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Cecell proliferanon and protein kinase C (PKC) isoform expression in cholestatic hamster hepatocytes and cholangiocytes
GASTROENTEROLOGY Vol. 118, No...
A908 AASLD ABSTRACTS
56 PI6 METHYLATION: AN EARLY EVENT IN HEPATOCELLULAR CARCINOGENESIS. Robert A. Ander s. Marc Bissonnette , Thom as A. Brasitus, M. Lisa Yaremko, Jim Koh, E. Brunt. John A. Hart. Univ of Chicago, Chicago , IL; Univ of Vermont, Burlington. VT; Saint Louis Univ, Saint Loius, MO. Backround: The p 16 INK4a tumor suppressor gene induces a cell cycle G 1 arrest. It is the second most commonly inactivated gene identified in human cancers after p53. The p16 gene has been shown to be inactivated in nearly all human colon cancer cell lines and half of colon cancers and adenomas. While the p16 gene has been demonstrated to be inactivated primarily by promoter DNA methylation in hepatocellular carcinoma s, there have been no studies examining p16 expression in premalignant liver lesions . Design: 17 macroregenerative and dysplastic nodules and 2 hepatocellular carcinomas from 15 hepatectomy specimens in patients undergoing transplan tation for hepatitis C cirrhosis were examined by immunohistochemistry and methylation sensitive polymerase chain reaction (PeR). The nodules were less than 2 cm in diameter and clinically undetected . Sections were stained with anti-human pl6 monoclonal antibody . Human sporadic colon adenocarci nomas served as positive controls . Staining for p16 was considered positive if nuclear staining was greater than cytoplasmic staining. Methylation sensitive PCR was performed on bisulfite-treated DNA extracted from microdissected paraffin sections. Results: No nuclear staining was detected in the macroregenerative or dysplastic nodules. Two nodules demonstrated weak cytoplasmic staining . The surrounding cirrhotic liver showed cytoplasmic and no nuclear staining . Bile ductules at the margins of the nodules showed positive staining and served as internal positive controls. The 2 hepatocellular carcinomas showed a distinct lack of nuclear or cytoplasmic staining. Negative controls lacked cytoplasmic and nuclear staining. One hepatocellular carcinoma and two macroregenerative nodules had methylated p 16 gene promoters , while two of the surrounding cirrhotic liver samples had unmethylated pl6 gene promoter s. One macroregenera tive nodule also showed evidence of both methylated and unmethylated pl6 forms. Conclusions: In patients with hepatitis C, clinically undetected macroregenerative and dysplastic nodules show an absence of pl6 staining. It is likely that pl6 gene methylation is responsible for suppression ofpl6 expression in these nodules. Thi s supports previous observations that a high percentage of hepatocellular carcinomas have inactive p16 genes and suggests that p16 inactivation occurs early in liver tumor progression.
57 OVEREXPRESSION OF CYCLIN A AND PS3 IN HEPATOCELLU· LAR CARCINOMAS. WILD P53 DOWNREGURA TE CYCLIN A PROMOTER ACTIVITY. Daiju Nakayama. Yasushi Magami , Shinichiro Kokuno, Masaya Furukawa. Yoshihisa Tsukioka , Toshiya Horibe, Tomoyuki Seki, Toshihiko Saitoh, Toshio Nikaido, Kazuhiko Kasuya, Akihiko Tsuchida, Yasuhisa Koyanagi, Tokyo Med Univ, Tokyo. Japan ; Shishu Univ, Matsumoto . Japan. [Background and purpose] Cyclin A is a prime cell cycle regulator and play a central role in to the control of both Sand G2-M phases of the cell cycle. Overexpression of cyclin A protein has been reported in some tumors. We examined the involvement of cyclin A and p53 in the development of human hepatocellular carcinomas (HCC) . Methods: Expression of cyclin A and p53 in HCC was examined irnmunohistochemically, revealing that 40% of HCC expressed both cyclin A and p53. In addition. cyclin A positive cells were topographically related to p53 positve cells. Secondly, we examined cyclin A promoter activity and the effect of the p53 gene on its activity. The HCC cell lines examined to determine cyclin A promoter activitywere HepG2. Huh7, HLE . The cell lines were transfected with human cyclin A promoter fused to luciferase cDNA using lipofectin. 24h after completion of transfection , the cyclin A promoter level was determined by luciferase assay. Results: Luciferase activity was observed at various levels in all cell lines. The cyclin A promoter activity of Huh7 was highest among the 3 cell lines. Concerning the promoter activity of various deletion constructs of cyclin A promoter region, lucifera se activity sharply decreased when the deletion construct lacked the ATF and three Sp1 binding sites in all cells. In the deletion experiment, the decrease of activity was most remarkable in Huh7 . In order to examine the effect of p53 on yclin A promoter activity. these cells were cotransfected with cyclin A promoter fused to the luciferase cDNA and a plasmid expressing p53 . Wild type p53. but no mutant type p53. remarkabl y suppressed cyelin A promoter activity in all cells. [conclusion] These findings suggest that overexpression of cyclin A and p53 is associated with growth and progression of hepatocellul ar carcinomas. 58 CECEH PROLIFERATION AND PROTEIN KINASE C (PKC) ISOFORM EXPRESSION IN CHOLESTATIC HAMSTER HEPA· TOCYTES AND CHOLANGIOCYTES. Man Le, Rachel Weston, Yasushi Matsuzaki , Mikio Doi, Bernard Bouscarel, The George Washington Univ, Washington. DC; Tsukuba Univ, Tsukub a City. Japan; Ibaraki Prefectural Institute of Public Health, Mitoshi, Japan. BACKGROUND : The proliferation of biliary ductules is predominant in many liver diseases. including cholestasis and cirrhosis in humans. Furthermore, PKC 13. 0 and E isofonns have been associated to cell prolifer-
ation. AIM:To compare the proliferation of hepatocytes and cholangiocytes with the respective cellular expression level of these PKC isofonns in cholestatic golden Syrian hamster. METHOD:Hepatocytes and cholang iocytes were isolated by the collagenase perfusion method two days after either bile duct ligation (BDL) or sham-operation . Cell proliferation was assessed by immunohistochemistry using an antibody against the proliferating cell nuclear antigen (PCNA). PKC expression was determined by Western blotting and quantitated as a ratio of that of J3-actin. RESULTS : After BDL, increased bile duct proliferation was accompanied by a significantly increased number ofPCNA-positive cholangiocytes. Under these conditions. the hepatocyte proliferation was also increased but significantly less than that of cholangiocyte s. The purity of the hepatocyte preparation , following isolation from both BDL and sham hamsters. was confirmed by the absence of cytokeratin 19. which is specific to epithelial cells. The PKC-.B2 expression was increased by over 82% in hepatocytes from BDL vs sham. However . this isofonn was not detected in choJangiocytes from either BDL or sham hamsters, thus, confirming the purity of the cholangiocyte preparation . PKC-o isoform expression was increased by 130% in BDL over that in sham hepatocytes , In contrast. cholangioc yte PKC-o expression was mostly of the dephosphoryl ated form and was decreased by over 60% after BDL. PKC-E expression was respectively increased by over 50% and 100% in hepatocytes and cholangiocytes isolated from BDL. above that observed in sham hamster. CONCLUSION : PKC-132 is expressed preferentially in hepatocytes and little or not at all in cholangiocytes. The inverse expression of PKC-I) and PKC-E is consistent with the increased cell proliferation observed by PCNA expression in cholangiocytes isolated from BDL hamster. The differential expression of PKC-132' I) and E in hepatocytes and cholangiocytes may be relevant to the pathophysiology of choJestasis.
59 LIVER NICOTINAMIDE METHYLATION: NICOTINAMIDE NMETHYLTRANSFERASE EXPRESSION IN CIRRHOSIS. Rossella Pumpo, Pasqualina Buono, Rosario Cuomo. Francesco Salvatore. Richard Weinshilboum, Gabriele Budillon, Universita ' degli Study Fed II • Gastroenterolog y 2. Naples. Italy; Universita' degli Study Fed II , DBBM . Naples. Italy; Mayo Clin, Rochester. MN. Nicotinam ide (NA) methylation followed by urinary excretion of Nvmethylnicotinamide (NMN) has been shown to be increased in cirrhotic patients under basal conditions and after NA oral loading in spite of the well known derangement of overall methylation processes during hepatic failure . Such a finding could depend on induction and activity increase of the specific hepatic enzyme nicotinamide N-methyltransferase (NNMT). Therefore we studied by Western blot analysis with polyclonal anti-NNMT antibody the expression ofNNMT in liver biopsies of 13 normal control s (5M. mean age 53 y) and 37 patients with liver diseases. 25 (14M, mean age 43 y) with C virus chronic hepatitis and 12 (9M. mean age 43 y) with liver cirrhosis (8 Child A; 4 B). Statistical analysis was performed with ANOVA and post-test for linear trend. Hepatic NNMT expression (as a ratio to a reference value) was shown to progressively decrease. with a significant {p<0.05) linear trend, from normal liver (1.08:t0.46) to hepatitis (0.44:tO.l3) and. finally. to cirrhosis (0.17:t 0. 11). The overall decrease of NNMT expression in cirrhotic versus nonn alliver amounted to 84%. These results may indicate that the increased NMN production in cirrhosis is dependent on a higher than normal turnover of NA to form NMN by NNMT in spite of its decreased hepatic tissue expression. The "hyperfunction" of this methylating pathway might playa protective role against the toxic effect of intracellular accumulation of NA resulting from the catabolic trend in cirrhotic patients.
60 EXPRESSION OF BRAIN·SPECIFIC ANGIOGENESIS INHIBI· TOR 1 IN HEPATOCELLULAR CARCINOMA. Fumihiko Komine, Kenzaburo Tan i, Xixiong Kang, Yuansong Bai, Hidenori Hase, Hajime Sugiyam a, Reiko Kitamura. Tsu yoshi Tanabe, Keisuke Takahashi, Yusuke Nakamura, Yasuyuki Arakawa. Shigetaka Asano, Univ of Tokyo. Tokyo, Japan ; Nihon Univ Sch of Medicine, Tokyo, Japan . Background/Aims : Brain-specific angiogenesis inhibitor I(BAIl ) was recently isolated as a p53 target gene specifically expressed in brain . BAil has been suggested to playa significant role in angiogenesis inhibition as amediator of p53 in glioma. However, it is not clear about the significance of BAIl expression in other tumors. We examined BAIl expre ssion inhepatocellular carcinoma (HCC)s. also known as hyperva scular tumor. and discussed about the significance of BAI-I expression in these tumor cells. Materials and Methods: We studied the expression of BAIl in 5 hepatoma cell lines (HepG2, Hep3B. PLCIPRF/5. HLF. HUH7) and 16 specimens of HCC patients (8 HCC nodules and 8 extranodular regions : liver cirrhosis) and 2 specimens of norrnal livers by Northern blot analysis. The samples were obtained by surgical resection of tumors and extra-tumor regions from these patients. Result s: The expression of BAIl mRNA was detected in all hepatoma cell lines (5 of 5: 100%) and 5 of 8 HCC nodules (62.5%) and 3 of 8 extranodular regions (37.5%). But the expression in extranodular regions were weaker than HCC nodules in the same patient The expression of BAIl mRNA was not detected in normal livers (0 of 2: 0%) as original manuscript reported (Nishimori et aI, Oncogene 1997;15: 2145-50). Conclusions : Our results showed that the BAIlmRNA was also expressed in the hepatoma cells as well as normal brain tissues . The expression of BAIl mRNA in hepatoma cells was detected in PLCIPRF/5 ,
A908 AASLD ABSTRACTS
56 PI6 METHYLATION: AN EARLY EVENT IN HEPATOCELLULAR CARCINOGENESIS. Robert A. Ander s. Marc Bissonnette , Thom as A. Brasitus, M. Lisa Yaremko, Jim Koh, E. Brunt. John A. Hart. Univ of Chicago, Chicago , IL; Univ of Vermont, Burlington. VT; Saint Louis Univ, Saint Loius, MO. Backround: The p 16 INK4a tumor suppressor gene induces a cell cycle G 1 arrest. It is the second most commonly inactivated gene identified in human cancers after p53. The p16 gene has been shown to be inactivated in nearly all human colon cancer cell lines and half of colon cancers and adenomas. While the p16 gene has been demonstrated to be inactivated primarily by promoter DNA methylation in hepatocellular carcinoma s, there have been no studies examining p16 expression in premalignant liver lesions . Design: 17 macroregenerative and dysplastic nodules and 2 hepatocellular carcinomas from 15 hepatectomy specimens in patients undergoing transplan tation for hepatitis C cirrhosis were examined by immunohistochemistry and methylation sensitive polymerase chain reaction (PeR). The nodules were less than 2 cm in diameter and clinically undetected . Sections were stained with anti-human pl6 monoclonal antibody . Human sporadic colon adenocarci nomas served as positive controls . Staining for p16 was considered positive if nuclear staining was greater than cytoplasmic staining. Methylation sensitive PCR was performed on bisulfite-treated DNA extracted from microdissected paraffin sections. Results: No nuclear staining was detected in the macroregenerative or dysplastic nodules. Two nodules demonstrated weak cytoplasmic staining . The surrounding cirrhotic liver showed cytoplasmic and no nuclear staining . Bile ductules at the margins of the nodules showed positive staining and served as internal positive controls. The 2 hepatocellular carcinomas showed a distinct lack of nuclear or cytoplasmic staining. Negative controls lacked cytoplasmic and nuclear staining. One hepatocellular carcinoma and two macroregenerative nodules had methylated p 16 gene promoters , while two of the surrounding cirrhotic liver samples had unmethylated pl6 gene promoter s. One macroregenera tive nodule also showed evidence of both methylated and unmethylated pl6 forms. Conclusions: In patients with hepatitis C, clinically undetected macroregenerative and dysplastic nodules show an absence of pl6 staining. It is likely that pl6 gene methylation is responsible for suppression ofpl6 expression in these nodules. Thi s supports previous observations that a high percentage of hepatocellular carcinomas have inactive p16 genes and suggests that p16 inactivation occurs early in liver tumor progression.
57 OVEREXPRESSION OF CYCLIN A AND PS3 IN HEPATOCELLU· LAR CARCINOMAS. WILD P53 DOWNREGURA TE CYCLIN A PROMOTER ACTIVITY. Daiju Nakayama. Yasushi Magami , Shinichiro Kokuno, Masaya Furukawa. Yoshihisa Tsukioka , Toshiya Horibe, Tomoyuki Seki, Toshihiko Saitoh, Toshio Nikaido, Kazuhiko Kasuya, Akihiko Tsuchida, Yasuhisa Koyanagi, Tokyo Med Univ, Tokyo. Japan ; Shishu Univ, Matsumoto . Japan. [Background and purpose] Cyclin A is a prime cell cycle regulator and play a central role in to the control of both Sand G2-M phases of the cell cycle. Overexpression of cyclin A protein has been reported in some tumors. We examined the involvement of cyclin A and p53 in the development of human hepatocellular carcinomas (HCC) . Methods: Expression of cyclin A and p53 in HCC was examined irnmunohistochemically, revealing that 40% of HCC expressed both cyclin A and p53. In addition. cyclin A positive cells were topographically related to p53 positve cells. Secondly, we examined cyclin A promoter activity and the effect of the p53 gene on its activity. The HCC cell lines examined to determine cyclin A promoter activitywere HepG2. Huh7, HLE . The cell lines were transfected with human cyclin A promoter fused to luciferase cDNA using lipofectin. 24h after completion of transfection , the cyclin A promoter level was determined by luciferase assay. Results: Luciferase activity was observed at various levels in all cell lines. The cyclin A promoter activity of Huh7 was highest among the 3 cell lines. Concerning the promoter activity of various deletion constructs of cyclin A promoter region, lucifera se activity sharply decreased when the deletion construct lacked the ATF and three Sp1 binding sites in all cells. In the deletion experiment, the decrease of activity was most remarkable in Huh7 . In order to examine the effect of p53 on yclin A promoter activity. these cells were cotransfected with cyclin A promoter fused to the luciferase cDNA and a plasmid expressing p53 . Wild type p53. but no mutant type p53. remarkabl y suppressed cyelin A promoter activity in all cells. [conclusion] These findings suggest that overexpression of cyclin A and p53 is associated with growth and progression of hepatocellul ar carcinomas. 58 CECEH PROLIFERATION AND PROTEIN KINASE C (PKC) ISOFORM EXPRESSION IN CHOLESTATIC HAMSTER HEPA· TOCYTES AND CHOLANGIOCYTES. Man Le, Rachel Weston, Yasushi Matsuzaki , Mikio Doi, Bernard Bouscarel, The George Washington Univ, Washington. DC; Tsukuba Univ, Tsukub a City. Japan; Ibaraki Prefectural Institute of Public Health, Mitoshi, Japan. BACKGROUND : The proliferation of biliary ductules is predominant in many liver diseases. including cholestasis and cirrhosis in humans. Furthermore, PKC 13. 0 and E isofonns have been associated to cell prolifer-
ation. AIM:To compare the proliferation of hepatocytes and cholangiocytes with the respective cellular expression level of these PKC isofonns in cholestatic golden Syrian hamster. METHOD:Hepatocytes and cholang iocytes were isolated by the collagenase perfusion method two days after either bile duct ligation (BDL) or sham-operation . Cell proliferation was assessed by immunohistochemistry using an antibody against the proliferating cell nuclear antigen (PCNA). PKC expression was determined by Western blotting and quantitated as a ratio of that of J3-actin. RESULTS : After BDL, increased bile duct proliferation was accompanied by a significantly increased number ofPCNA-positive cholangiocytes. Under these conditions. the hepatocyte proliferation was also increased but significantly less than that of cholangiocyte s. The purity of the hepatocyte preparation , following isolation from both BDL and sham hamsters. was confirmed by the absence of cytokeratin 19. which is specific to epithelial cells. The PKC-.B2 expression was increased by over 82% in hepatocytes from BDL vs sham. However . this isofonn was not detected in choJangiocytes from either BDL or sham hamsters, thus, confirming the purity of the cholangiocyte preparation . PKC-o isoform expression was increased by 130% in BDL over that in sham hepatocytes , In contrast. cholangioc yte PKC-o expression was mostly of the dephosphoryl ated form and was decreased by over 60% after BDL. PKC-E expression was respectively increased by over 50% and 100% in hepatocytes and cholangiocytes isolated from BDL. above that observed in sham hamster. CONCLUSION : PKC-132 is expressed preferentially in hepatocytes and little or not at all in cholangiocytes. The inverse expression of PKC-I) and PKC-E is consistent with the increased cell proliferation observed by PCNA expression in cholangiocytes isolated from BDL hamster. The differential expression of PKC-132' I) and E in hepatocytes and cholangiocytes may be relevant to the pathophysiology of choJestasis.
59 LIVER NICOTINAMIDE METHYLATION: NICOTINAMIDE NMETHYLTRANSFERASE EXPRESSION IN CIRRHOSIS. Rossella Pumpo, Pasqualina Buono, Rosario Cuomo. Francesco Salvatore. Richard Weinshilboum, Gabriele Budillon, Universita ' degli Study Fed II • Gastroenterolog y 2. Naples. Italy; Universita' degli Study Fed II , DBBM . Naples. Italy; Mayo Clin, Rochester. MN. Nicotinam ide (NA) methylation followed by urinary excretion of Nvmethylnicotinamide (NMN) has been shown to be increased in cirrhotic patients under basal conditions and after NA oral loading in spite of the well known derangement of overall methylation processes during hepatic failure . Such a finding could depend on induction and activity increase of the specific hepatic enzyme nicotinamide N-methyltransferase (NNMT). Therefore we studied by Western blot analysis with polyclonal anti-NNMT antibody the expression ofNNMT in liver biopsies of 13 normal control s (5M. mean age 53 y) and 37 patients with liver diseases. 25 (14M, mean age 43 y) with C virus chronic hepatitis and 12 (9M. mean age 43 y) with liver cirrhosis (8 Child A; 4 B). Statistical analysis was performed with ANOVA and post-test for linear trend. Hepatic NNMT expression (as a ratio to a reference value) was shown to progressively decrease. with a significant {p<0.05) linear trend, from normal liver (1.08:t0.46) to hepatitis (0.44:tO.l3) and. finally. to cirrhosis (0.17:t 0. 11). The overall decrease of NNMT expression in cirrhotic versus nonn alliver amounted to 84%. These results may indicate that the increased NMN production in cirrhosis is dependent on a higher than normal turnover of NA to form NMN by NNMT in spite of its decreased hepatic tissue expression. The "hyperfunction" of this methylating pathway might playa protective role against the toxic effect of intracellular accumulation of NA resulting from the catabolic trend in cirrhotic patients.
60 EXPRESSION OF BRAIN·SPECIFIC ANGIOGENESIS INHIBI· TOR 1 IN HEPATOCELLULAR CARCINOMA. Fumihiko Komine, Kenzaburo Tan i, Xixiong Kang, Yuansong Bai, Hidenori Hase, Hajime Sugiyam a, Reiko Kitamura. Tsu yoshi Tanabe, Keisuke Takahashi, Yusuke Nakamura, Yasuyuki Arakawa. Shigetaka Asano, Univ of Tokyo. Tokyo, Japan ; Nihon Univ Sch of Medicine, Tokyo, Japan . Background/Aims : Brain-specific angiogenesis inhibitor I(BAIl ) was recently isolated as a p53 target gene specifically expressed in brain . BAil has been suggested to playa significant role in angiogenesis inhibition as amediator of p53 in glioma. However, it is not clear about the significance of BAIl expression in other tumors. We examined BAIl expre ssion inhepatocellular carcinoma (HCC)s. also known as hyperva scular tumor. and discussed about the significance of BAI-I expression in these tumor cells. Materials and Methods: We studied the expression of BAIl in 5 hepatoma cell lines (HepG2, Hep3B. PLCIPRF/5. HLF. HUH7) and 16 specimens of HCC patients (8 HCC nodules and 8 extranodular regions : liver cirrhosis) and 2 specimens of norrnal livers by Northern blot analysis. The samples were obtained by surgical resection of tumors and extra-tumor regions from these patients. Result s: The expression of BAIl mRNA was detected in all hepatoma cell lines (5 of 5: 100%) and 5 of 8 HCC nodules (62.5%) and 3 of 8 extranodular regions (37.5%). But the expression in extranodular regions were weaker than HCC nodules in the same patient The expression of BAIl mRNA was not detected in normal livers (0 of 2: 0%) as original manuscript reported (Nishimori et aI, Oncogene 1997;15: 2145-50). Conclusions : Our results showed that the BAIlmRNA was also expressed in the hepatoma cells as well as normal brain tissues . The expression of BAIl mRNA in hepatoma cells was detected in PLCIPRF/5 ,