lines were obtained from the American Type Culture Collection. The cervical carcinoma cell line, CC19, was established as described (53 ). Lung tumor lines were obtained through the Colorado Lung Cancer SPORE Tissue Culture Core Laboratory. Normal cell lines included the human lymphoblastoid cell lines TL8229 and AG4103 and the E1A-immortalized human embryonic kidney cell line, E293. The somatic cell hybrids have been described previously (54 ). Libraries. The gridded chromosome 3-specific cosmid library (55 ) was obtained from Lawrence Livermore National Laboratories. The 850A6 YAC subclone library was previously described (16 ). Fetal brain and adult kidney cDNA libraries were obtained from Clontech. DNA and RNA isolations. DNA was isolated from cell lines by standard methods. Cosmid DNAs were isolated using alkaline lysis; preparations used for DNA sequence analysis were purified further by CsCl gradient centrifugation. DNA was isolated from single or pooled phage clones by the Grossberger method (56 ). RNA was isolated from cell lines when the cultures reached 90% of confluency using the RNA-STAT-60 kit from Tel-Test, Inc. (Friendswood, TX). Library screening, contig assembly and hybridization analysis. The gridded cosmid library was spotted onto filters at high density (1536 clones per filter) and hybridized using standard techniques with a 370 kb MluI fragment derived from YAC 74B2. The 850A6 YAC phage library (16 ) was screened using the same probe. Resulting clones were assembled into a contiguous segment by hybridization analysis using end probes and total inserts followed by analysis with the software tool SEGMAP. Complete and partial digestion analyses (57 ) were used to restriction map the central 170 kb of the contig prior to DNA sequence analysis.
Biotinylated primers were obtained from Research Genetics, Huntsville, AL. PCR amplifications were performed with 40 ng of template DNA utilizing hot start and touch down procedures. After separation on denaturing polyacrylamide gels, PCR products were detected using the New England BioLabs Phototopetm Detection Kit. Alleles were scored by visual inspection of band patterns.
RT-PCR was performed using primers FHIT-5' (5'-CTCGAA- TTCTTAGACCCTCTATAAAAGC-3') and FHIT-3' (5'-CTGATTCAGTTCCTCTTG-3') derived from non-coding exons 4 and 10, respectively. First strand synthesis was accomplished with 1-3 mg of total RNA and the Superscript II kit (Life Technologies). Subsequent amplification utilized one-fifth of the reverse transcriptase reaction together with the FHIT primers. Standard PCR conditions of 94oC denaturation (1 min), 55oC annealing (1 min) and 72oC elongation for 35 cycles were employed. PCR products were subcloned into the EcoRI site of pBluescript II SK+ using an introduced EcoRI site present in the FHIT-5' primer and a natural site located 21 bp downstream of the FHIT stop codon.
Cosmid and phage clones were sequenced using a random shotgun subcloning and end-sequencing strategy. Clone DNA was sonicated and size selected by LMP-agarose gel electrophoresis. Recovered fragments of 1-2 kb were end-repaired with Klenow fragment of Escherichia coli DNA polymerase I and T4 DNA polymerase, ligated into the phosphatased EcoRV site of pBluescript II and transformed into E.coli DH10B. AmpR/[beta]-Gal- subclones were grown in 3 ml of TB for isolation of sequencing templates. To eliminate vector sequences from the subclone library, phage inserts were amplified by long-range PCR. Inserts were gel purified, 32P labeled and hybridized to subclone libraries. Positive subclones were sequenced using an ABI 373 or 377 and the ABI Prism dye terminator cycle sequencing kit. Chromatograms were transferred to a SUN workstation, analyzed and assembled using PHRED and PHRAP (from Dr Phil Green). Gaps were closed by primer walking. Based on independently obtained overlapping contigs, as well as analysis of cosmid vector sequences in some subclones, sequencing accuracy was at least 99.6%.
Assembled sequences were analyzed for database similarities by BLASTN and BLASTX, searching the nr and dbest databases with default parameters. Strongly similar sequences (P <e-30) were retrieved from GenBank for further analysis. Homologies to repeat sequences were found using Pythia (32 ). Potential exons were identified using GRAIL2 (58 ) and GeneMark (31 ) programs. GeneMark was run using 5th order matrices trained on sequences with GC content similar to the sequences checked. Sequence alignments were prepared using the FASTA program from the Wisconsin Package (GCG).
This work was supported by DAMD17-94-J-4391 (HAD, RMG), CA51395 (HPK, MRM) and the DNA sequencing studies were supported by HG000358 (H.A.D., R.M.G.). We gratefully acknowledge support from the Colorado Cancer League (2531718) to F.B. Additional Core support was provided through the University of Colorado Cancer Center (CA 46934) and by Dr Wilbur Franklin through the Lung Cancer Spore.
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Sequencing of the CC19 deletion region has been completed with the finalization of lambda 94, a phage proximal to lambda 33. This 18 810 bp sequence maintains the same characteristics as the balance of FRA3B. It is 63% AT and 27% is comprised of known repeats with the L1 type elements predominating.
+Present address: CNRS URA1172, IBMIG, Universite de Poitiers, 40 Av. du Recteur Pineau, 86022 Poitiers Cédex, France
}These authors contributed equally to this work
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