ONCOGENOMICS Results Strongly Suggest That 3P21.3T and 3P21.3C Regions 2002)

Deletion mapping using quantitative real-time PCR identiﬁes two distinct 3p21.3 regions affected in most cervical carcinomas

Vera Senchenko1,2,7, Jian Liu1,7, Eleonora Braga1,3, Natalia Mazurenko4, Witaly Loginov3, Yury Seryogin3, Igor Bazov1,3, Alexei Protopopov1, Fedor L Kisseljov4, Vladimir Kashuba1, Michael I Lerman5, George Klein1 and Eugene R Zabarovsky*,1,6

1Microbiology and Tumor Biology Center, Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, 17177 Sweden; 2Center ‘Bioengineering’, Russian Academy of Sciences, Moscow, 117312 Russia; 3Russian State Genetics Center, Moscow, 113545 Russia; 4Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Moscow, 115478 Russia; 5Cancer-Causing Genes Section, Laboratory of Immunobiology, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA; 6Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia

We report chromosome 3p deletion mapping of 32 cervical Keywords: quantitative real-time PCR; human chromo- carcinoma (CC) biopsies using 26 microsatellite markers some 3p; tumor suppressor genes; NotI linking clone; located in frequently deleted 3p regions to detect loss of Loss of heterozygosity; cervical carcinoma heterozygosity and homozygous loss. In addition, two STS markers (NLJ-003 and NL3-001) located in the 3p21.3 telomeric (3p21.3T) and 3p21.3 centromeric (3p21.3C) regions, respectively, were used for quantitative Introduction real-time PCR as TaqMan probes. We show that quantitative real-time PCR is reliable and sensitive and Tumor suppressor genes (TSG(s)) represent one of the allows discriminating between 0, 1 and 2 marker copies main classes of cancer-associated genes and their per human genome. For the first time, frequent (five of 32 identification constitutes one of the major efforts in cases, i.e. 15.6%) homozygous deletions were demon- cancer research today. strated in CCs in both 3p21.3T and 3p21.3C regions. The Deletions in chromosome 3p are frequently associated smallest region homozygously deleted in 3p21.3C was with different epithelial tumors, and this chromosome is located between D3S1568 (CACNA2D2 gene) and harboring several TSG(s) (Zabarovsky et al., 2002). D3S4604 (SEMA3F gene) and contains 17 genes Many studies have shown abnormalities on the short previously defined as lung cancer candidate Tumor arm of this chromosome in carcinomas of the kidney, suppressor genes (TSG(s)). The smallest region homo- lung, breast, ovary, cervix, testis, head and neck and zygously deleted in 3p21.3T was flanked by D3S1298 and other (Kok et al., 1997; Braga et al., 1999; Lazo, 1999). NL1-024 (D3S4285), excluding DLEC1 and MYD88 as However, the search for resident TSG(s) was hampered candidate TSGs involved in cervical carcinogenesis. by the large size of the region that covers practically the Overall, this region contains five potential candidates, whole chromosome 3p (about 100 Mb). It is still not namely GOLGA4, APRG1, ITGA9, HYA22 and VILL, clear which TSG(s) located on chromosome 3 are cancer which need to be analysed. The data showed that type specific or common to different epithelial tumors. aberrations of either NLJ-003 or NL3-001 were detected According to several studies, the most frequently in 29 cases (90.6%) and most likely have a synergistic affected regions (FAR) in sporadic renal cell carcinoma effect (Po0.01). The study also demonstrated that (RCC) and small cell lung carcinoma (SCLC) are 3p13– aberrations in 3p21.3 were complex and in addition to p14 and 3p21.2–3p21.3 (Kok et al, 1997; Van den Berg deletions, may involve gene amplification as well. The and Buys, 1997; Alimov et al., 2000; Zabarovsky et al., ONCOGENOMICS results strongly suggest that 3p21.3T and 3p21.3C regions 2002). Recently, using deletion mapping we have shown harbor genes involved in the origin and/or development of that the 3p21.3 region is the most frequently deleted CCs and imply that those genes might be multiple TSG(s). region not only in RCC and SCLC but also in nonsmall Oncogene (2003) 22, 2984–2992. doi:10.1038/sj.onc.1206429 cell lung (NSCLC), breast and cervical carcinomas (CCs). Moreover, we have shown that this region could be subdivided into centromeric 3p21.3 (3p21.3C) and telomeric 3p21.3–p22 (3p21.3T) subparts (Alimov et al., *Correspondence: ER Zabarovsky, Microbiology and Tumor Biology 2000; Lerman et al., 2000; Braga et al., 2002). Others Center, Center for Genomics and Bioinformatics, Karolinska Institute, and we identified several candidate TSG(s) from the Stockholm, 17177 Sweden; E-mail: [email protected]; [email protected] centromeric 3p21.3 region, however, no clear candidate 7These authors contributed equally to this work TSG(s) from the telomeric 3p21.3 part have been Received 22 July 2002; revised 23 January 2003; accepted 28 January isolated yet (Zabarovsky et al., 2002). Importantly, 2003 both telomeric and centromeric 3p21.3 critical regions 3p deletion hot spots in cervical carcinomas V Senchenko et al 2985 contain several genes that justified further fine deletion ABI Primer Express Software (version 1.5). They were as mapping of these regions. follows: NLJ-003 forward 50-CAG AGT GCG TGT GCC Loss of heterozygosity (LOH) is frequently used as an GAC T-30, reverse 50-ACA ACT TCT CTG CGG GCG T-30 indicator of genetic losses associated with tumor and probe 50-CTG GCG GAG AGA CTG GGA GCG A-30 (125 bp amplicon); NL3-001 forward 50-CTT GCC ATC TGC development, and the microsatellite repeat analysis is 0 0 often the method of choice for LOH detection. LOH AAT TCC CT-3 , reverse 5 -CTC CAT GAG GCT GTG GGA AG-30 and probe 50-CCC CAG AAA CGC GCG GGC- studies, however, have several disadvantages discussed 30 (60 bp amplicon). The sequences of primers and TaqMan previously (Liu et al., 1999; Zabarovsky et al., 2002). probes for phospho-fructo-2 kinase (PF2K) gene were: Real-time PCR using TaqMan probes is a well-known, forward 50-ATG CCC TGG CCA ACT CA-30, reverse 50- precise, and reproducible quantitative nucleic acid assay TGC GAC TGG TCC ACA CCT T-30 and probe 50-FAM- with a high throughput and large dynamic range of TCA GTC CCA GGG CAT CAG CTC CC-TAMRA-30 applications (Livak et al., 1995). The method overcomes (Boulay et al., 1999). Beta-actin gene (ACTB) was used as a some limitations of LOH analysis, for example, it does reference (Applied Biosystems, Foster City, CA, USA). NLJ- not require polymorphism, so any unique marker can be 003, PF2K and ACTB probes were labeled with FAM (6- used. In contrast to LOH with microsatellite markers that carboxy-fluoroscein) and NL3-001 contained JOE (2,7-di- detect only allelic imbalance without discrimination methoxy-4,5-dichloro-6-carboxy-fluoroscein) as reporter dyes, located at the 50-ends. All of the reporters were quenched by between deletions and amplifications, real-time PCR TAMRA (6-carboxy-N,N,N0N0-tetramethyl-rhodamine), con- yields information about copy number changes. In this jugated to their 30-terminal nucleotides. All probes and primers study, real-time PCR was used to assess the presence and were purchased from Applied Biosystems. PCRs were carried frequency of homozygous deletions in the centromeric out in triplicate in 25 ml consisting of 1 Â PCR buffer A and telomeric 3p21.3 regions. (Applied Biosystems), 3.5 mm MgCl2, 0.2 mm dATP, dGTP, dCTP, and 0.4 mm dUTP, 100 nm TaqMan probe, forward and reverse primers in appropriate concentrations (150–200 nm), 0.025 U/ml Taq Gold DNA polymerase (Applied Biosystems), Materials and methods 0.01 U/ml AmpErase and 5 ml of DNA template (20–60 ng). PCRs were carried out according to the thermal profile: 2 min DNA samples and Southern blot analysis at 501C, 10 min at 951C, followed by 40 cycles of 15 s at 951C In all, 32 cervical squamous CCs were studied. For histological and 1 min at 601C. verification, top and bottom sections (5-mm thick) cut from Comparative CT method (DDCT method) was used for frozen tumor tissues were examined after staining with quantification of marker copy numbers. The parameter CT hematoxylin and eosin. Selected samples containing 60% or (threshold cycle) is defined as the cycle number required for more tumor cells, and matched normal tissues were stored at reporter dye fluorescence to become higher than background À701C. High-molecular-weight DNA was isolated with fluorescence level and automatically determined using ABI guanidinium thiocyanate followed by centrifugation through Prisms Model 7700 Sequence Detector (Applied Biosystems). a CsCl cushion (Samoylova et al., 1995). The method is based on the inverse exponential relation Female and male DNA samples for testing X chromosome that exists between the initial quantity (copy number) of copy numbers were obtained from the blood of healthy target sequence copies in the reactions and the corresponding individuals. High-molecular-weight DNA was isolated by CT determinations – the higher the starting copy number of overnight treatment with proteinase K at 501C followed by DNA target, the lesser the CT value. This method was phenol/chloroform extraction and precipitation with ethanol. used to determine target sequence copy number in The DNA preparations were examined by electrophoresis tumor DNA sample relative to the normal DNA from the using 0.8% agarose gels. same patient (calibrator) and relative to an endogenous Chromosome 3 specific NotI linking clones NLJ-003/ control sequence (reference) – ACTB in both samples. The D3S1642 and NL3-001/D3S3874 (mapped to 3p21.3T and to starting relative copy number DNA (at each locus in tumor ÀDDCT tumor normal 3p21.3C, respectively) were described previously (Kashuba sample) is given by 2 , where DDCT ¼ DCT ÀDCT target reference et al., 1999). and each DCT ¼ CT ÀCT . Data were analysed using Southern blotting and hybridization were performed ac- ABI Prisms 7700 Sequence Detection System software cording to the standard procedures. Band intensities were (version 1.7). The range given for the probes was determined ÀDD determined using a Molecular Dynamics Personal Densit- as 2 CT with DDCT +s and DDCT À s, where s ¼ the ometer SI (Sunnyvale, CA, USA) according to the manufac- standard deviation of the DDCT value. The absence of turer’s protocol. NotI linking clone NL1-290/D3S4293 nonspecific amplification was confirmed by analysing the mapped to the 3q13.3–q21 (Kashuba et al., 1999) was used PCR amplification by 2.5% agarose gel electrophoresis and as a reference control because this region usually does not ethidium bromide staining. reveal frequent allelic imbalances in CCs (Rader et al., 1996; For the DDCT calculation to be valid, the efficiency of the Lazo, 1999; Matthews et al., 2000). The ratio of band target amplification and the efficiency of the reference intensities showing copy number changes of the probe NLJ- amplification must be approximately equal. Before using the 003 was calculated according to the formula: PT Â RN/ DDCT method for quantitative assessment, a validation RT Â PN, where PT and PN were band intensities for the experiment was performed. The validation experiments NLJ-003 bands, and RT and RN represented band intensities (Figure 1) demonstrated that efficiencies of the targets and of NL1-290 in tumor and normal samples, respectively. reference are approximately equal for the chosen dilutions. The absolute value of the slope of log input amount vs DCT should be 0.1. For the NL3-001, the slope value is 0.0848, Real-time quantitative PCR o for NLJ-003 it is 0.0133 and for PF2K it is –0.0577. Thus, the Selection of primer and TaqMan probe sequences for NLJ- DDCT calculations can be used for the relative quantification 003/D3S1642 and NL3-001/D3S3874 was performed using the of targets without using standard curves.

Oncogene 3p deletion hot spots in cervical carcinomas V Senchenko et al 2986 Polymorphic and STS markers and microsatellite analysis In all, 26 polymorphic and two STS markers of 3p were applied. A total of 23 microsatellite markers were selected from genome- (GDB) and CHLC databases. Three polymorphic (NL1-024/D3S4285, D3S4604 and D3S4597) and two STS (NLJ-003/D3S1642 and NL3-001/D3S3874) markers were designed and located earlier (Braga et al., 1997; Kashuba et al., 1999; Wistuba et al., 2000). The order of all 28 selected markers according to the location database (http://www.ce- dar.genetics.soton.ac.uk/pub/chrom3/gmap), NCBI human GDB (http://www.ncbi.nlm.nih.gov/genome/guide/human) and our own data was as follows: 3p-tel-D3S2405/GGAT2A11- D3S1317-D3S1038-D3S1286-D3S3047/GATA85F02-D3S1283- D3S2420 / ATA25A07-NL1-024/D3S4285-NLJ-003/D3S1642- D3S1298-D3S3527-p33715/GAAT12D11-D3S2456/GATA63 E04-D3S1767/GATA7A01-D3S2409/ATA10H11-D3 S3615- D3S1573-D3S4597-D3S4604-NL3-001/D3S3874-D3S1568-D3 S3667-D3S1766/GATA6F06-D3S1300-D3S1481-D3S1285-D3 S2454/GATA52H09-D3S2406/GGAT2G03-3p-cen. The PCR primers were purchased from Life Technologies/ Invitrogen (Carlsbad, CA, USA). The PCR and PAG electrophoresis was carried out as described earlier (Braga et al., 1999). Band intensities for PCR products of high- and low- molecular-weight alleles (H- and L alleles) were compared using a Molecular Dynamics Personal Densitometer SI. The LOH was scored when a ratio of intensities for matched tumor and normal DNAs differed twofold or more.

Results

Testing sensitivity of real-time PCR using TaqMan probes

Figure 1 Relative efﬁciency plot of log input amount vs DCT for NL3-001 (a), NLJ-003 (b) and ACTB (c). For detailed description In previous experiments using LOH deletion mapping, see Applied Biosystems guidelines. The absolute value of the slopes we have shown that regions surrounding NLJ-003 and for all three probes is less than 0.1 NL3-001 are the most FARS in RCC (see Figure 2). It was also shown that these clones were homozygously deleted in SCLC and breast cancers (Murata et al., 1994;

Comparative CT method can be used in separate tubes and Wei et al., 1996; Sekido et al., 1998; Alimov et al., 2000; in the same tube. Control experiments (data not shown) Lerman et al., 2000). Recently, the same was demon- demonstrated that both methods gave the same results. strated for NSCLC, breast and cervical cancers (Braga Quantification of NLJ-003 and PF2K was carried out in et al., 2002). The data strongly suggest that both regions separate tubes and quantification of NL3-001 was carried out (telomeric 3p21.3T and centromeric 3p21.3C) contain in the same tube. For an accurate CT quantification using multiple tumor suppressor genes. The focus of the multiplex PCR in the same tube, it is important that two present study was to compare LOH data with another independent reactions do not compete. The primer limitation experiments were performed using a matrix of forward and independent deletion mapping method and to check reverse primer concentrations (15–100 nm). The chosen con- whether these regions are also homozygously deleted in CCs. Comparative genomic hybridization (CGH) data centrations were those that have shown a reduction in DRn but little effect on CT. The primer and probe concentrations for could verify and validate the LOH mapping (Alimov multiplex PCRs in the same tubes were found: for NL3-001– et al., 2000); however, the resolution of this method is 20 nm (probe), 90 nm (primers) and ACTB À 20 nm (probe), rather low. We decided to use a much more sensitive, 60 nm (primers). rapid and quantitative method, termed real-time PCR (Heid et al., 1996) to assay genetic changes in Sample sex determination using of PF2K gene dosage in X tumorigenesis. This technology does not require poly- chromosome morphic markers, and any marker is informative for any cancer case. Real-time PCR permits to evaluate and Real-time PCRs were performed on the 15 male samples and compare a set of samples at different loci to identify 15 female samples using two different sets of primers, one set for X-linked housekeeping gene PF2K (Boulay et al., 1999), deletions, retentions and amplifications. As described and another for ACTB gene used as a reference. The reactions in Material and methods, all control experiments were carried out in triplicate for each sample. For each demonstrated that primers and probes designed for the individual, CT value obtained for ACTB was subtracted from NLJ-003 and NL3-001 loci were appropriate for the that of PF2K, thus defining DCT. comparative CT method.

Oncogene 3p deletion hot spots in cervical carcinomas V Senchenko et al 2987 approach permits detection of the difference in X chromosome copy number between males (one copy) and females (two copies).

Analysis of two critical regions in 3p21.3 To test two critical sites in 3p for copy number changes and homozygous deletions, two TaqMan probes were designed: the NLJ-003 (3p21.3T), residing in the AP20 homozygous deletions site (Kashuba et al., 1995; Ishikawa et al., 1997; Protopopov et al., 2003) and NL3-001 (3p21.3C), mapped to the LUCA homozygous deletion site (Wei et al., 1996; Lerman et al., 2000, see Figure 2). Frequent aberrations were shown in these regions in previous studies (Alimov et al., 2000; Wistuba et al., 2000; Braga et al., 2002). The results of the experiments are shown in Table 1. According to histology analysis, contamination of tumor samples with normal stroma and lymphocytes can reach up to 30–40%. Therefore, alleles were taken as homozygously deleted if the highest value of standard deviation is below 0.5 and hemizygously deleted if this value is below 1.0. An allele was considered as amplified/ multiplied, if the lowest value of standard deviation is over 1.0. In this case, the results of quantitative PCR analysis suggest that seven samples (21.8%) contained amplified NLJ-003 marker and nine samples (28.1%) had amplified NL3-001. Homozygous deletions were detected in five cases (15.6%) for both markers, of which four cases were in the same tumor samples. Frequencies of simultaneous homozygous deletions in both loci were highly significant (P ¼ 0.0009). Therefore, some aberrations in 3p21.3T and 3p21.3C often occurred in the same tumor. This might mean that TSG(s) in these regions can have a synergistic effect. Hemizygous deletions of NLJ-003 were found in 14 samples (43.8%) and of NL3-001 in 10 (31.3%). Figure 2 Schematic maps of 3p21.3C and 3p21.3T regions. For homozygous deletions found in SCLC cell lines (Wei et al., 1996; Altogether, these data showed that copy number Ishikawa et al., 1997; Lerman et al., 2000; Protopopov et al., 2003) changes (homo- and hemizygous deletions and amplifi- and for flanking regions, the genes are represented by pointed cations) of either NLJ3-001 or NL3-001 were detected in arrows, indicating the orientations of transcription. Physical 29 cases (90.6%). This is significantly higher than it was position from 3p telomere is according to http://www.ncbi.nlm.- reported earlier for the whole 3p – around or some more nih.gov/cgi-bin/Entrez/hum_srch?chr ¼ hum_chr.inf&query (build 30) than 70% (Larson et al., 1997a; Wistuba et al., 1997; Braga et al., 1999; 2002; Helland et al., 2000). These results lend strong support to the conclusion that these two regions are really hot spots for rearrangements in Before doing deletion mapping of cervical carcino- CCs. Only in three cases (9.4%), no aberrations were mas, we tested real-time PCR with human X chromo- detected in either of these loci. some, thus requiring differentiation between one (male) This study has highlighted the complex character of and two (female) copies per genome. The DCT values chromosomal rearrangements in 3p during development obtained for 30 samples indicated that samples could be of CC. In addition to homo- and hemizygous deletions, divided into two nonoverlapping groups. The DCT value duplication/amplification of the whole chromosome 3 or for one group was 6.0170.40 (av.7s.d.) and it was some regions was also detected. One tentative explana- approximately one unit higher than the DCT for another tion of these phenomena might be that inactivated group: 4.9170.44. The difference of one unit reflects a TSG(s) were amplified together with neighboring twofold value of initial gene copy number for females oncogenes. compared to males. The amount of target (PF2K) To confirm that allelic imbalance could be because of for females, normalized to a reference (ACTB) and the amplification, we performed additional experiments relative to target (PF2K) for males was: with five RCC cell lines showing LOH and considered to NFemale=NMale ¼ 2ÀDDCT ¼ 2.14(2.08–2.20). Thus, this have 3p deletions (Alimov et al., 2000). However,

Oncogene 3p deletion hot spots in cervical carcinomas V Senchenko et al 2988 Table 1 Results of real-time PCR for cervical cancer samples tions in cases #10 and #15, but neighboring markers Relative DNA copy numberb showed retention. This disagreement can be explained by different factors. On the one hand, it can mean that Sample no.a NLJ-003 (3p21.3) NL3-001 (3p21.2–21.3) NLJ-003 is a hot spot for the rearrangements and 1 1.34 (1.24–1.45) 0.51 (0.34–0.67) flanking regions were unaffected; on the other, it is clear 2 1.16 (0.90–1.42) 0.71 (0.54–0.89) that not every copy number change will result in allelic 3 0.98 (0.91–1.06) 1.04 (0.99–1.09) imbalance. Moreover, application of real-time PCR 4 1.20 (1.13–1.27) 1.43 (1.19–1.67) 5 2.75 (2.46–3.04) 1.65 (1.53–1.77) allowed detecting homozygous deletions that is very 6 0.61 (0.50–0.71) 2.85 (2.48–3.22) difficult with microsatellite markers. From the data 7 1.56 (1.32–1.80) 1.15 (0.99–1.31) shown in Figure 3, it is possible to conclude that the 8 1.10 (0.97–1.23) 1.09 (0.83–1.35) smallest homozygously deleted region in 3p21.3T is 9 0.36 (0.28–0.43) 0.18 (0.10–0.26) bordered with NL1-024 (D3S4285) and D3S1298. 10 0.78 (0.66–0.90) 1.03 (0.98–1.08) 11 0.80 (0.61–1.00) 1.06 (0.97–1.15) Additional data with primers D3S2968, D3S4597 and 12 2.39 (2.19–2.59) 2.55 (2.50–2.60) D3S4604 (not shown) suggested that the similar region 13 0.79 (0.66–0.92) 1.45 (1.37–1.53) in 3p21.3C is limited with D3S4604 and D3S1568. It is 14 0.38 (0.30–0.46) 0.49 (0.43–0.55) important to mention that these two regions overlap 15 0.66 (0.61–0.71) 0.50 (0.42–0.58) 16 0.61 (0.51–0.71) 0.63 (0.52–0.75) with previously described homozygous deletions in lung 17 0.70 (0.56–0.85) 0.57 (0.52–0.63) and breast carcinomas (Murata et al., 1994; Wei et al., 18 0.17 (0.13–0.22) 0.06 (0.04–0.08) 1996; Sekido et al., 1998; Lerman et al., 2000). 19 0.15 (0.11–0.19) 0.04 (0.03–0.05) Owing to the limited amount of tumor material, only 20 0.78 (0.68–0.89) 1.51 (1.19–1.82) some samples could be tested by Southern hybridiza- 21 0.44 (0.38–0.51) 0.31 (0.26–0.36) 22 1.37 (1.25–1.50) 0.62 (0.58–0.66) tion. Results for the available cases are shown 23 2.74 (2.66–2.82) 2.80(2.38–3.21) in Figure 4. This figure clearly showed that the real- 24 0.98 (0.94–1.03) 1.26 (1.00–1.51) time PCR data were consistent with the Southern 25 0.50 (0.40–0.61) 2.03 (1.82–2.24) data. 26 0.20 (0.19–0.22) 0.10 (0.09–0.11) 27 0.61 (0.55–0.66) 1.07 (0.98–1.17) 28 0.88 (0.87–0.89) 1.54 (1.08–2.01) 29 1.08 (0.98–1.14) 0.60 (0.53–0.67) Discussion 30 2.79 (2.44–3.13) 1.05 (1.00–1.09) 31 0.48 (0.39–0.58) 1.14 (0.99–1.29) According to LOH and CGH data (Kisseljov et al., 32 0.57 (0.54–0.61) 0.80 (0.65–0.95) 1996; Rader et al., 1996, 1998; Lazo, 1999; Mazurenko aGray samples did not show any aberrations and bold contained et al., 1999; Matthews et al., 2000) chromosome arms homozygous deletions in one or both of the loci bSamples showed 3p, 6p and 11q were most frequently affected in multiplication are in bold italics and samples with white letters squamous CC of the uterine cervix. Moreover, 3p and contained hemizygous deletions 6p were also involved at the early precancer stages and during progression of cervical intraepithelial neoplasia to invasive cervical cancer (Larson et al., 1997b; Wistuba et al., 1997; Fouret et al., 1998; Guo et al., cytogenetic analysis demonstrated that among them 1998, 2000, 2001; Umayahara et al., 2002). Different only A498 had two copies of chromosome 3 and four studies reported various 3p LOH rates in the range 50– lines had from three to five copies (KRC/Y, HN51, 76% (Wistuba et al., 1997; Braga et al., 1999; Guo et al., CAKI1 and TK164). Nevertheless, all these five lines 2000; Helland et al., 2000; Herzog et al., 2001; Acevedo revealed only one marker in LOH analysis arguing that et al., 2002). Several reports suggested that the most one 3p arm was lost. These data proved that amplifica- frequently affected in CC regions were located in tion associated with 3p deletion is a common event in 3p21.3–p22 region and near FHIT gene in 3p14.2 cancer cells. (Wistuba et al., 1997; Muller et al., 1998; Helland Real-time PCR data for NLJ-003 and NL3-001 loci et al., 2000; Herzog et al., 2001; Acevedo et al., 2002). were compared with allelic alterations observed for We have found recently that two regions in 3p21.3 neighboring polymorphic markers (Figure 3). In all, 20 (centromeric – C or LUCA and telomeric – T or AP20) cancer samples were analysed in this work. Significant were frequently deleted in major human malignancies correlation between real-time PCR and LOH data was (Braga et al., 2002). Homozygous deletions are excellent observed in both loci for many cancer samples. For indicators of the locations of TSG(s); however, example, relative copy number values for NLJ-003 locus microsatellite deletion mapping is not very well suited in CC samples #6, #17 and #25 suggested allelic for the detection of such deletions because of normal imbalance, and flanking microsatellite markers sup- cell contamination. Balance between two alleles does ported this finding. In cases #2, #8 and #24 quantitative not change in such cases and deletion would go real-time PCR suggested for NLJ-003 retention and this undetected. In fact, this can lead to paradoxical results result coincided with the LOH in neighboring poly- when LOH in homozygously deleted regions may be less morphic markers. frequent than in surrounding regions. Furthermore, However, in other cases discordance was apparent. this approach in fact detects only allelic imbalance For instance, NLJ-003 demonstrated hemizygous dele- that could result from multiplication as well as

Oncogene 3p deletion hot spots in cervical carcinomas V Senchenko et al 2989

Figure 3 Combination of LOH and real-time PCR data for 3p deletion mapping. Black squares represent LOH and homozygous deletions; white squares, retention of heterozygosity and normal copy number; gray squares, multiplication and dashed boxes are noninformative cases. L and H inside black squares denote the type of allele lost. It was shown (Liu et al., 1999) that the H-allele is less sensitive to normal cell contamination than L-allele

from deletions. Real-time PCR in contrast to the LOH can detect precisely copy number changes (Cairns et al., 1998; Chiang et al., 1999; Braga et al., 2002) and in this study we performed detailed analysis of these two critical loci combining real-time PCR with LOH. Several candidate TSG(s) were identified in these two regions; however, none of them showed a frequent mutation rate in tumor samples (Daigo et al., 1999; Lerman et al., 2000; Zabarovsky et al., 2002). Epigenetic inactivation by methylation and homozygous deletions are alter- native pathways for TSG inactivation (Baylin and Herman, 2000). Indeed, for example, hypermethylation of promoter region was demonstrated for RASSF1A gene from 3p21.3C (LUCA) region in various epithelial carcinomas (Dammann et al., 2000; Burbee et al., 2001; Dreijerink et al., 2001; Kuzmin et al., 2002). We designed two probes for real-time PCR, one from each of these regions. Clone NL3-001 is located closely to candidate TSG semaphorin 3B (SEMA3B), while NLJ- 003 is between the integrin alpha 9 (ITGA9) gene involved in cell adhesion and border of homozygous deletion in ACC-LC5 SCLC cell line (Murata et al., 1994). To our knowledge, real-time quantitative PCR was Figure 4 Comparison of real-time PCR and Southern hybridiza- applied here for the first time in a detailed study of copy tion results. DNA was digested with BamHI and transferred to the number changes in chromosome 3p loci in solid tumors, nylon filters. Hybridization was carried out simultaneously with and we demonstrated frequent (15.6%) homozygous two probes NLJ-003 (3p21.3T) and NL1-290 (3q13-q21). Upper numbers show the ratio according band intensities determined by deletions in both 3p21.3C and 3p21.3T critical regions in scanning nylon filters and bottom numbers represent results of real- squamous cell CCs. Earlier homozygous deletions were time PCR detected in CC only around the FHIT region, and these

Oncogene 3p deletion hot spots in cervical carcinomas V Senchenko et al 2990 results were based on application of LOH and multiplex et al., 2001). This result further confirmed the usefulness PCR (Larson et al., 1997a; Muller et al., 1998). of quantitative real-time PCR in the mapping of Moreover, our results strongly argue that these two important regions in cancer genome research. Amplifi- regions in 3p21.3 are hot spots for rearrangements in cation of 3p loci in CC was shown here for the first time. cervical cancer and therefore most likely contain TSG(s) Earlier increase in chromosome copy number and loss of and other cancer-associated genes. Indeed, aberrations one allele with concomitant duplication of the second in NLJ-003 locus were detected in 81.3% and in NL3- allele was shown in 3p only for lung cancer. Combina- 001 in 71.9% cases. Furthermore, aberrations in at least tion of LOH and FISH was applied in that study one of these loci were detected in 90.6% cases that is (Varella-Garcia et al., 1998). Thus, allelic imbalance can significantly higher than was reported before for 3p21.3 frequently lead to an erroneous conclusion that the (23–57%) and FHIT (30–58%) (Wistuba et al., 1997; other allele had suffered deletion while indeed another Muller et al., 1998; Helland et al., 2000; Herzog et al., allele was multiplied (Zabarovsky et al., 2002). Human 2001; Acevedo et al., 2002). chromosome 3p probably contains many genes that can According to histology analysis, contamination of play a dominant oncogenic role. MST1 receptor (RON) tumor samples with normal stroma and lymphocytes and its ligand MST1 gene are located in 3p21 and their was not more than 40%. Quantitative data are heavily overproduction can result in autocrine stimulation and affected by the degree of contamination of tumor uncontrolled proliferation (Angeloni and Lerman, samples. Intratumoral heterogeneity related to clonal 2001). Thus, amplification of some 3p21.3 regions can or subclonal events was reported earlier (Larson et al., lead to oncogene activation following the deletion of 1997a) and observed here by LOH (data not shown). resident TSG(s). Therefore, quantitative copy number changes deter- To confirm that allelic imbalance may result from mined by real-time PCR could be even more significant. frequent multiplication/amplification in tumor cells, we The smallest region homozygously deleted in 3p21.3C performed additional experiments with RCC cell lines. is bordered by D3S1568 and D3S4604. Therefore, the RCC showed very high level of 3p deletions by LOH centromeric border is inside the CACNA2D2 gene (cases studies (>90%) and it has been shown that 3p21.3T and #18 and #19) that is consistent with the data for SCLCs, 3p21.3C are the most frequently deleted regions in both for example, GLC20 SCLC cell line (Wei et al., 1996; RCC and cervical cancers (see for example Braga et al., Lerman et al., 2000). This implies that the TSG(s) 2002). For five RCC cell lines we have previously shown located in 3p21.3C could be involved in the development 3p deletions by LOH analysis (Alimov et al., 2000). of different tumors and genes located centromeric to However, cytogenetic analysis demonstrated that among CACNA2D2 (Lerman et al., 2000) could be excluded them only one had two copies of chromosome 3 and from the list of candidate cervical TSG(s). The telomeric four lines had from three to five copies. These data border is inside the SEMA3F gene (cases #18, #19 and proved that amplification associated with 3p deletion is #26) and this supported suggestion that in addition to a common event and therefore, without additional RASSF1A, both SEMA genes in this region could be independent data, it is more correct to speak about TSG(s) (Zabarovsky et al., 2002). allelic disbalance. The smallest region homozygously deleted in 3p21.3T Interestingly, homozygous deletions were detected in is flanked by D3S1298 and NL1-024 (D3S4285). The five cases (15.6%) for both markers, and in four cases centromeric border is close to D3S1298 (cases #9 and both markers were simultaneously homozygously de- #26) and this most probably suggests that DLEC1 and leted in the same tumor (P ¼ 0.0009). This most MYD88 genes (Daigo et al., 1999; Protopopov et al., probably means that TSG(s) in these regions could 2003) could not be considered as candidate TSG(s) have synergistic effects. The estimation of possible involved in cervical cancer. The telomeric border was interdependency between all aberrations in loci NL3- not precisely determined in this study and included two 001 and NLJ-003 as different events was carried out candidate TSG(s), namely MLH1 and ITGA9. Keeping using a permutation test (Mantel, 1967). This test in mind that telomeric border of the homozygous demonstrated a significant correlation between different deletion in SCLC cell line ACC-LC5 was determined aberrations in these two loci (Po0.01). However, earlier (Ishikawa et al., 1997), we could identify here further research is necessary to clarify this situation. only five known candidate TSG(s): trans-Golgi p230 Finally, the exceptionally high level of chromosome (GOLGA4), gene deleted in AP20-region 1 (APRG1), aberrations in NLJ-003 and NL3-001 loci suggests that ITGA9, gene homologous to S. pombe YA22 (actually a cervical TSG(s) could be located very near to these human ortholog referred to as HYA22) and villin-like markers. VILL genes (Daigo et al., 1999, Protopopov et al., 2003). Additional studies are needed to reveal which of these genes has TSG activity. Abbreviations Our study also demonstrated that aberrations in CC, cervical carcinoma; CGH, comparative genome hybridi- 3p21.3 have a complex character and in addition to zation; GDB, genome data base; H allele, high-molecular- deletions, amplification also could happen in this region weight allele; ACTB, human b-actin gene; L allele, low- with duplication/multiplication of the second allele, molecular-weight allele; LDB, location database; LOH, loss of which can accompany allelic loss (De Nooij-van Dalen heterozygosity; NSCLC, non-small cell lung carcinoma; PF2K, et al., 1998; Varella-Garcia et al., 1998; Thiagalingam phospho-fructo-2 kinase gene; RCC, renal cell carcinoma;

Oncogene 3p deletion hot spots in cervical carcinomas V Senchenko et al 2991 STS, sequence-tagged site; SCLC, small cell lung carcinoma; Ingabritt och Arne Lundbergs Forskningsstiftelse, Pharmacia TSG, tumor suppressor gene. Corporation, Karolinska Institute, the Russian National Human Genome Program and the Russian Foundation for Basic Research (Grants 01-04-48028 and 01-04-49086). MIL was funded in toto with funds from the National Cancer Acknowledgements Institute, National Institutes of Health, under Contract No. This work was supported by research grants from the Swedish NO1-CO-56000. Cancer Society, the Swedish Research Council, STINT,

References

Acevedo CM, Henriquez M, Emmert-Buck M and Chuaqui Heid CA, Stevens J, Livak KJ and Williams PM. (1996). RF. (2002). Cancer, 94, 793–802. Genome Res., 6, 986–994. Alimov A, Kost-Alimova M, Liu J, Li C, Bergerheim U, Imreh Helland A, Kraggerud SM, Kristensen GB, Holm R, Abeler S, Klein G and Zabarovsky ER. (2000). Oncogene, 19, VM, Huebner K, Borresen-Dale AL and Lothe RA. (2000). 1392–1399. Int. J. Cancer, 88, 217–222. Angeloni D and Lerman MI (2001). Human Lung cancer: Herzog CR, Keith AC, Sabourin CLK, Kelloff GJ, Boone CW, Cancer-Causing Genes, Environmental Factors. Sourcebook Stoner GD and You M. (2001). Mol. Carcinog., 30, 159–168. on Asbestos Diseases, Vol. 23. Peters GA, Peters BJ (eds). Ishikawa S, Kai M, Tamari M, Takei Y, Takeuchi K, Bandou Philadelphia: LexisNexis Press, pp 169–209. H, Yamane Y, Ogawa M and Nakamura Y. (1997). DNA Baylin SB and Herman JG. (2000). Trends Genet., 16, 168–174. Res., 4, 35–43. Boulay J-L, Reuter J, Ristchard R, Terracciano L, Herrmann Kashuba VI, Gizatullin RZ, Protopopov AI, Li J, Vorobieva R and Rochlist C. (1999). BioTechniques, 27, 228–232. NV, Fedorova L, Zabarovska VI, Muravenko OV, Kost- Braga EA, Kotova EI, Pugacheva EM, Gizatullin RZ, Alimova M, Domninsky DA, Kiss C, Allikmets R, Kashuba VI, Lerman MI, Aksenova MG, Efremov IA, Zakharyev VM, Braga EA, Sumegi J, Lerman M, Wahles- Zabarovsky ER and Kiselev LL. (1997). Mol. Biol. (Mosk.), tedt C, Zelenin AV, Sheer D, Winberg G, Grafodatsky A, 31, 985–996. Kisselev LL, Klein G and Zabarovsky ER. (1999). Gene, Braga E, Pugacheva E, Bazov I, Ermilova V, Kazubskaya T, 239, 259–271. Mazurenko N, Kisseljov F, Liu J, Garkavtseva R, Kashuba VI, Szeles A, Allikmets RL, Nilsson AS, Bergerheim Zabarovsky E and Kisselev L. (1999). FEBS Lett., 454, SR, Modi W, Grafodatsky A, Dean M, Stanbridge EJ, 215–219. Winberg G, Klein G and Zabarovsky ER. (1995). Cancer Braga E, Senchenko V, Bazov I, Loginov W, Liu J, Ermilova Genet. Cytogenet., 81, 144–150. V, Kazubskaya T, Garkavtseva R, Mazurenko N, Kisseljov Kisseljov F, Semionova L, Samoylova E, Mazurenko N, F, Lerman MI, Klein G, Kisselev L and Zabarovsky ER. Komissarova E, Zourbitskaya V, Gritzko T, Kozachenko V, (2002). Int. J. Cancer, 100, 534–541. Netchushkin M, Petrov S, Smirnov A and Alonso A. (1996). Burbee DG., Forgacs E, Zochbauer-Muller S, Shivakumar L, Int. J. Cancer, 69, 484–487. Fong K, Gao B, Randle D, Virmani A, Bader S, Sekido Y, Kok K, Naylor SL and Buys CH. (1997). Adv. Cancer Res., 71, Latif F, Milchgrub S, Gazdar AF, Lerman MI, Zabarovsky 27–92. E, White M and Minna JD. (2001). J. Natl. Cancer Inst., 93, Kuzmin I, Gillespie JW, Protopopov A, Geil L, Dreijerink K, 691–699. Yang Y, Vocke CD, Duh F-M, Zabarovsky E, Rhim JS, Cairns JP, Chiang P-W, Ramamoorthy S, Kurnit DM and Emmert-Buck MR, Linehan WM and Lerman ML. (2002). Sidransky D. (1998). Clin. Cancer Res., 4, 441–444. Cancer Res., 62, 3498–3502. Chiang P-W, Beer DG, Wei W-L, Orringer MB and Kurnit Larson AA, Kern S, Curtiss SH, Gordon R, Cavenee W and DM. (1999). Clin. Cancer Res., 5, 1381–1386. Hampton GM. (1997a). Cancer Res., 57, 4082–4090. Daigo Y, Isomura M, Nishiwaki T, Tamari M, Ishikawa S, Larson AA, Liao S-Y, Stanbridge EJ, Cavenee WK and Kai M, Murata Y, Takeuchi K, Yamane Y, Hayashi R, Hampton GM. (1997b). Cancer Res., 57, 4171–4176. Minami M, Fujino MA, Hojo Y, Uchiyama I, Takagi T and Lazo PA. (1999). Br. J. Cancer, 80, 2008–2019. Nakamura Y. (1999). DNA Res., 6, 37–44. Lerman M, Minna J, Sekido Y, Bader S, Burbee D, Fong K, Dammann R, Li C, Yoon J-H, Chin P L, Bates S and Pfeifer Forgacs E, Gao B, Garner H, Gazdar AF, Girard L, GP. (2000). Nat. Genet., 25, 315–319. Kamibayashi C, Kondo M, Pande A, Ramalingam V, De Nooij-vanDalen AG, van Buuren-van Seggelen VHA, Randle D, Tomizawa Y, Virmani A, Wistuba I, Macartney Lohman PHM and Giphart-Gassler M. (1998). Genes D, Honorio S, Maher E, Latif F, Kashuba V, Protopopov Chromosomes Cancer, 21, 30–38. A, Li J, Klein G, Zabarovsky E, Duh F-M, Wei M-H, Geil Dreijerink K, Braga E, Kuzmin I, Geil L, Duh F-M, Angeloni L, Kuzmin I, Ivanov S, Angeloni D, Ivanova A, Zbar B and D, Zbar B, Lerman MI, Stanbridge EJ, Minna JD, Johnson B.E. (2000). Cancer Res., 60, 6116–6133. Protopopov A, Li J, Kashuba V, Klein G and Zabarovsky Liu J, Zabarovska VI, Braga E, Alimov A, Klein G and ER. (2001). Proc. Natl. Acad. Sci. USA, 98, 7504–7509. Zabarovsky ER. (1999). FEBS Lett., 462, 121–128. Fouret PJ, Dabit D, Mergui JL and Uzan S. (1998). Livak KJ, Flood SJ, Marmaro J, Giusti W and Deets K. Pathobiology, 66, 306–310. (1995). PCR Methods Appl., 4, 357–362. Guo Z, Wilander E, Sallstrom J and Ponten J. (1998). Mantel N. (1967). Cancer Res., 27, 209–220. Anticancer Res., 18, 707–712. Matthews CP, Shera KA and McDougall JK. (2000). Proc. Guo Z, Hu X, Aﬁnk G, Ponten F, Wilander E and Ponten J. Soc. Exp. Biol. Med., 223, 316–321. (2000). Int. J. Cancer, 86, 518–523. Mazurenko N, Attaleb M, Gritsko T, Semjonova L, Pavlova Guo Z, Wu F, Asplund A, Hu X, Mazurenko N, Kisseljov F, L, Sakharova O and Kisseljov F. (1999). Oncol. Rep., 6, Ponten J and Wilander E. (2001). Mod. Pathol., 14, 54–61. 859–863.

Oncogene 3p deletion hot spots in cervical carcinomas V Senchenko et al 2992 Muller CY, O’Boyle JD, Fong KM, Wistuba II, Biesterveld E, Umayahara K, Numa F, Suehiro Y, Sakata A, Nawata S, Ahmadian M, Miller DS, Gazdar AF and Minna JD. (1998). Ogata H, Suminami M, Sasaki K and Kato H. (2002). Genes J. Natl. Cancer Inst., 90, 433–439. Chromosomes Cancer, 33, 98–102. Murata Y, Tamari M, Takahashi T, Horio Y, Hibi K, Van den Berg A and Buys CH. (1997). Genes Chromosomes Yokoyama S, Inazawa J, Yamakawa A, Takahashi T and Cancer, 19, 59–76. Nakamura Y. (1994). Hum. Mol. Genet., 3, 1341–1344. Varella-Garcia M, Gemmill RM, Rabenhorst SH, Lotto A, Protopopov A, Kashuba VI, Zabarovska VI, Muravenko OV, Drabkin HA, Archer PA and Franklin WA. (1998). Cancer Lerman MI, Klein G and Zabarovsky ER. (2003). Cancer Res., 58, 4701–4707. Res., 63, 404–412. Wei MF, Latif F, Bader S, Kashiba V, Chen JY, Duh FM, Rader JS, Gerhard DS, O’Sullivan MJ, Li Y, Li L, Sekido Y, Lee C, Geil L, Kuzmin I, Zabarovsky E, Klein G, Liapis H and Huettner PC. (1998). Genes Chrom. Cancer, Zbar B, Minna JD and Lerman MI. (1996). Cancer Res., 56, 22, 57–65. 1487–1492. Rader JS, Kamarasova T, Huettner PC, Li L, Li Yand Wistuba II, Behrens C, Virmani AK, Meme G, Milchgrub S, Gerhard DS. (1996). Oncogene, 13, 2737–2741. Girard L, Fondon III JW, Garner HR, McKay B, Latif F, Samoylova E, Shihaev G, Petrov S, Kisseljova N and Kisseljov Lerman MI, Lam S, Gazdar AF and Minna JD. (2000). F. (1995). Int. J. Cancer, 61, 337–341. Cancer Res., 60, 1949–1960. Sekido Y, Ahmadian M, Wistuba II, Latif F, Bader S, Wei M- Wistuba II, Montellano FD, Milchgrub S, Virmani AK, H, Duh F-M, Gazdar AF, Lerman MI and Minna JD. Behrens C, Chen H, Ahmadian M, Nowak JA, Muller C, (1998). Oncogene, 16, 3151–3157. Minna JD and Gazdar AF. (1997). Cancer Res., 57, Thiagalingam S, Laken S, Willson JKV, Markowitz SD, 3154–3158. Kinzler KW, Vogelstein B and Lengauer Ch. (2001). Proc. Zabarovsky ER, Lerman MI and Minna JD. (2002). Onco- Natl. Acad. Sci. USA, 98, 2698–2702. gene, 21, 6915–6935.

Oncogene