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304 PCNA Expression in human hepatocellular carcinoma: An in vivo and in vitro proteomic approach
S 118
POSTERS
viability; acute hypoxia (similar PO2 for <30 minutes) did not induce a similar suppression of respiration. This study sought to measure the alterations in the supply and demand of ATP during this hypoxic conformance, and to determine whether viability is maintained by preferentially suppressing non-essential processes while sustaining processes essential for maintaining cell homeostasis. In addition, the rate of recovery of oxygen consumption and ATP production following reoxygenation after prolonged and acute hypoxia/anoxia was compared. Methods: An in-vitro model of moderate hypoxia in primary rat hepatocytes was used. Cells were exposed to normoxia and prolonged moderate hypoxia (PO 2 20 tort for 3 h), and the following parameters were measured and compared: (a) O2 consumption (VO2); (b) adenine nucleotide concentrations; (c) lactic acid production; (d) cellular ATPase activities as a measure of metabolic demand, defined as essential (Na-K ATPase) and non-essential (acetaminophen [APAP] conjugation related ATPases). Results: Measurements of oxygen consumption (VO2) and ATP production decreased during prolonged hypoxia compared with acute hypoxia. The decrease in VO2 during hypoxia was not associated with an increase in the rate of lactic acid production, indicating a suppression of ATP utilization. However, ouabain-inhibitable respiration did not decrease during prolonged hypoxia, indicating that membrane Na + K+-ATPase activity, an essential process for cell viability, was maintained. In contrast, ATPdependent glucuronidation and sulfation of acetaminophen, deemed 'nonessential' processes, were decreased significantly compared with normoxic cells. Following reoxygenation, cells exposed to prolonged moderate hypoxia demonstrated a more rapid recovery of respiration compared to acute hypoxia/anoxia. Conclusion: This observation of 'hepatic hibernation' of metabolism during prolonged moderate hypoxia may represent an anticipatory adaptation which seeks to maintain cell viability while delaying or preventing the onset of lethal hypoxia, and facilitates a rapid recovery following the resumption of normoxia.
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PCNA EXPRESSION IN HUMAN HEPATOCELLULAR CARCINOMA: AN IN VIVO AND IN VITRO PROTEOMIC APPROACH
A. Venturi 1.2., L. Gramantieri 1.2 C. Giovannini2, R. Righini 1.2 p. Chieco 2, L. Bolondi 1'2. 1Department of Internal Medicine and Gastroenterology,
University of Bologna, Italy," 2Center for Applied Biomedical Research (CRBA), S. Orsola-Malpighi University Hospital, Bologna, Italy Background and Aims: Proliferating Cell Nuclear Antigen (PCNA) is a 36 kDa protein involved in several cellular mechanisms, including cell cycle regulation, chromatin remodeling, DNA synthesis and repair (as DNA-bound fraction). In neoplastic transformation, the progressive accumulation of DNA damage in cancer cells could be linked to an alteration in PCNA structure or function. The aim of the present study was to evaluate PCNA expression in hepatocellular carcinoma (HCC) and cirrhotic tissues as well as in HCC cell lines by using a proteomic approach. Methods: Neoplastic and cirrhotic matched tissues from nine patients undergoing surgery for HCC were analysed together with HepG2 (p53+/+) and Hep3B (p53 / ) cell lines. Protein extracts were subjected to 2-dimensional gel electrophoresis (2D SDS-PAGE) and PCNA protein expression was assessed by subsequent Western Blot analysis. In cell lines, to release the DNA-bound fraction of PCNA, cellular pellets were digested with DNAse-I prior to 2D SDS-PAGE and both the soluble and DNAbound protein fractions were analysed. Results: In neoplastic samples, 2D SDS-PAGE and Western Blot analysis revealed the existence of two different PCNA expression patterns. In the first one, assessed in four of nine HCC tissues and in both cell lines, PCNA was present in three acidic forms, probably corresponding to PCNA monomers, dimers and trimers, and in a basic isoform. In the second pattern, displayed by the remaining HCCs, only a monomeric
PCNA form was detected in association with multiple basic modified isoforms. Interestingly, in none of cirrhotic tissues a PCNA basic form was demonstrated. Finally, in cell lines after DNAse-I digestion only a 36 kDa acidic PCNA form was detected. Conclusions: This preliminary report exploring PCNA expression pattern in HCC shows that PCNA is differently expressed in neoplastic and cirrhotic tissues, in terms of both quantity and isoforms, suggesting functional implications in carcinogenetic process. Future mass spectrometry analysis will clarify the nature of PCNA isoforms, providing useful information about PCNA involvement in HCC development.
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ADENOVIRUS-MEDIATED TRANSFECTION OF CASPASE 8 SENSITIZES HEPATOCELLULAR CARCINOMA TO TRAILAND CHEMOTHERAPEUTIC AGENTS-INDUCED CELL DEATH
Y. Yamaguchi, K. Shiraki, H. Fuke, T. Inoue, K. Miyashita, Y. Yamanaka, T. Nakano. First Department of Internal Medicine, Mie University School
of Medicine, Edobashi, Japan Baekgronnd and Aims: Caspase 8 is a member of the cysteine protease family that plays a critical role in death receptor-mediated apoptosis. Cellular FLICE/caspase-8-inhibitory protein (cFLIP) is crucial for modulation of the cell death signal, inhibiting procaspase-8 processing at DISC. We have demonstrated that cFLIP plays an important role of an endogenous signaling pathway switching modulator to either an apoptosis induction or cell proliferation by activated nuclear factor (NF)-~;B, and we consider that strategy to inhibit cFLIP expression is a potential tool in the useful treatment of human HCCs. We investigated whether adenovirus-mediated transfection of caspase 8 suppresses function of cFLIP and sensitizes HCC cells to TRAIL-and chemotherapeutic agents-induced cell death. Methods: Recombinant adenovirus expressing human caspase 8 (AxCALNL-hCaspase-8, Adv-Casp8) utilized the Cre/loxP system and recombinant adenovirus expressing Cre recombinase (AxCANCre, AdvCre) were coinfected into HCC cell lines, SK-Hepl, HLE, and HepG2. Expression of caspase 8 and apoptosis related proteins were analyzed by Western blotting. DAPI staining was used for detection of apoptosis. Cell viability after infection of adenovirus vectors and incubation of absent or present TRAIL or chemotherapeutic agents (camptothecin, doxorubicin, cisplatin, taxol, or 5-FU) were assessed by MTT assay. Cell Fractionation Kit was utilized for investigation of mitochondrial pathway. Results: Expression levels of procaspase 8 and cleaved caspase 8 was increased and viability of infected cells with Adv-Casp8 was decreased in a MOI-dependent manner. Adv-Casp8 infected cells showed typical apoptotic features including nuclear condensation and nuclear fragmentation with DAPI staining. Adv-Casp8 augmented TRAIL- or chemotherapeutic agents-induced cell death. Adv-Casp8 suppressed expression of FLIP in all cell lines, and XIAE survivin, and Bcl-xL in HLE cell line. In addition,_Adv-Casp8 tends to promote release of Smac and cytochrome c from mitochondria to cytosol. Conclusions: Adv-Casp8 sensitizes HCC cells to TRAIL- or chemotherapeutic agents-induced cell death_with participation of mitochondrial pathway.
POSTERS
viability; acute hypoxia (similar PO2 for <30 minutes) did not induce a similar suppression of respiration. This study sought to measure the alterations in the supply and demand of ATP during this hypoxic conformance, and to determine whether viability is maintained by preferentially suppressing non-essential processes while sustaining processes essential for maintaining cell homeostasis. In addition, the rate of recovery of oxygen consumption and ATP production following reoxygenation after prolonged and acute hypoxia/anoxia was compared. Methods: An in-vitro model of moderate hypoxia in primary rat hepatocytes was used. Cells were exposed to normoxia and prolonged moderate hypoxia (PO 2 20 tort for 3 h), and the following parameters were measured and compared: (a) O2 consumption (VO2); (b) adenine nucleotide concentrations; (c) lactic acid production; (d) cellular ATPase activities as a measure of metabolic demand, defined as essential (Na-K ATPase) and non-essential (acetaminophen [APAP] conjugation related ATPases). Results: Measurements of oxygen consumption (VO2) and ATP production decreased during prolonged hypoxia compared with acute hypoxia. The decrease in VO2 during hypoxia was not associated with an increase in the rate of lactic acid production, indicating a suppression of ATP utilization. However, ouabain-inhibitable respiration did not decrease during prolonged hypoxia, indicating that membrane Na + K+-ATPase activity, an essential process for cell viability, was maintained. In contrast, ATPdependent glucuronidation and sulfation of acetaminophen, deemed 'nonessential' processes, were decreased significantly compared with normoxic cells. Following reoxygenation, cells exposed to prolonged moderate hypoxia demonstrated a more rapid recovery of respiration compared to acute hypoxia/anoxia. Conclusion: This observation of 'hepatic hibernation' of metabolism during prolonged moderate hypoxia may represent an anticipatory adaptation which seeks to maintain cell viability while delaying or preventing the onset of lethal hypoxia, and facilitates a rapid recovery following the resumption of normoxia.
I'~
PCNA EXPRESSION IN HUMAN HEPATOCELLULAR CARCINOMA: AN IN VIVO AND IN VITRO PROTEOMIC APPROACH
A. Venturi 1.2., L. Gramantieri 1.2 C. Giovannini2, R. Righini 1.2 p. Chieco 2, L. Bolondi 1'2. 1Department of Internal Medicine and Gastroenterology,
University of Bologna, Italy," 2Center for Applied Biomedical Research (CRBA), S. Orsola-Malpighi University Hospital, Bologna, Italy Background and Aims: Proliferating Cell Nuclear Antigen (PCNA) is a 36 kDa protein involved in several cellular mechanisms, including cell cycle regulation, chromatin remodeling, DNA synthesis and repair (as DNA-bound fraction). In neoplastic transformation, the progressive accumulation of DNA damage in cancer cells could be linked to an alteration in PCNA structure or function. The aim of the present study was to evaluate PCNA expression in hepatocellular carcinoma (HCC) and cirrhotic tissues as well as in HCC cell lines by using a proteomic approach. Methods: Neoplastic and cirrhotic matched tissues from nine patients undergoing surgery for HCC were analysed together with HepG2 (p53+/+) and Hep3B (p53 / ) cell lines. Protein extracts were subjected to 2-dimensional gel electrophoresis (2D SDS-PAGE) and PCNA protein expression was assessed by subsequent Western Blot analysis. In cell lines, to release the DNA-bound fraction of PCNA, cellular pellets were digested with DNAse-I prior to 2D SDS-PAGE and both the soluble and DNAbound protein fractions were analysed. Results: In neoplastic samples, 2D SDS-PAGE and Western Blot analysis revealed the existence of two different PCNA expression patterns. In the first one, assessed in four of nine HCC tissues and in both cell lines, PCNA was present in three acidic forms, probably corresponding to PCNA monomers, dimers and trimers, and in a basic isoform. In the second pattern, displayed by the remaining HCCs, only a monomeric
PCNA form was detected in association with multiple basic modified isoforms. Interestingly, in none of cirrhotic tissues a PCNA basic form was demonstrated. Finally, in cell lines after DNAse-I digestion only a 36 kDa acidic PCNA form was detected. Conclusions: This preliminary report exploring PCNA expression pattern in HCC shows that PCNA is differently expressed in neoplastic and cirrhotic tissues, in terms of both quantity and isoforms, suggesting functional implications in carcinogenetic process. Future mass spectrometry analysis will clarify the nature of PCNA isoforms, providing useful information about PCNA involvement in HCC development.
I-~
ADENOVIRUS-MEDIATED TRANSFECTION OF CASPASE 8 SENSITIZES HEPATOCELLULAR CARCINOMA TO TRAILAND CHEMOTHERAPEUTIC AGENTS-INDUCED CELL DEATH
Y. Yamaguchi, K. Shiraki, H. Fuke, T. Inoue, K. Miyashita, Y. Yamanaka, T. Nakano. First Department of Internal Medicine, Mie University School
of Medicine, Edobashi, Japan Baekgronnd and Aims: Caspase 8 is a member of the cysteine protease family that plays a critical role in death receptor-mediated apoptosis. Cellular FLICE/caspase-8-inhibitory protein (cFLIP) is crucial for modulation of the cell death signal, inhibiting procaspase-8 processing at DISC. We have demonstrated that cFLIP plays an important role of an endogenous signaling pathway switching modulator to either an apoptosis induction or cell proliferation by activated nuclear factor (NF)-~;B, and we consider that strategy to inhibit cFLIP expression is a potential tool in the useful treatment of human HCCs. We investigated whether adenovirus-mediated transfection of caspase 8 suppresses function of cFLIP and sensitizes HCC cells to TRAIL-and chemotherapeutic agents-induced cell death. Methods: Recombinant adenovirus expressing human caspase 8 (AxCALNL-hCaspase-8, Adv-Casp8) utilized the Cre/loxP system and recombinant adenovirus expressing Cre recombinase (AxCANCre, AdvCre) were coinfected into HCC cell lines, SK-Hepl, HLE, and HepG2. Expression of caspase 8 and apoptosis related proteins were analyzed by Western blotting. DAPI staining was used for detection of apoptosis. Cell viability after infection of adenovirus vectors and incubation of absent or present TRAIL or chemotherapeutic agents (camptothecin, doxorubicin, cisplatin, taxol, or 5-FU) were assessed by MTT assay. Cell Fractionation Kit was utilized for investigation of mitochondrial pathway. Results: Expression levels of procaspase 8 and cleaved caspase 8 was increased and viability of infected cells with Adv-Casp8 was decreased in a MOI-dependent manner. Adv-Casp8 infected cells showed typical apoptotic features including nuclear condensation and nuclear fragmentation with DAPI staining. Adv-Casp8 augmented TRAIL- or chemotherapeutic agents-induced cell death. Adv-Casp8 suppressed expression of FLIP in all cell lines, and XIAE survivin, and Bcl-xL in HLE cell line. In addition,_Adv-Casp8 tends to promote release of Smac and cytochrome c from mitochondria to cytosol. Conclusions: Adv-Casp8 sensitizes HCC cells to TRAIL- or chemotherapeutic agents-induced cell death_with participation of mitochondrial pathway.