- Email: [email protected]
A rapid new detection method for K-ras point mutations. Point-EXACCT
19 071 A RAPID NKW DE-ON METHOD FOR K-RAS POINT MUTATIONS. POINT-EXACCT Thunnissen FEJM, Murtagh jr JJ, Somers V, Moerkerk P, Wage-
naar Sj. Department of Pathology, University of Limburg, Maastricht, The Netherlands and Pulmonary and Critical Care Medicine, Atlanta VA Medical center, Decatur, GA, USA K-ras point mutations occur in adenocarcinomas of tbe lung. Recently, a rapid, sensitive and efficient method was described for determination of specific PCR products (EXACCT, Nucl Acid Res 21,; 3905, 1993). After exonuclease digestion, hybridisation with a capture and a detection primer and subsequent immunochemical staining of the detection primer PCR fragments are reliably determined. For the detection of mutations this procedure was extended using ligase and K-ras codon 12 mutation specific primers (Point-EKACCT). PCR for K-ras was performed using primers outside the codon 12 region. After exormclease digestion a biotin labeled primer containing the point mutation at the 3’ side and a digoxigenin labeled primer were hybridised with the single stranded DNA. DNA of HL60, A549 and CALUl cellines were used as control for wild type, mutation AGT and TGT on K-ras codon 12, respectively. PCR products of HL60 hybridised wild type primers showed a distinct staining. In contrast I-IL60 DNA hybridised with mutation primers did not give staining above the background values. PCR products of A549 hybridised with mutation specific primer yielded a distinct staining pattern. For CALUl similar results were obtained. This procedure can be performed in one day. Conclusion The Point EKACCT method is reliable and rapid. This procedure can be used to detect K-ras point mutations in DNA of tumors and non-malignant epithelial cells in patients at risk for lung cancer. l’hi~researchW&S funded in part by the Sdentitic Council of Smoking and Health, Tbe Netherlands.
072
073
ANALYSIS OF 3p TUMOR SUPPRESSOR LOCI IN HUMAN LUNG CANCER. R. Gemmill’, J. Rock&d. S. Todd’, W. Franklin’,F. B&log’, M. Swmm’. S. Nayld, K. Kdr’. Charles Boys’ and II. Drabkin’. %Jniversityof Colorsdo Cancer Center and the Eleanor Roosevelt I&tote. Denver, CO. USA, %e University of Texas. San Antonio. ‘The Univexsityof Gmiqen. The Netberlmds. 4UniversiteC. Bernard, Villewbame C&x. Framx Cytogaetic and loss of betemzygmity &OH) analyses fmm several laborator& suggest &at then may be 3-4 mar snppressorgenes located on 3p. Thesame3pngionshavellsobeenimplieatedinothermatignantdiseases in&ding kidmy. brat and comical carcinomas. The most likely candidate regions are: 3~21 - a site of maming banozygous deletions in SCLC, the 3~14.2 !nmlocadon bmkpoint ssswiated with a form of hereditaryrenal cancer, and a 3~13 hcmmygous deletion ideatifii in the SCLC cell line, U2020. We have identifii overkppii 3~21 homozygous deletions in the SCLC cell liis. H740, GLCU) and X1450 end a telomexic deletion break&u cluster region (dim). Intemtiagly. this is also the site of a spmtmeous tra~slocationidentifii in the somatic call hybrid 2A3a. The bmkpoiot in the GLC20 dekticm has been cloned and conmved DNA sequemes identifii suggesdng the pnsence of a dbcr g=. At the 3~142 bmkpoint, we have identifii a me tmxript, HRCA-1. whose expression is absent in preliminary slwJie.sinvolvimghmor cell lines associaixd with frequent 3p MH. In cootrest. HRCA-I is expressed in tumorcell lines not usually a.wociated with 3p loss. Consistent with the possible involvement of HRCA-1 is a very high rate of homozygosity using adjacentmicrosatellite loci in SCLC cell lines. The U2Mo homoeygous dektion has been clmed in YACs and encompasses approximettly 8 Mb. HighIy polymxphic microsateUiteloci have demonstmkd selective LOH fox this ngion io other 1~ cancer cell lines. Evidence to date indiites that the critical region excludes the most centmmeric portion of the deL%icm.FurtherLOII studies are ongoing to better d&m the segment. In semanry. candidate tumor suppma gmes. poteatially involved in the deveIopmat or pmgmssion of long cancer. have been identifii at the sites of 3p rearrangements. Furthershxiies me required to confirm and assess the importance of these new genes.
FINE MAPPING OF NON-SMALL CELL LUNG CANCER LOCI ON CHROMOSOME 11~13 AND llp15.5. G. Bepler. Departmentsof Medicine and Molecular Cancer Biology,
Duke University Medical Center, Durham, North Carolina, U.S.A. We have mcently reported frequent loss of heterozygosity (LOH) on chromosome 1lp in human non-small cell lung cancer (NSCLC). LOH was found in 57% of tumors on band llp13,71% of tumors proximal on band llp15.5, and 45% of tumors distally on band 1lp15.5. Here, we report further line mapping of these regions using polymorphic markers mapped to 11~13and 11~15.5. Six markers (CAT, DllS325, WTI, DIlS323, DIlS16, and DllSI.51) were evaluated on 11~13. The smallest common area of deletion was between DllS323 and DIIS151, an area of approximately 2.5 Mb in size and clearly distal to the tumor suppressor gene WTI. Thit.__.. markers (STS, DIIS861, DlIS932, HBBp DllS12, DIlS860, DllS988, DlIS454, TH, INS, IGF2. HRAS, and DRD4) were studied on 11~15.5. LOH in the proximal region was confined to Dl IS12 and bordered by HBB and Dl lS86OM8. LOH in the distal region was conflned to HIRATand bordered by IGF2 and DRZM. It is estimated that the size of each of these regions is approximately2 Mb. Currently, studies are underway to assess if the ntalitmant boy ohenotvne of NSCLCcell lines can be reversed mine senetic comolememation’witb subcbrcnn~maI transfemblefragment “&aming DNA from the deleted regions.
naar Sj. Department of Pathology, University of Limburg, Maastricht, The Netherlands and Pulmonary and Critical Care Medicine, Atlanta VA Medical center, Decatur, GA, USA K-ras point mutations occur in adenocarcinomas of tbe lung. Recently, a rapid, sensitive and efficient method was described for determination of specific PCR products (EXACCT, Nucl Acid Res 21,; 3905, 1993). After exonuclease digestion, hybridisation with a capture and a detection primer and subsequent immunochemical staining of the detection primer PCR fragments are reliably determined. For the detection of mutations this procedure was extended using ligase and K-ras codon 12 mutation specific primers (Point-EKACCT). PCR for K-ras was performed using primers outside the codon 12 region. After exormclease digestion a biotin labeled primer containing the point mutation at the 3’ side and a digoxigenin labeled primer were hybridised with the single stranded DNA. DNA of HL60, A549 and CALUl cellines were used as control for wild type, mutation AGT and TGT on K-ras codon 12, respectively. PCR products of HL60 hybridised wild type primers showed a distinct staining. In contrast I-IL60 DNA hybridised with mutation primers did not give staining above the background values. PCR products of A549 hybridised with mutation specific primer yielded a distinct staining pattern. For CALUl similar results were obtained. This procedure can be performed in one day. Conclusion The Point EKACCT method is reliable and rapid. This procedure can be used to detect K-ras point mutations in DNA of tumors and non-malignant epithelial cells in patients at risk for lung cancer. l’hi~researchW&S funded in part by the Sdentitic Council of Smoking and Health, Tbe Netherlands.
072
073
ANALYSIS OF 3p TUMOR SUPPRESSOR LOCI IN HUMAN LUNG CANCER. R. Gemmill’, J. Rock&d. S. Todd’, W. Franklin’,F. B&log’, M. Swmm’. S. Nayld, K. Kdr’. Charles Boys’ and II. Drabkin’. %Jniversityof Colorsdo Cancer Center and the Eleanor Roosevelt I&tote. Denver, CO. USA, %e University of Texas. San Antonio. ‘The Univexsityof Gmiqen. The Netberlmds. 4UniversiteC. Bernard, Villewbame C&x. Framx Cytogaetic and loss of betemzygmity &OH) analyses fmm several laborator& suggest &at then may be 3-4 mar snppressorgenes located on 3p. Thesame3pngionshavellsobeenimplieatedinothermatignantdiseases in&ding kidmy. brat and comical carcinomas. The most likely candidate regions are: 3~21 - a site of maming banozygous deletions in SCLC, the 3~14.2 !nmlocadon bmkpoint ssswiated with a form of hereditaryrenal cancer, and a 3~13 hcmmygous deletion ideatifii in the SCLC cell line, U2020. We have identifii overkppii 3~21 homozygous deletions in the SCLC cell liis. H740, GLCU) and X1450 end a telomexic deletion break&u cluster region (dim). Intemtiagly. this is also the site of a spmtmeous tra~slocationidentifii in the somatic call hybrid 2A3a. The bmkpoiot in the GLC20 dekticm has been cloned and conmved DNA sequemes identifii suggesdng the pnsence of a dbcr g=. At the 3~142 bmkpoint, we have identifii a me tmxript, HRCA-1. whose expression is absent in preliminary slwJie.sinvolvimghmor cell lines associaixd with frequent 3p MH. In cootrest. HRCA-I is expressed in tumorcell lines not usually a.wociated with 3p loss. Consistent with the possible involvement of HRCA-1 is a very high rate of homozygosity using adjacentmicrosatellite loci in SCLC cell lines. The U2Mo homoeygous dektion has been clmed in YACs and encompasses approximettly 8 Mb. HighIy polymxphic microsateUiteloci have demonstmkd selective LOH fox this ngion io other 1~ cancer cell lines. Evidence to date indiites that the critical region excludes the most centmmeric portion of the deL%icm.FurtherLOII studies are ongoing to better d&m the segment. In semanry. candidate tumor suppma gmes. poteatially involved in the deveIopmat or pmgmssion of long cancer. have been identifii at the sites of 3p rearrangements. Furthershxiies me required to confirm and assess the importance of these new genes.
FINE MAPPING OF NON-SMALL CELL LUNG CANCER LOCI ON CHROMOSOME 11~13 AND llp15.5. G. Bepler. Departmentsof Medicine and Molecular Cancer Biology,
Duke University Medical Center, Durham, North Carolina, U.S.A. We have mcently reported frequent loss of heterozygosity (LOH) on chromosome 1lp in human non-small cell lung cancer (NSCLC). LOH was found in 57% of tumors on band llp13,71% of tumors proximal on band llp15.5, and 45% of tumors distally on band 1lp15.5. Here, we report further line mapping of these regions using polymorphic markers mapped to 11~13and 11~15.5. Six markers (CAT, DllS325, WTI, DIlS323, DIlS16, and DllSI.51) were evaluated on 11~13. The smallest common area of deletion was between DllS323 and DIIS151, an area of approximately 2.5 Mb in size and clearly distal to the tumor suppressor gene WTI. Thit.__.. markers (STS, DIIS861, DlIS932, HBBp DllS12, DIlS860, DllS988, DlIS454, TH, INS, IGF2. HRAS, and DRD4) were studied on 11~15.5. LOH in the proximal region was confined to Dl IS12 and bordered by HBB and Dl lS86OM8. LOH in the distal region was conflned to HIRATand bordered by IGF2 and DRZM. It is estimated that the size of each of these regions is approximately2 Mb. Currently, studies are underway to assess if the ntalitmant boy ohenotvne of NSCLCcell lines can be reversed mine senetic comolememation’witb subcbrcnn~maI transfemblefragment “&aming DNA from the deleted regions.