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Tissue transglutaminase in A1−antitrypsin deficiency liver disease
AASLDAl445
April 2000
alopathy was found with similar frequencie s in patients with or without impaired oxygenation, it was more frequent in hypoxemic compared to normoxemic patients (12/13 [92%] vs 29/46 [63%), p=0,043). Discussion: In patients with cirrhosis mild cerebral dysfunction and hyperventilation are common but not interrelated findings. Contrary , arterial hypoxemia may represent an important contributing cause of mild cerebral dysfunct ion but not of hyperventilation.
6553 HYPERURICEMIA ASSOC IATED WITH RIBAVIRIN T REATMENT OF CH RONIC HEPATI TI S C INDICATI NG IMPD H INHIBITION: A POSS IBLE MO DE OF ANT IVIRAL ACTIVITY? Akeel Halai, Gerond V. Lake-Bak aar, Vamc, Northport, NY. Ribavirin is a guanosine analogue and probably exerts its antiviral activity against Hepatitis C by interfering with RNA synthesis. Possible modes of action include inhibition of viral RNA polymerase, inhibition of guanyIytransferase and methyltransferase and competition with guanosine for incorporation into the 5' terminus of viral RNA. Ribavirin also competitively inhibits IMPDH (inosine monophosphate dehydrogenase) , the enzyme necessary for synthesis of guanosine monophosphate, which could result in depletion of intracellular nucleotide pools of guanosine triphosphate. Inhibition ofIMPDH by Ribavirin would also increase production of hypoxanthine and xanthine, which are precursors of uric acid. We hypoth esized that significant inhibition ofIMPDH by ribavirin during treatment of hepatitis C would result in increased serum uric acid levels. We followed during a fourteen day period , the sequence of changes in serum uric acid in patients during treatment with high dose interferon plus Ribavirin 1000 mg (n= 4)and compared these to change s in patients on high dose interferon alone (n = 4). Serum uric acid levels were measured through the routine chem istry laboratory. RESULTS : Changes in daily serum uric acid levels were minimal and inconsistent during the 14 days of treatment in patients on interferon alone. By contrast, uric acids levels increased gradually to a peak at I to 10 days in the patients on ribavirin, and then gradually declined back to baseline levels The mean maximal uric acid levels in the interferon alone group 8.85 :!: 1.05, was lower than the group treated with interferon and ribavirin, 10.6 :!: 3.8 (p = 0.09). Conclusion: These results suggest that inhibition of IMPDH may be one possible mechanism of antiviral activity of ribavirin. However the effect on uric acid metabolism is short-lived, which suggests that this may not be the only mode of action of ribavirin.
6554 MUTATION ANALYS IS OF THE PSllP63 GENE IN HUM AN HEPATO CE LLULAR CARC INOMA. Kunihiro Hamada, Toru Koyama, Kimihiro Shimizu , Susumu Kawate, Jun Yokota, Susumu Ohwada , Yasuo Morishita, Gunma Univ Sch of Medicine, Maebashi, Japan; Bioi Div , Natl Cancer Ctr Research Inst, Tokyo, Japan . Background: The p5l1p63 gene, a novel family member of human p53 gene, is a recently identified candidate tumor suppressor gene mapped at chromosome 3q28. Like p53, p51 was found to activate p21 and to induce apoptosis, To investigate the prevalence of p51 mutations in human hepatocarcinogenesis, mutation analysis of the p51 gene was performed in human hepatocellular carcinoma (HCC) . Materials and Methods : Highmolecular-weight DNA was prepared from 3 cell lines (2 HCC and 1 hepatoblastoma) , 51 HCC tumors and adjacent non-cancerous tissues. All samples were examined for mutations in the entire coding sequence of the p51 gene (exons 2-15 and exon 3') as well as the core domain of the p53 gene (exon 4-8) by PCR-SSCP analysis. PCR fragments showing different mobilities were directly sequenced . Results: Although we analyzed the entire coding sequence as well as splicing donor and acceptor sites in introns, no tumor specific aberrant bands were detected. We detected three polymorphisms in intron 5, 8 and 13. All the polymorphisms detected in this study were base substitutions. We found p53 mutations in eight (15% ) of the tumors. Conclusion : This result indicates that p51 mutations do not playa major role in human hepatocarcinogenesis. Although the p51 gene is not mutated in HCC, it is still possible that it is inactivated by other molecular mechanisms, such as aberrant hypermethylation leading to transcriptional repression. Thus, further studies are necessary to fully understand the role of the p51 gene in human hepatocarcinogenesis.
6555 A CAUTION TO USE TUMOR MA RKERS IN ASSESS ING THERAPEUTIC EFFECTS OF HEPATOCELLULULAR CARC INOMA (HCC). Keisuke Hamamura, Shuichiro Shiina, Takuma Tera tani, Shuntaro Obi, Yukihiro Koike, Yoshiyuki Dan, Ryosuke Tateishi, Haruhiko Yoshida, Yasushi Shiratori , Masao Ornata, Dept Gastroenterolo gy Univ Tokyo. Tokyo. Japan. (Aim) alpha-fetoprotein is believed to correlate to the extent of HCC. However. we have in 1998 AGA reported that AFP hardly represent therapeutic effect for HCC. indicating that those with AFP respondin g to treatment do not always have better survivals. In this study we carried out more detailed analysis, including des-gamma carboxy prothrombin (DCP) and lectin-reactive AFP fraction (AFP-L3), and show that only AFP does not reflect therapeutic effect well, especialy in the early period of the disease. (Patients & methods) We studied 209 patients who underwent HCC treatment. October 1997 to December 1998, 152 males and 57 females, 42 to 86 year's old. This population included 83 initial and 126 recurrent cases. AFP. DCP, and AFP-L3 were assessed before and after treatment. Cut-off values were set at 100 nglml for AFP, 40 mAU (arbitrary units) for DCP. and 15% for AFP-L3 . When tumor marker levels, exceeding given cut offs, responded to treatment , and decreased below the cut offs, we assigned the patients to responders. Thus, we compared Kaplan-Meier' s survival rates for the patients , according to whether tumor markers responded to treatment or not. (Results) AFP respond ed to therapy for 26/197 (13.2%) patients. DCP and AFP-L3 responces were seen for 18/193 (9.1%) and 8/143 (5.6%) patients, respectively. Survival rates were raised in the table. Except for AFP initial. cases, responders show better survivals than non responder s. AFP response for initial cases only had difficulty in predicting survivals. (Conclusion) Patients with DCP and AFP-L3 responding to treatment have better survival rates. In contrast. only AFP had difficulty in foreseeing patient prognosis in initial treatmen t. l -year survivalrates(%)
AFP (n=197) DCP(n=193) AFP·L3(n=143)
Responsetotreatment
All cases
Inillalcases
Responder Nonresponder Responder Non responder Responder Non responder
95.0 79.8
91.7 87.3 100.0 87.1 100.0
94.4 806 1000 80.6
84.3
6556 TISSUE TRANSGLUTAMINASE IN A. _ANTITRYPSIN DEFICIENCY LIVER DISEASE. Moneera N. Haque, Julie Collins, Patricia Thomas, Karen SantaCruz, Univ of Kansas Med ce , Kansas City, KS. The serpin family of protease inhibitors, to which ai_antitrypsin belongs, is a common autosomal recessive genetic deficiency that principally targets elastase released by inflammatory leucocytes, and is associated with early development of of emphysema, liver cirrhosis, and hepatocellular carcinoma. ai-antitrypsin has a notable mobile reactive center loop. Mutations in the proximal hinge of this critical region of the molecule, that favors the spontaneous opening of the main sheet of the molecule. can precipit ate untimely changes in conformati on with subsequent abnormalities in folding and accompanying polymer formation, which elucidate the plasma deficiency and liver inclusions associated witht the common z variant mutation. The z-variant of a t.antitrypsin results of blockage in the final stage of processing secretion in the hepatocyte. which manifests in a severe decrease in the plasma level of the inhibitor. Consequentl y, the accumulation of the z-antitrypsin is accompanied by liver damage that lead to hepatocellular damage and cirrhosis. Tissue transglutamina se (TIG), is a multifunctiona l enzyme which belongs to the family of transg1utaminase enzymes. that has been identified as a novel G-protein , can be inhibited by GTP,is also involved in cell proliferation, cellular differentiati on. and apoptosis. One of the functions of TIG is to form a protein polymer in dying apoptotic cells by epsilon lysine and specifically gamma-glutamylpolyamine links. TIG in liver cells undergoing apoptosis determ ines extensive cross-linking of cellular proteins. Since, TIG, a retinoid-inducible enzyme. can increase cellular adhesiveness and is capable of crosslinking large multi-domain extracellular glycoproteins, these data demonstrate in increase in TIG gene experession, suggesting a possible role for this transglutaminase enzyme in catalyzing the loop-sheet abnormal crosslinking of (l' ,antitrypsin deposition within the smooth endoplasmi c reticulum. Moreover, this increase may represent one of the mechanisms by which cell injury induces TIG transcription and thus potentiates the process.
April 2000
alopathy was found with similar frequencie s in patients with or without impaired oxygenation, it was more frequent in hypoxemic compared to normoxemic patients (12/13 [92%] vs 29/46 [63%), p=0,043). Discussion: In patients with cirrhosis mild cerebral dysfunction and hyperventilation are common but not interrelated findings. Contrary , arterial hypoxemia may represent an important contributing cause of mild cerebral dysfunct ion but not of hyperventilation.
6553 HYPERURICEMIA ASSOC IATED WITH RIBAVIRIN T REATMENT OF CH RONIC HEPATI TI S C INDICATI NG IMPD H INHIBITION: A POSS IBLE MO DE OF ANT IVIRAL ACTIVITY? Akeel Halai, Gerond V. Lake-Bak aar, Vamc, Northport, NY. Ribavirin is a guanosine analogue and probably exerts its antiviral activity against Hepatitis C by interfering with RNA synthesis. Possible modes of action include inhibition of viral RNA polymerase, inhibition of guanyIytransferase and methyltransferase and competition with guanosine for incorporation into the 5' terminus of viral RNA. Ribavirin also competitively inhibits IMPDH (inosine monophosphate dehydrogenase) , the enzyme necessary for synthesis of guanosine monophosphate, which could result in depletion of intracellular nucleotide pools of guanosine triphosphate. Inhibition ofIMPDH by Ribavirin would also increase production of hypoxanthine and xanthine, which are precursors of uric acid. We hypoth esized that significant inhibition ofIMPDH by ribavirin during treatment of hepatitis C would result in increased serum uric acid levels. We followed during a fourteen day period , the sequence of changes in serum uric acid in patients during treatment with high dose interferon plus Ribavirin 1000 mg (n= 4)and compared these to change s in patients on high dose interferon alone (n = 4). Serum uric acid levels were measured through the routine chem istry laboratory. RESULTS : Changes in daily serum uric acid levels were minimal and inconsistent during the 14 days of treatment in patients on interferon alone. By contrast, uric acids levels increased gradually to a peak at I to 10 days in the patients on ribavirin, and then gradually declined back to baseline levels The mean maximal uric acid levels in the interferon alone group 8.85 :!: 1.05, was lower than the group treated with interferon and ribavirin, 10.6 :!: 3.8 (p = 0.09). Conclusion: These results suggest that inhibition of IMPDH may be one possible mechanism of antiviral activity of ribavirin. However the effect on uric acid metabolism is short-lived, which suggests that this may not be the only mode of action of ribavirin.
6554 MUTATION ANALYS IS OF THE PSllP63 GENE IN HUM AN HEPATO CE LLULAR CARC INOMA. Kunihiro Hamada, Toru Koyama, Kimihiro Shimizu , Susumu Kawate, Jun Yokota, Susumu Ohwada , Yasuo Morishita, Gunma Univ Sch of Medicine, Maebashi, Japan; Bioi Div , Natl Cancer Ctr Research Inst, Tokyo, Japan . Background: The p5l1p63 gene, a novel family member of human p53 gene, is a recently identified candidate tumor suppressor gene mapped at chromosome 3q28. Like p53, p51 was found to activate p21 and to induce apoptosis, To investigate the prevalence of p51 mutations in human hepatocarcinogenesis, mutation analysis of the p51 gene was performed in human hepatocellular carcinoma (HCC) . Materials and Methods : Highmolecular-weight DNA was prepared from 3 cell lines (2 HCC and 1 hepatoblastoma) , 51 HCC tumors and adjacent non-cancerous tissues. All samples were examined for mutations in the entire coding sequence of the p51 gene (exons 2-15 and exon 3') as well as the core domain of the p53 gene (exon 4-8) by PCR-SSCP analysis. PCR fragments showing different mobilities were directly sequenced . Results: Although we analyzed the entire coding sequence as well as splicing donor and acceptor sites in introns, no tumor specific aberrant bands were detected. We detected three polymorphisms in intron 5, 8 and 13. All the polymorphisms detected in this study were base substitutions. We found p53 mutations in eight (15% ) of the tumors. Conclusion : This result indicates that p51 mutations do not playa major role in human hepatocarcinogenesis. Although the p51 gene is not mutated in HCC, it is still possible that it is inactivated by other molecular mechanisms, such as aberrant hypermethylation leading to transcriptional repression. Thus, further studies are necessary to fully understand the role of the p51 gene in human hepatocarcinogenesis.
6555 A CAUTION TO USE TUMOR MA RKERS IN ASSESS ING THERAPEUTIC EFFECTS OF HEPATOCELLULULAR CARC INOMA (HCC). Keisuke Hamamura, Shuichiro Shiina, Takuma Tera tani, Shuntaro Obi, Yukihiro Koike, Yoshiyuki Dan, Ryosuke Tateishi, Haruhiko Yoshida, Yasushi Shiratori , Masao Ornata, Dept Gastroenterolo gy Univ Tokyo. Tokyo. Japan. (Aim) alpha-fetoprotein is believed to correlate to the extent of HCC. However. we have in 1998 AGA reported that AFP hardly represent therapeutic effect for HCC. indicating that those with AFP respondin g to treatment do not always have better survivals. In this study we carried out more detailed analysis, including des-gamma carboxy prothrombin (DCP) and lectin-reactive AFP fraction (AFP-L3), and show that only AFP does not reflect therapeutic effect well, especialy in the early period of the disease. (Patients & methods) We studied 209 patients who underwent HCC treatment. October 1997 to December 1998, 152 males and 57 females, 42 to 86 year's old. This population included 83 initial and 126 recurrent cases. AFP. DCP, and AFP-L3 were assessed before and after treatment. Cut-off values were set at 100 nglml for AFP, 40 mAU (arbitrary units) for DCP. and 15% for AFP-L3 . When tumor marker levels, exceeding given cut offs, responded to treatment , and decreased below the cut offs, we assigned the patients to responders. Thus, we compared Kaplan-Meier' s survival rates for the patients , according to whether tumor markers responded to treatment or not. (Results) AFP respond ed to therapy for 26/197 (13.2%) patients. DCP and AFP-L3 responces were seen for 18/193 (9.1%) and 8/143 (5.6%) patients, respectively. Survival rates were raised in the table. Except for AFP initial. cases, responders show better survivals than non responder s. AFP response for initial cases only had difficulty in predicting survivals. (Conclusion) Patients with DCP and AFP-L3 responding to treatment have better survival rates. In contrast. only AFP had difficulty in foreseeing patient prognosis in initial treatmen t. l -year survivalrates(%)
AFP (n=197) DCP(n=193) AFP·L3(n=143)
Responsetotreatment
All cases
Inillalcases
Responder Nonresponder Responder Non responder Responder Non responder
95.0 79.8
91.7 87.3 100.0 87.1 100.0
94.4 806 1000 80.6
84.3
6556 TISSUE TRANSGLUTAMINASE IN A. _ANTITRYPSIN DEFICIENCY LIVER DISEASE. Moneera N. Haque, Julie Collins, Patricia Thomas, Karen SantaCruz, Univ of Kansas Med ce , Kansas City, KS. The serpin family of protease inhibitors, to which ai_antitrypsin belongs, is a common autosomal recessive genetic deficiency that principally targets elastase released by inflammatory leucocytes, and is associated with early development of of emphysema, liver cirrhosis, and hepatocellular carcinoma. ai-antitrypsin has a notable mobile reactive center loop. Mutations in the proximal hinge of this critical region of the molecule, that favors the spontaneous opening of the main sheet of the molecule. can precipit ate untimely changes in conformati on with subsequent abnormalities in folding and accompanying polymer formation, which elucidate the plasma deficiency and liver inclusions associated witht the common z variant mutation. The z-variant of a t.antitrypsin results of blockage in the final stage of processing secretion in the hepatocyte. which manifests in a severe decrease in the plasma level of the inhibitor. Consequentl y, the accumulation of the z-antitrypsin is accompanied by liver damage that lead to hepatocellular damage and cirrhosis. Tissue transglutamina se (TIG), is a multifunctiona l enzyme which belongs to the family of transg1utaminase enzymes. that has been identified as a novel G-protein , can be inhibited by GTP,is also involved in cell proliferation, cellular differentiati on. and apoptosis. One of the functions of TIG is to form a protein polymer in dying apoptotic cells by epsilon lysine and specifically gamma-glutamylpolyamine links. TIG in liver cells undergoing apoptosis determ ines extensive cross-linking of cellular proteins. Since, TIG, a retinoid-inducible enzyme. can increase cellular adhesiveness and is capable of crosslinking large multi-domain extracellular glycoproteins, these data demonstrate in increase in TIG gene experession, suggesting a possible role for this transglutaminase enzyme in catalyzing the loop-sheet abnormal crosslinking of (l' ,antitrypsin deposition within the smooth endoplasmi c reticulum. Moreover, this increase may represent one of the mechanisms by which cell injury induces TIG transcription and thus potentiates the process.