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ONCOGENOMICS Results Strongly Suggest That 3P21.3T and 3P21.3C Regions 2002) Oncogene (2003) 22, 2984–2992 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc 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).
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