- Email: [email protected]
The impact of RNAses in post-trancriptional control of RNA population after reactive nitrogen species attack
212 cal difference between the mean number of mutations in the two groups (p>O.O5). The mean level of ferritin in cases with mutations had no statistically significant difference with that of patients without mutations (P>O.O5). Conclusion: The results do not support the hypothesis that the occurrence of the HFE gene mutations in thalassemia patients is more frequent than that in normal population. Finding no impact of the mutations on the level of ferritin is not in line with some previous studies.
ulatory sequences the level of expression declined with time. Among the chimeric promoters analyzed, the alpha 1-antitrypsin promoter combined with the albumin enhancer was found to be the strongest and more stable regulatory sequence. Conclusion: We have developed a collection of liver-specific promoters of different activity in liver cells. A sustained, long-lasting and high level of expression of therapeutic genes could be achieved using the alpha lantitrypsin promoter fused to the albumin enhancer
I734
I736
THE IMPACT OF RNASES CONTROL NITROGEN
IN POST-TRANCRIPTIONAL
OF RNA POPULATION SPECIES
AFTER REACTIVE
AlTACK
The central role of ribonucleases (RNases) in the control of the splicing processes and response to changing cell environments make them oldest regulatory‘housekeeping enzymes”. Since RNase activity is further controlled by the presence of naturally-occurring inhibitor, the aim of the study was to evaluate effect of peroxynitrite on liver RNase activity in relation to quantitative and spectral RNA changes. Authentic peroxynitrite, synthesized by a quench-flow technique (O.lml of 30mmol solution) was infused intraventricularly in rats under pentobarbital sodium anesthesia. Animals were sacrified 24h later. The formation of end products (NO2 and N03) in plasma increased (81.94&5.1.5 vs control 45.47&18.80mmol/l p
CHIMERIC
LIVER SPECIFIC
GENE EXPRESSION
VECTOR
EXPRESSION
POTENTIAL
R. Pavlovic2, D. Pavlovic’, R. Kocic3, G. Kocic’, G. Bjelakovic’, T. Jevtovic’, G. Nikolic2, I. Stojanovic’. ‘Department Of Biochemistry Medical Faculty, Nis, Yugoslavia; 2Department Of Chemistry Medical Faculty, Nis, Yugoslavia; ‘Clinic For Endocrinology Medical Faculty, Nis, Yugoslavia
I735
ADENOVIRAL TRANSGENE
PROMOTERS
FOR LONG-TERM
IN THE LIVER
M.G. Kramer, C. Gomar, M. Barajas, M. Zabala, C. Qian, .I. Prieto. University Of Navarra, School Of Medicine, Department Of Internal Medicine, Division Of Hepatology And Gene Therapy, Pamplona, Spain Background and Aims: Therapy of some genetic or acquired diseases using gene transfer procedures may require promoters that restrict gene expression to the liver and ensure a durable synthesis of the needed protein. Our aim was to construct a collection of liver specific promoters with different activity to be used when a certain amount of the gene product is sought in hepatic cells. Methods: We have generated chimeric constructs by combining promoter and enhancer regions of the albumin, alpha 1-antitrypsin, hepatitis B virus core protein and hemopexin genes to drive the expression of the human alpha 1-antitrypsin (hAAT) gene as a reporter. The serum levels of hAAT were determined during a period of 2 months in mice whose liver has been transfected with plasmid DNA using the hydrodynamics-based procedure. Results: Between 10.20% of liver cells incorporate the DNA vector using the hydrodynamics-based methodology. Peak serum concentration of hAAT varied according to the promoter used and with some of these reg-
DESIGNED
FOR P53 DEPENDENT
AND REPLICATION
TOOL FOR CANCER
AS A
GENE THERAPY
F. Kuehnel, L. Zender, T. Wirth, B. Schulte, M.P. Manns, S. Kubicka. Dept Of Gastroenterology, Hepatology And Endocrinology, MHH, Hannoves Get-many The major obstacle of adenoviral gene therapy of cancer is the restriction of therapeutic gene expression or viral replication to malignant cells to avoid severe side effects on healthy tissue. In this study we present the bicistronic adenoviral vector Adp53dpR for gene therapy of cancer cells with altered p53-status. Initially a luciferase reporter version of Adp53dpR was constructed. One transgene cistron of this vector consists of a luciferase gene under control of an artificial GAL4-binding CMV-promoter. Cotransfection experiments in hepatoma cell lines revealed that this promoter was strongly repressed by the repressor GAL4-KRAB. The second cistron of Adp53dpR (Luc) contains the gene for GAL4-KRAB under control of the artificial p53-dependent promoter prMinRGC. This promoter was shown to be strongly upregulated following activation of endogenous ~53 in HepG2 or by cotransfection of p53wt in Hep3B cells whereas the promoter was almost inactive in absence of p53wt. For generating the adenoviral vector both cistrons were cloned into the backbone of an El/E3-deleted adenovirus. The control virus AdR (Luc) was comparably constructed but was lacking the ~53 dependent promoter. Infection experiments in HepG2 (HuH7 as control) followed by luciferase measurements showed that the luciferase expression in Adp53dpR- (Luc) -infected HepG2 was repressed 65fold compared to AdR (luc) -infected cells. This repression could be enhanced up to 200fold when ~53 was preactivated by Doxorubicin. For the construction of a replication competent vector the luciferase gene was replaced by the adenoviral early regions ElA/ElB. In Doxorubicin treated HepG2 cells infected by Adp53dpR viral replication was inhibited compared to the control virus AdR. In ~53 mutated Huh7 cells both vector types showed an equivalent replication rate. Together these data suggest that Adp53dpR provides an effective tool for a specific transgene expression in p53-altered cancer cells and might help to reduce deleterious side effects on differentiated tissue if used for the application of therapeutic genes. Furthermore the replication competent form of this system is capable to replicate selectively in ~53 altered cancer cells.
I737
COLLAGEN
TYPE IA1 GENE SPl
BONE DISEASE
POLYMORPHISM
AND
IN PRIMARY BILIARY CIRRHOSIS
P.L. Lakatos’, E. Bajnok’, A. Folhoffer’, A. Horvath’, I. Tomai2, l? Lakatos I, F. Szalay I. ’1stDepartment Of Medicine, Semmelweis University, Budapest, Hungary; 22nd Department Of Internal Medicine, Debrecen University, Debrecen, Hungary Genetic factors have been implicated in the pathogenesis of osteoporosis, a common disorder in primary biliary cirrhosis (PBC). Collagen type Ial (COLIAl) is the most important structural protein of bone matrix. Spl polymorphism of COLIAl gene was found to be associated with reduced bone mineral density (BMD) in osteoporosis and in the only one study done in PBC. We investigated COLIAl gene Spl polymorphism and BMD in Hungarian PBC patients. Patients and Methods: 47 women with PBC (mean age: 56.9 yrs, range: 36-8 1 yrs, each AMA M2 positive, stage I-IV) and 82 age-matched healthy
ulatory sequences the level of expression declined with time. Among the chimeric promoters analyzed, the alpha 1-antitrypsin promoter combined with the albumin enhancer was found to be the strongest and more stable regulatory sequence. Conclusion: We have developed a collection of liver-specific promoters of different activity in liver cells. A sustained, long-lasting and high level of expression of therapeutic genes could be achieved using the alpha lantitrypsin promoter fused to the albumin enhancer
I734
I736
THE IMPACT OF RNASES CONTROL NITROGEN
IN POST-TRANCRIPTIONAL
OF RNA POPULATION SPECIES
AFTER REACTIVE
AlTACK
The central role of ribonucleases (RNases) in the control of the splicing processes and response to changing cell environments make them oldest regulatory‘housekeeping enzymes”. Since RNase activity is further controlled by the presence of naturally-occurring inhibitor, the aim of the study was to evaluate effect of peroxynitrite on liver RNase activity in relation to quantitative and spectral RNA changes. Authentic peroxynitrite, synthesized by a quench-flow technique (O.lml of 30mmol solution) was infused intraventricularly in rats under pentobarbital sodium anesthesia. Animals were sacrified 24h later. The formation of end products (NO2 and N03) in plasma increased (81.94&5.1.5 vs control 45.47&18.80mmol/l p
CHIMERIC
LIVER SPECIFIC
GENE EXPRESSION
VECTOR
EXPRESSION
POTENTIAL
R. Pavlovic2, D. Pavlovic’, R. Kocic3, G. Kocic’, G. Bjelakovic’, T. Jevtovic’, G. Nikolic2, I. Stojanovic’. ‘Department Of Biochemistry Medical Faculty, Nis, Yugoslavia; 2Department Of Chemistry Medical Faculty, Nis, Yugoslavia; ‘Clinic For Endocrinology Medical Faculty, Nis, Yugoslavia
I735
ADENOVIRAL TRANSGENE
PROMOTERS
FOR LONG-TERM
IN THE LIVER
M.G. Kramer, C. Gomar, M. Barajas, M. Zabala, C. Qian, .I. Prieto. University Of Navarra, School Of Medicine, Department Of Internal Medicine, Division Of Hepatology And Gene Therapy, Pamplona, Spain Background and Aims: Therapy of some genetic or acquired diseases using gene transfer procedures may require promoters that restrict gene expression to the liver and ensure a durable synthesis of the needed protein. Our aim was to construct a collection of liver specific promoters with different activity to be used when a certain amount of the gene product is sought in hepatic cells. Methods: We have generated chimeric constructs by combining promoter and enhancer regions of the albumin, alpha 1-antitrypsin, hepatitis B virus core protein and hemopexin genes to drive the expression of the human alpha 1-antitrypsin (hAAT) gene as a reporter. The serum levels of hAAT were determined during a period of 2 months in mice whose liver has been transfected with plasmid DNA using the hydrodynamics-based procedure. Results: Between 10.20% of liver cells incorporate the DNA vector using the hydrodynamics-based methodology. Peak serum concentration of hAAT varied according to the promoter used and with some of these reg-
DESIGNED
FOR P53 DEPENDENT
AND REPLICATION
TOOL FOR CANCER
AS A
GENE THERAPY
F. Kuehnel, L. Zender, T. Wirth, B. Schulte, M.P. Manns, S. Kubicka. Dept Of Gastroenterology, Hepatology And Endocrinology, MHH, Hannoves Get-many The major obstacle of adenoviral gene therapy of cancer is the restriction of therapeutic gene expression or viral replication to malignant cells to avoid severe side effects on healthy tissue. In this study we present the bicistronic adenoviral vector Adp53dpR for gene therapy of cancer cells with altered p53-status. Initially a luciferase reporter version of Adp53dpR was constructed. One transgene cistron of this vector consists of a luciferase gene under control of an artificial GAL4-binding CMV-promoter. Cotransfection experiments in hepatoma cell lines revealed that this promoter was strongly repressed by the repressor GAL4-KRAB. The second cistron of Adp53dpR (Luc) contains the gene for GAL4-KRAB under control of the artificial p53-dependent promoter prMinRGC. This promoter was shown to be strongly upregulated following activation of endogenous ~53 in HepG2 or by cotransfection of p53wt in Hep3B cells whereas the promoter was almost inactive in absence of p53wt. For generating the adenoviral vector both cistrons were cloned into the backbone of an El/E3-deleted adenovirus. The control virus AdR (Luc) was comparably constructed but was lacking the ~53 dependent promoter. Infection experiments in HepG2 (HuH7 as control) followed by luciferase measurements showed that the luciferase expression in Adp53dpR- (Luc) -infected HepG2 was repressed 65fold compared to AdR (luc) -infected cells. This repression could be enhanced up to 200fold when ~53 was preactivated by Doxorubicin. For the construction of a replication competent vector the luciferase gene was replaced by the adenoviral early regions ElA/ElB. In Doxorubicin treated HepG2 cells infected by Adp53dpR viral replication was inhibited compared to the control virus AdR. In ~53 mutated Huh7 cells both vector types showed an equivalent replication rate. Together these data suggest that Adp53dpR provides an effective tool for a specific transgene expression in p53-altered cancer cells and might help to reduce deleterious side effects on differentiated tissue if used for the application of therapeutic genes. Furthermore the replication competent form of this system is capable to replicate selectively in ~53 altered cancer cells.
I737
COLLAGEN
TYPE IA1 GENE SPl
BONE DISEASE
POLYMORPHISM
AND
IN PRIMARY BILIARY CIRRHOSIS
P.L. Lakatos’, E. Bajnok’, A. Folhoffer’, A. Horvath’, I. Tomai2, l? Lakatos I, F. Szalay I. ’1stDepartment Of Medicine, Semmelweis University, Budapest, Hungary; 22nd Department Of Internal Medicine, Debrecen University, Debrecen, Hungary Genetic factors have been implicated in the pathogenesis of osteoporosis, a common disorder in primary biliary cirrhosis (PBC). Collagen type Ial (COLIAl) is the most important structural protein of bone matrix. Spl polymorphism of COLIAl gene was found to be associated with reduced bone mineral density (BMD) in osteoporosis and in the only one study done in PBC. We investigated COLIAl gene Spl polymorphism and BMD in Hungarian PBC patients. Patients and Methods: 47 women with PBC (mean age: 56.9 yrs, range: 36-8 1 yrs, each AMA M2 positive, stage I-IV) and 82 age-matched healthy