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Regulation of p53 protein expression in human cancer
Abstracts
NONRANDOM LOSS OF HUMAN CHROMOSOME 3 FRAGMENTS IN SCID TUMORS OF MOUSE/HUMAN MICROCELL HYBRIDS. A POTENTIAL METHOD FOR TUMOR SUPPRESSOR OENE ISOLATION? S.lmrgh , I.Khciodnyok, E.J.Stanbridge, E.R.Zabarovsky & G.Klein Dept. of Tumor Biology, Karolinska Inst. Box. 60400, S-171 77 Stockholm, Sweden, Fax.08-330498 Cytogenetic and moleculw (RFLP) studies have detected frequent deletions with or without tranelocatlon in the short arm of chromosome 3. The tumors include renal call, lung, naeopharynguel, thyroid, testlcular and breast carcinomas. Rearrangements or losses of hatorozygosis in 3q are often found in tumors of the hematopoiatlc system. A gene located in 3p25 is frequently rearranged or lost in yon Hlppel Llndau disease. A reciprocal t(3;B)(p14.2;p24.1} was closely linked to the renal cell carcinoma (RCC) in a high risk family described by Cohen at el (1979). It was reported (Shimizu st el, 1990, Satoh, 1993, Stenbrldge at el unpubl.) that chromosome 3 transfer via microcell fusion can retard the tumorigenicity of the YRC, A 5 4 9 and KRCY renal and lung carcinoma lines. Killory st el, (1992), reported tumor suppression in nude mouse tumors arising sfter the inoculation of A 9 microoail hybrids containing a fragment of 3p21. In the search for relevant suppressor genes located on 3p, we have generated Notl linking and jumping libraries and have mapped eight genes to 3p and two to 3q (Zaborovsky st el, 1993). At least 15 more clones showed a high percentage of DNA sequence homology with known rat and chicken genes. We tested a set of A9 microcell hybrids containing chromosomes 3, 3 del(p24-p141(q21-q26), 3 del(p22-p14), 3 del(p25-p21), as the only human chromosome, for tumorigenicity in SCID mice. FISH-painting and PCR analysis showed that the intact human chromosome was maintained in the case of 3 del(p24-p14)(q21-q26}, 3 del(p22-p14), 3 del(p25p21) but not when a whole chromosome 3 has been introduced. In such cases and also in the microcell hybrids that maintained the deleted chromosomes, multiple chimeric monse-human chromosomes were detected. These were generated by the translocetion of human chromosome segments to the terminal end of mouse chromosomes, as a rule. Interstitial translocations were only rarely seen. The chimeric chromosomes could be classified in 25 categories on the basis of the morphology of mouse recipient chromosome and the size of trsnslocatad chr 3 element. The retained human chromosome segments were identified (after FiSH-painting) by PCR analysis using 20 probes covering the entire chromosome 3. PCR markers have shown that the entire short arm was regularly eliminated in the tumors generated by intact chromosome 3 containing microcell hybrids. The possibilities of further restriction of putative tumor suppressor region is discussed.
REGULATION OF p53 PROTEIN EXPRESSION IN HUMAN CANCER Steven M. Pickslez, Ted R. Hupp, Xin Lu, Carol A. Midgley, Borivoj Vojtesek and David P. Lane; Department of Biochemistry, University of Dundee, Dundee DDI 4HN, U.K. Mutation of the p53 tumour suppressor is a common occurrence in a wide variety of human cancers, and is often, but not always, associated with elevated levels of p53 expression. The elevated levels of p53 expression are readily detected by antibodies such as CM] and DO-I, but are not detectable in normal tissue. Recent work would suggest that mutation per se is not sufficient to lead to high levels of p53 expression, but rather that the cellular environment plays a critical role in the regulation of p53 expression. This issue will be discussed in context with the mechanisms known to modulate p53 function.
15 3
A MAJOR SUSCEPTIBILITY LOCUS TO MURINE LUNG CARCINOGENESIS MAPS ON CHROMOSOME 6 M.A. P i e r o t t i , M. G a r i b o l d i , Falvella, G. D e l l a P o r t a , S .
G. M a n e n t i , F. C a n s ~ a n , F . S . Slnelli 1, & T.A. Dragani
Division of Experimental Ontology A, Ietituto Nazionale Tumori and IDepartment of Genetics and Microbiology, University of Milan, Milan, Italy In humans, the identification of possible predisposing genetic factors in the pathogenesis of lung tumors is difficult, because of their 10w penetranoe. We took advantage of murine strains, genetically susceptible and resistant to lung tumor development, to map murine genes associated with lung tumor development. An F2 population obtained by a cross from the lung tumor susceptible A/J strain and the resistant C3H/He strain was treated with a single dose of urethane and observed until 40 (males) or 65 (fQmales) weeks of age. Lung tumor susceptible phenotype was quantltatively evaluated in each mouse by the total lung tumor volume. To locate genes controlling lung tumor development, in 87 male mice we mapped 83 genetic markers dispersed over all autosomes. A chromosome 6 distal region, spanning 35 oentimorgans, accounted for up to 40% of the phenotypic variability of the character and, therefore, contained a major putative lung tumor susceptlbility locus. The best association between lung tumor susceptibility phenotype and genetic markers was found near Kras-2. No other chromosomal region was significantly associated with lung tumor development. The mapping of a locus responsible for the genetic susceptibility to lung carcinogenesis in the mouse chromosome 6 (near Krae-2) would suggest the opportunity to test genetic markers localized in the corresponding human chromosomal region (12p12) for posslble linkage with the lung adenooarclnoma development.
G E R M L I N E M U T A T I O N S IN T H E V O N H I P P E L - L I N D A U T U M O R S U P P R E S S O R G E N E A N D P R E C L I N I C A L D I A G N O S I S OF G E N E CARRIERS. Hiltrud Brauch O.Ma{ek, F. Pausch, M.I. Lerman, B. Zbar, H. H6fler. iLaboratory of Molecular Pathology, T e c h n i c a l U n i v e r s i t y Munich, F.R.G., 2 G e n e r a l H o s p i t a l Ilava, R.S., 3 L a b o r a t o r y of I m m u n o b i o l o g y , NCI-FCRDC, Frederick, M D 21702, U.S.A. von Hippel-Lindau disease {VHL) is an a u t o s o m a l dominant inheritable syndrome in which gene carriers are at r i s k to d e v e l o p m u l t i p l e organ tumors. The tumor suppressor gene causing VHL d i s e a s e maps to the r e g i o n 3p25-26 a n d was r e c e n t l y i d e n t i f i e d (Latif et el., S c i e n c e 1993). W e i d e n t i f i e d two s l o v a k i a n p e d i g r e e s in w h i c h V H L t u m o r s w e r e c l i n i c a l l y d i a g n o s e d in 4/29 and 2/18 family members. Since early detection of V H L d i s e a s e by p e r i o d i c a l c l i n i c a l e x a m i n a t i o n s is not a v a i l a b l e to non s y m p t o m a t i c o f f s p r i n g in S l o v a k i a we a i m e d to i d e n t i f y gene c a r r i e r s by m o l e c u l a r DNA analysis. All family members were screened by S o u t h e r n b l o t t i n g w i t h a p a r t i a l c D N A that d e t e c t s germline rearrangements in a b o u t 12% of V H L g e n e carriers. A n a b n o r m a l f r a g m e n t was i d e n t i f i e d in EcoRI and HindIII cut DNA of t h e 6 affected i n d i v i d u a l s i n d i c a t i n g a g e r m l i n e d e l e t i o n of about i0 k i l o b a s e s . The presence of t h i s abnormal f r a g m e n t in 5 n o n s y m p t o m a t i c o f f s p r i n g i d e n t i f i e d these individuals as V H L gene carriers. The a b n o r m a l fragment m i g r a t e d at the same d i s t a n c e in both families suggesting t h a t the V H L m u t a t i o n m i g h t be i d e n t i c a l a n d w a s introduced" by a c o m m o n ancestor. O u r p r e c l i n i c a l m o l e c u l a r d i a g n o s i s of VHL gene carriers will have an i m p a c t on t h e t h e r a p e u t i c r e g i m e n a n d w i l l h e l p to p r e v e n t these patients from blindness, neurological impairment a n d m e t a s t a t i c disease.
NONRANDOM LOSS OF HUMAN CHROMOSOME 3 FRAGMENTS IN SCID TUMORS OF MOUSE/HUMAN MICROCELL HYBRIDS. A POTENTIAL METHOD FOR TUMOR SUPPRESSOR OENE ISOLATION? S.lmrgh , I.Khciodnyok, E.J.Stanbridge, E.R.Zabarovsky & G.Klein Dept. of Tumor Biology, Karolinska Inst. Box. 60400, S-171 77 Stockholm, Sweden, Fax.08-330498 Cytogenetic and moleculw (RFLP) studies have detected frequent deletions with or without tranelocatlon in the short arm of chromosome 3. The tumors include renal call, lung, naeopharynguel, thyroid, testlcular and breast carcinomas. Rearrangements or losses of hatorozygosis in 3q are often found in tumors of the hematopoiatlc system. A gene located in 3p25 is frequently rearranged or lost in yon Hlppel Llndau disease. A reciprocal t(3;B)(p14.2;p24.1} was closely linked to the renal cell carcinoma (RCC) in a high risk family described by Cohen at el (1979). It was reported (Shimizu st el, 1990, Satoh, 1993, Stenbrldge at el unpubl.) that chromosome 3 transfer via microcell fusion can retard the tumorigenicity of the YRC, A 5 4 9 and KRCY renal and lung carcinoma lines. Killory st el, (1992), reported tumor suppression in nude mouse tumors arising sfter the inoculation of A 9 microoail hybrids containing a fragment of 3p21. In the search for relevant suppressor genes located on 3p, we have generated Notl linking and jumping libraries and have mapped eight genes to 3p and two to 3q (Zaborovsky st el, 1993). At least 15 more clones showed a high percentage of DNA sequence homology with known rat and chicken genes. We tested a set of A9 microcell hybrids containing chromosomes 3, 3 del(p24-p141(q21-q26), 3 del(p22-p14), 3 del(p25-p21), as the only human chromosome, for tumorigenicity in SCID mice. FISH-painting and PCR analysis showed that the intact human chromosome was maintained in the case of 3 del(p24-p14)(q21-q26}, 3 del(p22-p14), 3 del(p25p21) but not when a whole chromosome 3 has been introduced. In such cases and also in the microcell hybrids that maintained the deleted chromosomes, multiple chimeric monse-human chromosomes were detected. These were generated by the translocetion of human chromosome segments to the terminal end of mouse chromosomes, as a rule. Interstitial translocations were only rarely seen. The chimeric chromosomes could be classified in 25 categories on the basis of the morphology of mouse recipient chromosome and the size of trsnslocatad chr 3 element. The retained human chromosome segments were identified (after FiSH-painting) by PCR analysis using 20 probes covering the entire chromosome 3. PCR markers have shown that the entire short arm was regularly eliminated in the tumors generated by intact chromosome 3 containing microcell hybrids. The possibilities of further restriction of putative tumor suppressor region is discussed.
REGULATION OF p53 PROTEIN EXPRESSION IN HUMAN CANCER Steven M. Pickslez, Ted R. Hupp, Xin Lu, Carol A. Midgley, Borivoj Vojtesek and David P. Lane; Department of Biochemistry, University of Dundee, Dundee DDI 4HN, U.K. Mutation of the p53 tumour suppressor is a common occurrence in a wide variety of human cancers, and is often, but not always, associated with elevated levels of p53 expression. The elevated levels of p53 expression are readily detected by antibodies such as CM] and DO-I, but are not detectable in normal tissue. Recent work would suggest that mutation per se is not sufficient to lead to high levels of p53 expression, but rather that the cellular environment plays a critical role in the regulation of p53 expression. This issue will be discussed in context with the mechanisms known to modulate p53 function.
15 3
A MAJOR SUSCEPTIBILITY LOCUS TO MURINE LUNG CARCINOGENESIS MAPS ON CHROMOSOME 6 M.A. P i e r o t t i , M. G a r i b o l d i , Falvella, G. D e l l a P o r t a , S .
G. M a n e n t i , F. C a n s ~ a n , F . S . Slnelli 1, & T.A. Dragani
Division of Experimental Ontology A, Ietituto Nazionale Tumori and IDepartment of Genetics and Microbiology, University of Milan, Milan, Italy In humans, the identification of possible predisposing genetic factors in the pathogenesis of lung tumors is difficult, because of their 10w penetranoe. We took advantage of murine strains, genetically susceptible and resistant to lung tumor development, to map murine genes associated with lung tumor development. An F2 population obtained by a cross from the lung tumor susceptible A/J strain and the resistant C3H/He strain was treated with a single dose of urethane and observed until 40 (males) or 65 (fQmales) weeks of age. Lung tumor susceptible phenotype was quantltatively evaluated in each mouse by the total lung tumor volume. To locate genes controlling lung tumor development, in 87 male mice we mapped 83 genetic markers dispersed over all autosomes. A chromosome 6 distal region, spanning 35 oentimorgans, accounted for up to 40% of the phenotypic variability of the character and, therefore, contained a major putative lung tumor susceptlbility locus. The best association between lung tumor susceptibility phenotype and genetic markers was found near Kras-2. No other chromosomal region was significantly associated with lung tumor development. The mapping of a locus responsible for the genetic susceptibility to lung carcinogenesis in the mouse chromosome 6 (near Krae-2) would suggest the opportunity to test genetic markers localized in the corresponding human chromosomal region (12p12) for posslble linkage with the lung adenooarclnoma development.
G E R M L I N E M U T A T I O N S IN T H E V O N H I P P E L - L I N D A U T U M O R S U P P R E S S O R G E N E A N D P R E C L I N I C A L D I A G N O S I S OF G E N E CARRIERS. Hiltrud Brauch O.Ma{ek, F. Pausch, M.I. Lerman, B. Zbar, H. H6fler. iLaboratory of Molecular Pathology, T e c h n i c a l U n i v e r s i t y Munich, F.R.G., 2 G e n e r a l H o s p i t a l Ilava, R.S., 3 L a b o r a t o r y of I m m u n o b i o l o g y , NCI-FCRDC, Frederick, M D 21702, U.S.A. von Hippel-Lindau disease {VHL) is an a u t o s o m a l dominant inheritable syndrome in which gene carriers are at r i s k to d e v e l o p m u l t i p l e organ tumors. The tumor suppressor gene causing VHL d i s e a s e maps to the r e g i o n 3p25-26 a n d was r e c e n t l y i d e n t i f i e d (Latif et el., S c i e n c e 1993). W e i d e n t i f i e d two s l o v a k i a n p e d i g r e e s in w h i c h V H L t u m o r s w e r e c l i n i c a l l y d i a g n o s e d in 4/29 and 2/18 family members. Since early detection of V H L d i s e a s e by p e r i o d i c a l c l i n i c a l e x a m i n a t i o n s is not a v a i l a b l e to non s y m p t o m a t i c o f f s p r i n g in S l o v a k i a we a i m e d to i d e n t i f y gene c a r r i e r s by m o l e c u l a r DNA analysis. All family members were screened by S o u t h e r n b l o t t i n g w i t h a p a r t i a l c D N A that d e t e c t s germline rearrangements in a b o u t 12% of V H L g e n e carriers. A n a b n o r m a l f r a g m e n t was i d e n t i f i e d in EcoRI and HindIII cut DNA of t h e 6 affected i n d i v i d u a l s i n d i c a t i n g a g e r m l i n e d e l e t i o n of about i0 k i l o b a s e s . The presence of t h i s abnormal f r a g m e n t in 5 n o n s y m p t o m a t i c o f f s p r i n g i d e n t i f i e d these individuals as V H L gene carriers. The a b n o r m a l fragment m i g r a t e d at the same d i s t a n c e in both families suggesting t h a t the V H L m u t a t i o n m i g h t be i d e n t i c a l a n d w a s introduced" by a c o m m o n ancestor. O u r p r e c l i n i c a l m o l e c u l a r d i a g n o s i s of VHL gene carriers will have an i m p a c t on t h e t h e r a p e u t i c r e g i m e n a n d w i l l h e l p to p r e v e n t these patients from blindness, neurological impairment a n d m e t a s t a t i c disease.