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Non-radioactive detection and identification of p53 mutations in fixed and frozen tissues from human cancers
Abstracts
16 7
N O N - R A D I O A C T I V E D E T E C T I O N A N D I D E N T I F I C A T I O N OF P53 M U T A T I O N S IN F I X E D AND F R O Z E N T I S S U E S F R O M H U M A N CANCERS. D o l o r e s Bautista, P a s c a l Chaubert, Luc Bron and Jean B e n h a t t a r , I n s t i t u t U n i v e r s i taire de P a t h o l o g i e , B u g n o n 25, C H - 1 0 1 1 Lausanne, Switzerland.
DETECTION OF THE EPISOMAL, LINEAR, AND INTEGRATED EPSTEIN-BARR VIRUS GENOME IN HUMAN INFECTED CELLS Valia S. Lestou. Hehnut Gadner, Peter F. Ambros; CCRI, St. Anna Children's Hospital, Kinderspitalgasse 6, A-1090 Vienna
We have d e v e l o F e d non-radioactive visualization t e c h n i q u e s of SSCP (Single S t r a n d C o n f o r m a t i o n P o l y m o r p h i s m ) and d i r e c t s e q u e n c i n g for safe, r a p i d and s e n s i t i v e a n a l y s i s of p53 gene in fixed and frozen t i s s u e s from h u m a n cancers. Exons 5 to 9 of the p53 gene were PCR a m p l i f i e d u s i ~ p r i m e r - p a i r s s p e c i f i c for each exon. In o r d e r to d e t e c t g e n e t i c a l t e r a t i o n s w i t h i n the p53 gene, we p e r f o r m e d a s c r e e n i n g p r o c e d u r e by a n o n - r a d i o a c t i v e v a r i a n t of the SSCP u s i n g a s t a n d a r d s i l v e r - s t a i n i n g d e t e c t i o n technique. The c a s e s w h i c h s h o w e d an a l t e r e d m o b i l i t y p a t t e r n by SSCP were d i r e c t l y s e q u e n c e d as d o u b l e s t r a n d e d PCR product, u s i n g b i o t i n l a b e l e d primers. The s e q u e n c i n g images were v i s u a l i z e d by both a c h e m i l u m i n e s c e n t and a color reactions. This s t r a t e g y was successEully a p p l i e d for the d e t e c t i o n and i d e n t i f i c a t i o n of p53 m u t a t i o n s on h u m a n breast, colon, g a s t r i c and head and neck cancers. This p r o t o c o l allows the study of tumors' series in a few days.
The present study was performed to clarify the reported inconsistencies regarding the Epstein-Barr virus (EBV) genome status in infected cells and to develop criteria for biological and possibly clinical interpretation of viral infection processes in human cells. We used qualitative and quantitative methods for detection of the EBV-genome on chromosomes, interphase nuclei, and spread DNA of LCL, BL, IM, and HD. The following methods were used: (i) non isotopic in situ hybridization on cytopreparations (NISH), (ii) fluorecence in situ hybridization on chromosomes and interphase nuclei (FISH), and (iii) Southern blotting techniques. To compare the results with molecular biological data, we performed Southern blot analysis with two 32p-labeled terminal EBV fragments, termed Xhola and EcoRI 1 on differend cell lines analyzed with ISH techniques. Furthermore these two fragments were also used as markers of clonal cell proliferation. For NISH, and FISH we used several biotinylated and/or digoxigenated labeled EBV-DNA fragments. We observed with the ISH technique from one up to hundred EBV signals per cell, and two qualitatively different sized hybridization signals, reflecting the two modes of EBV infection: Small, sharply defined signals often occuring in duplicate could be intepretated as integrated viral forms whereas, the diffuse and large signal type most likely represents the episomal form. A fine mapping of the integrated viral genome will be also reported. We believe that the EBV genome status within the infected cells may have a biological significance.
VISUALIZATION OF cHROMOSO~ NOI~ AND NEOPLASTIC CELLS SITU HYBRIDIZATION METHODS.
L08S 0P CONSTITUTIONAL EETEROZYGOSITY IN A BECKNITE NIEDEMANN SYNDROME PATIENT OI~PLAYING A WILNS' TUMOUR AND NEPEROGENIC RESTS. Paul R, Ruben, Jim Eeighva?, Jill Birch, Bert Baker, P ~ Morris Jones and Anna M, Kelsay, Dept. of Pathology. RoyalManchester Children's Hospital, Peudlehury, Manchester, M27 IRA.
BY
SUBREGIONS PCR-MEDIATED
IN IN
P.J. K r i h t e n b u r q , J.C. Alers, and H. v a n Dekken, Dept. of Pathology, E r a s m u s U n i v e r s i t y R o t t e r d a m , dr. M o l e w a t e r p l e i n 50, 3000 D R R o t t e r d a m , The Netherlands. W e h a v e u s e d the p o l y m e r a s e c h a i n r e a c t i o n (PER) w i t h p r i m e r s for i n t e r s p e r s e d s e q u e n c e s (interalu-PCR) and r a n d o m s e q u e n c e m o t i f s (DOP-PCR) to a m p l i f y h u m a n D N A i n s e r t s of y e a s t a r t i f i c i a l chromosomes (YAC). The reaction product was b i o t i n - l a b e l e d a n d u s e d for in situ h y b r i d i z a t i o n (ISH) to m e t a p h a s e and interphase cells. Also direct labeling was applied by adding biotinylated deo~nucleotides to the PeR reaction. H y b r i d i z a t i o n s i g n a l s w e r e c o m p a r a b l e , but d i r e c t l y l a b e l e d p r o b e s s h o w e d m u c h h i g h e r aspecific hybridization due to long probe fra~ents. For removal of this phenomenon additional DNase treatment was needed. F l u o r e s c e n t ISH on n o r m a l b l o o d l y m p h o c y t e s w i t h c l o n e d YAC D N A ' s for the 6p21 and 17p13 r e g i o n s was compared with the inter-alu-PCR products. The inter-alu-PCR mediated probes yielded signifantly s t r o n g e r s i g n a l s on m e t a p h a s e c h r o m o s o m e s and in i n t e r p h a s e nuclei. T h e 17p13 p r o b e w a s also u s e d for d e l e t i o n a n a l y s i s of a p r o s t a t i c c a n c e r cell line (I_~CaP). ISH w i t h the D O P - P C R p r o d u c t of a p u l s e d field gel i s o l a t e d YAC clone, h a r b o u r i n g . a 80 kb insert of t h e 8p region, resulted in s i g n a l s on m e t a p h a s e c h r o m o s o m e s of H L r O l e u k e m i a c e l l s t h a t c o u l d n o t be d e t e c t e d by i n t e r - a l u PeR. C u r r e n t l y t h e s u i t a b i l i t y of t h e s e m e t h o d s for i n t e r f a s e a n a l y s i s of HL60 cells, L N C a P c e l l s a n d h i s t o l o g i c t u m o r s e c t i o n s is i n v e s t i g a t e d .
an 11 menth 01d baby girl was shown to have Beckwith Wiedeesnn Syndrome {BWS).Thissyndromehas been linked to chr0n0n0me11p15.5.and prodlsp0sns to rare e~ry0nal tumenrs includingWiles' tumenr, hnpat0blast0esand rhabdoayosarcom. In the reportedcase the kidneydisplaysa Wiles'tn~Jur, nephrogenic rests and a subcapsnlar nodule of tulour with residual nephrogenicrests at the periphery.It has been suggestedthat nephrogenlc rests ere lesions fromwhich tu~urs my develop.We aimedto address this question by screening for genetic changes at the .nlecular level in nephrogenic rest, noresl kidney end tunour. RFLP analysishas performed on ONA extracted fromtnJmur, nephrogenicrest and norml kidney.Using PCR based polpmrphises, loss of constitutional heterozygoait? (LDCE) has demonstrated from mrkers along the length of chromosome ii, He-ras {llpl5.5), IGPII (llpiS.5),DllS325(llpl3}and PRADI (llql3} suggesting cn,pletnloss of the maternal chrolosomeII in the tnmeur and nephrogenic rest. Partial LOCH in the form of allele imbalancesat eachof theseloci was delonstrated in noresl kidney suggesting genetic mosaicism of chromosome II in the norml kidneytissue of this patient.As nephrogenic rests are thought to represent e visible manifestation of the first metational event in tumeur development,the LOCH chromesomeII sequences my he the primary event in tomerigenesis. Tumenr development however, alesst certainlyrequiresa second(or severalother}geneticevent.
16 7
N O N - R A D I O A C T I V E D E T E C T I O N A N D I D E N T I F I C A T I O N OF P53 M U T A T I O N S IN F I X E D AND F R O Z E N T I S S U E S F R O M H U M A N CANCERS. D o l o r e s Bautista, P a s c a l Chaubert, Luc Bron and Jean B e n h a t t a r , I n s t i t u t U n i v e r s i taire de P a t h o l o g i e , B u g n o n 25, C H - 1 0 1 1 Lausanne, Switzerland.
DETECTION OF THE EPISOMAL, LINEAR, AND INTEGRATED EPSTEIN-BARR VIRUS GENOME IN HUMAN INFECTED CELLS Valia S. Lestou. Hehnut Gadner, Peter F. Ambros; CCRI, St. Anna Children's Hospital, Kinderspitalgasse 6, A-1090 Vienna
We have d e v e l o F e d non-radioactive visualization t e c h n i q u e s of SSCP (Single S t r a n d C o n f o r m a t i o n P o l y m o r p h i s m ) and d i r e c t s e q u e n c i n g for safe, r a p i d and s e n s i t i v e a n a l y s i s of p53 gene in fixed and frozen t i s s u e s from h u m a n cancers. Exons 5 to 9 of the p53 gene were PCR a m p l i f i e d u s i ~ p r i m e r - p a i r s s p e c i f i c for each exon. In o r d e r to d e t e c t g e n e t i c a l t e r a t i o n s w i t h i n the p53 gene, we p e r f o r m e d a s c r e e n i n g p r o c e d u r e by a n o n - r a d i o a c t i v e v a r i a n t of the SSCP u s i n g a s t a n d a r d s i l v e r - s t a i n i n g d e t e c t i o n technique. The c a s e s w h i c h s h o w e d an a l t e r e d m o b i l i t y p a t t e r n by SSCP were d i r e c t l y s e q u e n c e d as d o u b l e s t r a n d e d PCR product, u s i n g b i o t i n l a b e l e d primers. The s e q u e n c i n g images were v i s u a l i z e d by both a c h e m i l u m i n e s c e n t and a color reactions. This s t r a t e g y was successEully a p p l i e d for the d e t e c t i o n and i d e n t i f i c a t i o n of p53 m u t a t i o n s on h u m a n breast, colon, g a s t r i c and head and neck cancers. This p r o t o c o l allows the study of tumors' series in a few days.
The present study was performed to clarify the reported inconsistencies regarding the Epstein-Barr virus (EBV) genome status in infected cells and to develop criteria for biological and possibly clinical interpretation of viral infection processes in human cells. We used qualitative and quantitative methods for detection of the EBV-genome on chromosomes, interphase nuclei, and spread DNA of LCL, BL, IM, and HD. The following methods were used: (i) non isotopic in situ hybridization on cytopreparations (NISH), (ii) fluorecence in situ hybridization on chromosomes and interphase nuclei (FISH), and (iii) Southern blotting techniques. To compare the results with molecular biological data, we performed Southern blot analysis with two 32p-labeled terminal EBV fragments, termed Xhola and EcoRI 1 on differend cell lines analyzed with ISH techniques. Furthermore these two fragments were also used as markers of clonal cell proliferation. For NISH, and FISH we used several biotinylated and/or digoxigenated labeled EBV-DNA fragments. We observed with the ISH technique from one up to hundred EBV signals per cell, and two qualitatively different sized hybridization signals, reflecting the two modes of EBV infection: Small, sharply defined signals often occuring in duplicate could be intepretated as integrated viral forms whereas, the diffuse and large signal type most likely represents the episomal form. A fine mapping of the integrated viral genome will be also reported. We believe that the EBV genome status within the infected cells may have a biological significance.
VISUALIZATION OF cHROMOSO~ NOI~ AND NEOPLASTIC CELLS SITU HYBRIDIZATION METHODS.
L08S 0P CONSTITUTIONAL EETEROZYGOSITY IN A BECKNITE NIEDEMANN SYNDROME PATIENT OI~PLAYING A WILNS' TUMOUR AND NEPEROGENIC RESTS. Paul R, Ruben, Jim Eeighva?, Jill Birch, Bert Baker, P ~ Morris Jones and Anna M, Kelsay, Dept. of Pathology. RoyalManchester Children's Hospital, Peudlehury, Manchester, M27 IRA.
BY
SUBREGIONS PCR-MEDIATED
IN IN
P.J. K r i h t e n b u r q , J.C. Alers, and H. v a n Dekken, Dept. of Pathology, E r a s m u s U n i v e r s i t y R o t t e r d a m , dr. M o l e w a t e r p l e i n 50, 3000 D R R o t t e r d a m , The Netherlands. W e h a v e u s e d the p o l y m e r a s e c h a i n r e a c t i o n (PER) w i t h p r i m e r s for i n t e r s p e r s e d s e q u e n c e s (interalu-PCR) and r a n d o m s e q u e n c e m o t i f s (DOP-PCR) to a m p l i f y h u m a n D N A i n s e r t s of y e a s t a r t i f i c i a l chromosomes (YAC). The reaction product was b i o t i n - l a b e l e d a n d u s e d for in situ h y b r i d i z a t i o n (ISH) to m e t a p h a s e and interphase cells. Also direct labeling was applied by adding biotinylated deo~nucleotides to the PeR reaction. H y b r i d i z a t i o n s i g n a l s w e r e c o m p a r a b l e , but d i r e c t l y l a b e l e d p r o b e s s h o w e d m u c h h i g h e r aspecific hybridization due to long probe fra~ents. For removal of this phenomenon additional DNase treatment was needed. F l u o r e s c e n t ISH on n o r m a l b l o o d l y m p h o c y t e s w i t h c l o n e d YAC D N A ' s for the 6p21 and 17p13 r e g i o n s was compared with the inter-alu-PCR products. The inter-alu-PCR mediated probes yielded signifantly s t r o n g e r s i g n a l s on m e t a p h a s e c h r o m o s o m e s and in i n t e r p h a s e nuclei. T h e 17p13 p r o b e w a s also u s e d for d e l e t i o n a n a l y s i s of a p r o s t a t i c c a n c e r cell line (I_~CaP). ISH w i t h the D O P - P C R p r o d u c t of a p u l s e d field gel i s o l a t e d YAC clone, h a r b o u r i n g . a 80 kb insert of t h e 8p region, resulted in s i g n a l s on m e t a p h a s e c h r o m o s o m e s of H L r O l e u k e m i a c e l l s t h a t c o u l d n o t be d e t e c t e d by i n t e r - a l u PeR. C u r r e n t l y t h e s u i t a b i l i t y of t h e s e m e t h o d s for i n t e r f a s e a n a l y s i s of HL60 cells, L N C a P c e l l s a n d h i s t o l o g i c t u m o r s e c t i o n s is i n v e s t i g a t e d .
an 11 menth 01d baby girl was shown to have Beckwith Wiedeesnn Syndrome {BWS).Thissyndromehas been linked to chr0n0n0me11p15.5.and prodlsp0sns to rare e~ry0nal tumenrs includingWiles' tumenr, hnpat0blast0esand rhabdoayosarcom. In the reportedcase the kidneydisplaysa Wiles'tn~Jur, nephrogenic rests and a subcapsnlar nodule of tulour with residual nephrogenicrests at the periphery.It has been suggestedthat nephrogenlc rests ere lesions fromwhich tu~urs my develop.We aimedto address this question by screening for genetic changes at the .nlecular level in nephrogenic rest, noresl kidney end tunour. RFLP analysishas performed on ONA extracted fromtnJmur, nephrogenicrest and norml kidney.Using PCR based polpmrphises, loss of constitutional heterozygoait? (LDCE) has demonstrated from mrkers along the length of chromosome ii, He-ras {llpl5.5), IGPII (llpiS.5),DllS325(llpl3}and PRADI (llql3} suggesting cn,pletnloss of the maternal chrolosomeII in the tnmeur and nephrogenic rest. Partial LOCH in the form of allele imbalancesat eachof theseloci was delonstrated in noresl kidney suggesting genetic mosaicism of chromosome II in the norml kidneytissue of this patient.As nephrogenic rests are thought to represent e visible manifestation of the first metational event in tumeur development,the LOCH chromesomeII sequences my he the primary event in tomerigenesis. Tumenr development however, alesst certainlyrequiresa second(or severalother}geneticevent.