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vHNF-1 ratio is strongly linked to histological differentiation of hepatocellular carcinoma and hepatoblastoma
Posters / International Hepatology Communications 3 Suppl. (1995) $37-S169
P-424
~ E N E n C ALTERATION OF P16 (MTS1) AND
CYCLIN E IN HCC AND CCC R.Kita, N.Nishida, Y.Matsuoka, T.Komeda, T.Sando, M.Furukawa, Y.Fukuda, K. Nakao] mid K.Ishizakiz Dep.Med.Clin.Sci.Kyoto.Univl, Dep.Rad.Aichi.Cancer.Centerz Recently the association of cell cycle regulatory genes with the carcinogenesis of several tumors has been spotlighted . We have previously studied about cyclinD1, CDK4 and CDK4-inhibitor (MTS1/pl6) and have shown the amplification of the cyclinD1 gene in 11% of HCC, the absence of amplification of CDK4, and deletion mcl mutation of pl6 in some eases of HCC. Herein, we further examined about pl6 and newly examined about cyclinE.(Method) Fiftysix HCCs, 5 CCCs, and 6 cell lines derived from hopatocyte were examined. All samples were examined for exonl and exon2 of pl6 by PCR and PCR-SSCP and some cases presenting abnormal bands were studied by sequence analysis. For cyclinE, genomic DNA was digested with EcoRI and was studied by Southern blot hybridization. (Resul0 The expected products for exonl of pl6 were detected in all samples except one case by PCR and no abnormal band could be detected by SSCP. For exon2, all samples showed the expected size of product by PCR, but 3 samples showed abnormal bands by SSCP. These 3 samples showed point-mutations leading to amino acid transition in cedon 75 or 94. Neither gene amplification nor rearrangement could be detected for cyclinE in evaluable samples. (Conclusion) Not only cyclinD1 but also pl6(MTSI) may play a role in carcinogenesis of HCC.
P-426
C Y C L I N A EXPRESSION IN HEPATOCELLULAR CARCINOMA AND POSSIBLE MECHANISM OF ITS OVEREXPRESSION N. Masaki, L Ogata, K. Kurokawaj), Y. Bandai, M. Makuuchi2), K. Fujiwara3) 1)lst Dept. of Internal Medicine and 2)2rid Dept. of Surgery, Univ. of Tokyo, Tokyo, 3)3rd Dept. of Internal Medicine, Saitama Medical School, Saitama, Japan
Cyclin A, one of cell cycle regulatory proteins, has been reported to be overexpressed in a variety of malignancy. As for hepatocellular carcinomas (HCC), however, little is known about this issue, except a report that one hepatitis B virus (HBV)-related HCC stably expressed undegradable cyclin A protein due to integration of HBV-DNA into cyclin A gene. It has also been reported that cyclin A gene possesses a tumor-suppressor gene product p53-binding consensus sequence in its promotor region. Under these circumstances, we attempted to study cyc]in A expression in HCCs of various etiology and to explore the mechanism of its overexpression in relation to p53. METHOD I. Total RNA was extracted from nontumorous liver tissues and cancer tissues of 14 patients with HCC (1 non B-non C, 1 HBV and 12 HCV-related). Cyclin A mRNA expression was analyzed by Northern blot hybridization. II. Gel shift assay was performed to evaluate the binding between a synthetic oligonucleotide based on the aucleotide sequence of cyclin A promotor region containing a p53-binding consensus sequence and nuclear extracts obtained flom cancer tissues by the method of Gorski K et al.. RESULTS L Cyclin A mRNA was not detected in any non-tumorous tissues, while it was found in 6 out of 14 HCCs (well-, moderately-, poorly-differentiated: 1/3, 3/7, 2/4, respectively). II. In HCC with cyclin A mRNA overexpression, the binding was completely lost, whereas in HCC withoul its expression the binding was present and attenuated by treatment with an anti-p53 monoclonal antibody. CONCLUSION Some HCCs overexpressed cyclin A. Loss of transcriptional regulation by p53 might cause this phenomenon.
$143
T'S A ~ q b ~
J[~-~'t~,~ EXPRESSION OF HNF-1/vHNF-1 RATIO IS STRONGLY LINKED TO HISTOLOGICAL DIFFERENTIATION OF HEPATOCELLULAR CARCINOMA AND HEPATOBLASTOMA *T.Ninomiya,**Y.Hayashi,**K.Ohta,#K.Saijoh,*Y.Seo,*M.Sugano *S.Yoon, **H.Itoh,*M.Kasuga *2nd Dep. of Medicine and **lst Dep.of Pathology , Kobe Univ. of Medicine, Kobe, Japan, #Dep. of Hygiene, Kanazawa Univ. of Medicine, Kanazawa, Japan Various liver-specific protein genes have multiple cis- and trans-acting elements, yet the elements accountable for hepatocytic differentiation are unclear. We have previously shown that the hepatic transcription of human albumin and o~-fetoprotein (AFP) genes is controlled by a complex set of regulatory elements, and that an AT-rich core sequence (AT motif) is essential as a cis-acting element for the hepatic transcription. Homologous proteins hepatocyte nuclear factor- 1 (HNF- 1), variant HNF- 1 (vHNF- 1), and AT motif-binding factor 1 (ATBF1) bind to the AT motif. To clarify the role of these three different trans-acting factors, we examined the mRNA and the expression ratio of the HNF-I/vHNF-1 mRNA in various liver tissues. Our competitive reverse transcriptional polymerase chain reaction (RT-PCR) method demonstrates that this ratio is linked to their histological differentiation, and that it is higher in well-differentiated hepatocellular carcinoma (HCC) than in poorly-differentiated and undifferentiated HCCs; non-neoplastic liver tissues, however, have a low expression ratio of HNF-I/ vHNF- 1 mRNA. This is the first study that indicates the importance of the HNF-t/vHNF-1 mRNA ratio in determining differentiation of hepatocytic neoplasms and suggests that measuring the ratio would be a useful diagnostic method.
P-427
RESTRICTION LANDMARK GENOME SCANNING ANALYSIS OF C H R O M O S O M A L D N A IN HEPATOCELLULAR CARCINOMA T. H a n a f u s a 1), Y. Y u m o t o 1), H. H a d a 2l, T. S h i n j i 2), T. M o r i t a z~, H. S h i r a h a x), T. T a m u r a x~, Y. I w a s a k i 2~, N. K o i d e 2), H. N a g a i s), K. M a t s u b a r a a), a n d T. Tsuji el 1)Isotope Center, OkayamaUniv., Okayama, Japan. 2)1st Dept. of Medicine, Okayama Univ. Medical School, Okayam a , J a p a n . 3) I n s t . f o r Mol. a n d Cell. Biol., O s a k a U n i v . , Osaka, Japan Restriction landmark genome scanning (RLGS) analysis was applied to detect chromosomal aberrations i n h e p a t o c e l l u l a r c a r c i n o m a (HCC). V a r i o u s t y p e s o f HO2 s a m p l e s , i n c l u d i n g t w o HCCs w i t h m u l t i - n o d u l a r t y p e , o n e HCC s h o w i n g n o d u l e i n n o d u l e p a t t e r n , o n e m e t a s t a s i z e d t o d i f f e r e n t t i s s u e s , t h r e e s m a l l (<2.0 c m ) HCCs, a n d f o u r l a r g e (>5.0 c m ) HCCs, w e r e e x a m i n e d b y RLGS m e t h o d w i t h c o r r e s p o n d i n g non-HCC tissues. Res t r i c t i o n e n z y m e Not I w a s u s e d a s a l a n d m a r k e n z y m e , Eco RV w a s u s e d as a f r a g m e n t a t i o n e n z y m e , a n d HJn f I was used as a digestion enzyme in gel for two dimensional electorophoresis. We observed spot aberrations b e t w e e n HCCs f r o m d i f f e r e n t n o d u l e s , b e t w e e n m e t a s t a s i z e d a n d o r i g i n a l HCC. S o m e s p e c i f i c s p o t a b e r r a t i o n s i n HCCs w h i c h h a v e n o m u t a t i o n in p53 and Rb genes w e r e i d e n t i f i e d . W e s h o w e d RLGS m e t h o d is a n e f f e c t i v e tool for identifying the chromosomal aberrations in HCC, a n d s u g g e s t e d s o m e a s p e c t s o f HCC d e v e l o p m e n t w i l t be revealed by this method.
P-424
~ E N E n C ALTERATION OF P16 (MTS1) AND
CYCLIN E IN HCC AND CCC R.Kita, N.Nishida, Y.Matsuoka, T.Komeda, T.Sando, M.Furukawa, Y.Fukuda, K. Nakao] mid K.Ishizakiz Dep.Med.Clin.Sci.Kyoto.Univl, Dep.Rad.Aichi.Cancer.Centerz Recently the association of cell cycle regulatory genes with the carcinogenesis of several tumors has been spotlighted . We have previously studied about cyclinD1, CDK4 and CDK4-inhibitor (MTS1/pl6) and have shown the amplification of the cyclinD1 gene in 11% of HCC, the absence of amplification of CDK4, and deletion mcl mutation of pl6 in some eases of HCC. Herein, we further examined about pl6 and newly examined about cyclinE.(Method) Fiftysix HCCs, 5 CCCs, and 6 cell lines derived from hopatocyte were examined. All samples were examined for exonl and exon2 of pl6 by PCR and PCR-SSCP and some cases presenting abnormal bands were studied by sequence analysis. For cyclinE, genomic DNA was digested with EcoRI and was studied by Southern blot hybridization. (Resul0 The expected products for exonl of pl6 were detected in all samples except one case by PCR and no abnormal band could be detected by SSCP. For exon2, all samples showed the expected size of product by PCR, but 3 samples showed abnormal bands by SSCP. These 3 samples showed point-mutations leading to amino acid transition in cedon 75 or 94. Neither gene amplification nor rearrangement could be detected for cyclinE in evaluable samples. (Conclusion) Not only cyclinD1 but also pl6(MTSI) may play a role in carcinogenesis of HCC.
P-426
C Y C L I N A EXPRESSION IN HEPATOCELLULAR CARCINOMA AND POSSIBLE MECHANISM OF ITS OVEREXPRESSION N. Masaki, L Ogata, K. Kurokawaj), Y. Bandai, M. Makuuchi2), K. Fujiwara3) 1)lst Dept. of Internal Medicine and 2)2rid Dept. of Surgery, Univ. of Tokyo, Tokyo, 3)3rd Dept. of Internal Medicine, Saitama Medical School, Saitama, Japan
Cyclin A, one of cell cycle regulatory proteins, has been reported to be overexpressed in a variety of malignancy. As for hepatocellular carcinomas (HCC), however, little is known about this issue, except a report that one hepatitis B virus (HBV)-related HCC stably expressed undegradable cyclin A protein due to integration of HBV-DNA into cyclin A gene. It has also been reported that cyclin A gene possesses a tumor-suppressor gene product p53-binding consensus sequence in its promotor region. Under these circumstances, we attempted to study cyc]in A expression in HCCs of various etiology and to explore the mechanism of its overexpression in relation to p53. METHOD I. Total RNA was extracted from nontumorous liver tissues and cancer tissues of 14 patients with HCC (1 non B-non C, 1 HBV and 12 HCV-related). Cyclin A mRNA expression was analyzed by Northern blot hybridization. II. Gel shift assay was performed to evaluate the binding between a synthetic oligonucleotide based on the aucleotide sequence of cyclin A promotor region containing a p53-binding consensus sequence and nuclear extracts obtained flom cancer tissues by the method of Gorski K et al.. RESULTS L Cyclin A mRNA was not detected in any non-tumorous tissues, while it was found in 6 out of 14 HCCs (well-, moderately-, poorly-differentiated: 1/3, 3/7, 2/4, respectively). II. In HCC with cyclin A mRNA overexpression, the binding was completely lost, whereas in HCC withoul its expression the binding was present and attenuated by treatment with an anti-p53 monoclonal antibody. CONCLUSION Some HCCs overexpressed cyclin A. Loss of transcriptional regulation by p53 might cause this phenomenon.
$143
T'S A ~ q b ~
J[~-~'t~,~ EXPRESSION OF HNF-1/vHNF-1 RATIO IS STRONGLY LINKED TO HISTOLOGICAL DIFFERENTIATION OF HEPATOCELLULAR CARCINOMA AND HEPATOBLASTOMA *T.Ninomiya,**Y.Hayashi,**K.Ohta,#K.Saijoh,*Y.Seo,*M.Sugano *S.Yoon, **H.Itoh,*M.Kasuga *2nd Dep. of Medicine and **lst Dep.of Pathology , Kobe Univ. of Medicine, Kobe, Japan, #Dep. of Hygiene, Kanazawa Univ. of Medicine, Kanazawa, Japan Various liver-specific protein genes have multiple cis- and trans-acting elements, yet the elements accountable for hepatocytic differentiation are unclear. We have previously shown that the hepatic transcription of human albumin and o~-fetoprotein (AFP) genes is controlled by a complex set of regulatory elements, and that an AT-rich core sequence (AT motif) is essential as a cis-acting element for the hepatic transcription. Homologous proteins hepatocyte nuclear factor- 1 (HNF- 1), variant HNF- 1 (vHNF- 1), and AT motif-binding factor 1 (ATBF1) bind to the AT motif. To clarify the role of these three different trans-acting factors, we examined the mRNA and the expression ratio of the HNF-I/vHNF-1 mRNA in various liver tissues. Our competitive reverse transcriptional polymerase chain reaction (RT-PCR) method demonstrates that this ratio is linked to their histological differentiation, and that it is higher in well-differentiated hepatocellular carcinoma (HCC) than in poorly-differentiated and undifferentiated HCCs; non-neoplastic liver tissues, however, have a low expression ratio of HNF-I/ vHNF- 1 mRNA. This is the first study that indicates the importance of the HNF-t/vHNF-1 mRNA ratio in determining differentiation of hepatocytic neoplasms and suggests that measuring the ratio would be a useful diagnostic method.
P-427
RESTRICTION LANDMARK GENOME SCANNING ANALYSIS OF C H R O M O S O M A L D N A IN HEPATOCELLULAR CARCINOMA T. H a n a f u s a 1), Y. Y u m o t o 1), H. H a d a 2l, T. S h i n j i 2), T. M o r i t a z~, H. S h i r a h a x), T. T a m u r a x~, Y. I w a s a k i 2~, N. K o i d e 2), H. N a g a i s), K. M a t s u b a r a a), a n d T. Tsuji el 1)Isotope Center, OkayamaUniv., Okayama, Japan. 2)1st Dept. of Medicine, Okayama Univ. Medical School, Okayam a , J a p a n . 3) I n s t . f o r Mol. a n d Cell. Biol., O s a k a U n i v . , Osaka, Japan Restriction landmark genome scanning (RLGS) analysis was applied to detect chromosomal aberrations i n h e p a t o c e l l u l a r c a r c i n o m a (HCC). V a r i o u s t y p e s o f HO2 s a m p l e s , i n c l u d i n g t w o HCCs w i t h m u l t i - n o d u l a r t y p e , o n e HCC s h o w i n g n o d u l e i n n o d u l e p a t t e r n , o n e m e t a s t a s i z e d t o d i f f e r e n t t i s s u e s , t h r e e s m a l l (<2.0 c m ) HCCs, a n d f o u r l a r g e (>5.0 c m ) HCCs, w e r e e x a m i n e d b y RLGS m e t h o d w i t h c o r r e s p o n d i n g non-HCC tissues. Res t r i c t i o n e n z y m e Not I w a s u s e d a s a l a n d m a r k e n z y m e , Eco RV w a s u s e d as a f r a g m e n t a t i o n e n z y m e , a n d HJn f I was used as a digestion enzyme in gel for two dimensional electorophoresis. We observed spot aberrations b e t w e e n HCCs f r o m d i f f e r e n t n o d u l e s , b e t w e e n m e t a s t a s i z e d a n d o r i g i n a l HCC. S o m e s p e c i f i c s p o t a b e r r a t i o n s i n HCCs w h i c h h a v e n o m u t a t i o n in p53 and Rb genes w e r e i d e n t i f i e d . W e s h o w e d RLGS m e t h o d is a n e f f e c t i v e tool for identifying the chromosomal aberrations in HCC, a n d s u g g e s t e d s o m e a s p e c t s o f HCC d e v e l o p m e n t w i l t be revealed by this method.