- Email: [email protected]
[379] EVOLVING CYTOGENETIC ANOMALIES IN LONG-TERM CULTURES OF TELOMERASE-IMMORTALIZED HUMAN FETAL HEPATOCYTES
040. MOLECULAR AND CELLULAR BIOLOGY
~
D ) LIVER REGENERATION AND EXPERIMENTAL ONCOLOGY
Methods: gene expression was analyzed in 2 independent lines of SV40 immortalized mouse hepatocytes untreated, treated with desferioxamine l00mcM and FeAmCit 150mcM (4 replicates x 3 conditions x 2 lines) by high-density oligonucleotide microarrays (Affymetrix), qRT-PCR and Western blotting; cell proliferation was evaluated by 3H-thymidine and growth curves; primary human monocytes were isolated by HFE wt/wt and C282Y/C282Y subjects (n= 10) by aCD14-microbeads.
(:-act+
H M CESZY/C28iV 1'1.3;
',,t;W'
:n-?!
HFE status affects Mdm2 and p53 expression in primary human monocytes. Results: FeAmCit downregulated and Dfo upregulated the expression of Mdm-2, the ubiquitin-ligase involved in p53 transcriptional inactivation and degradation, of its coactivator cyclin-G1, in both cell lines. Downregulation of Mdm-2 in the presence of iron was confirmed by Western blotting, and was associated with upregulation of p53 protein levels, and of the p53 target antioxidant genes aldehyde dehydrogenase4al and Sod2. Conversely, antioxidants (NAcCys and Vitamin E) inhibited the iron dependent increase in p53. FeAmCit induced hepatocyte proliferation independently of growth factors, suggesting that iron did not induce apoptosis in immortalized cells. Mdm-2 expression was higher and p53 expression lower in C282Y-'-, characterized by lower intracellular free iron vs. wt/wt primary human monocytes. Conclusions: iron status regulates Mdm-2, p53 and target antioxidant genes in hepatocytes and human monocytes. Mdm-2 down-regulation may be involved in the management of iron-dependent oxidative stress in normal and neoplastic cells.
13791 EVOLVING CYTOGENETIC ANOMALIES IN LONG-TERM CULTURES OF TELOMERASE-IMMORTALIZED HUMAN FETAL HEPATOCYTES B. Haker', S. Fuchs2, J. Dierlamm3, T.H. B r ~em m en d o r f H. ~, Were'. 'Department of Gastrorntrrologv and Hrpatology; 'Drpurtment of Hunzun Genetics; 3Departnzent q j Oncology and Hematologv, linioersity Medical Center Hum burg-E p m i o y f , ; Hum burg, Germany E-mail: [email protected]
Background and Aims: Expansion of human fetal liver cells is limited by replication-associated telomere erosion. We demonstrated that human fetal hepatocytes are amenable to immortalization by ectopic expression of telomerase reverse transcriptase, the catalytic and rate-limiting subunit of the telomerase holoenzyme. Since high levels of telomerase activity are a hallmark of many cancer entities, including hepatocellular carcinoma, and telomerase activation has been described as early step in carcinogenesis, we evaluated permissiveness of telomerase-immortalized human fetal hepatocytes for oncogenic lcaryotype changes. Methods: Telomere length was measured by telomere flow-FISH, a combination of telomere-specific FISH and flow cytometry. Conventional G-banding and multicolor FISH were performed to monitor cytogenetic integrity in two independently immortalized and expanded populations. Oncogenic transformation was determined by soft-agar assays, an established in vitro indicator for malignant growth, and assessment of functional growth control via p53.
S147
Results: Characterization of telomerase-immortalized human fetal hepatocytes revealed maintenance of robust levels of telomerase activity with telomere stabilization above 8 kb despite extended proliferation. Cytogenetic investigations confirmed a stable diploid karyotype beyond the expected stage of replicative senescence. However, following 80 to 100 population doublings (PD), we detected clones with aneuploidy, for example, trisomy 7, 8, and 14. Interestingly, trisomy 7 evolved as characteristic karyotype aberration in two separately cultured populations. Furthermore, cells with trisomy 7 dominated our cultures at later time points (40% of cells at PD 100 and 90% of cells at PD 150), thus, demonstrating a possible selective advantage for cells with trisomy 7. Multicolor FISH confirmed our G-banding results, but did not show additional structural lcaryotype changes. The detected karyotype changes did not result in oncogenic transformation reflected by anchorage-independent growth andor loss of effective p53-mediated DNA damage response. Conclusions: While telomerase reconstitution immortalizes human fetal hepatocytes, these cells do per se not display characteristics of transformed cells in long-term culture. However, telomere maintenance is not sufficient to prevent cytogenetic changes. Moreover, karyotype abnormalities arising in long-term culture resemble some of the cytogenetic events observed in clinical samples. Therefore, telomerase-immortalized human liver cells might serve as culture model to examine cytogenetic changes driving hepatocarcinogenesis.
13801 RADIOFREQUENCY THERMAL ABLATION OF HEPATOCELLULAR CARCINOMA MAY ACTIVATE REGULATORY T CELLS BLOCKING ANTI-TUMOR T-CELL RESPONSES A . Zerbini' , M . Pilli' , G. Pelosi', G. Menozzi' , D. Laccabue' , S. Cerioni', M.G. Pizzi', S. Schivazappa', F. Fagnoni2, C. Ferrari', G. Missale' . 'Lahorutory of Viral lmmunopathology,Azirndu Ospedaliero, Uniurr~xituriadi Purma, P u m a ; 2Luhoratory of Exprrimrntal Oncology, Fonduzionr Saluutorr Muugeri, Clinicu Del Luuoro e Della Riahilituzione, IRCCS, Paoiu, Ituly E-mail: [email protected]
Background and Aim: Radiofrequency thermal ablation (RFA) of HCC liver nodules has been shown to enhance tumor-specific T cell responses in man. Similar studies in animal models have shown that combination of RFA with intralesional injection of DC or with blocking of regulatory T-cells (Treg) by anti-CTLA-4 antibodies may protect mice from tumor rechallenge. Aim of our study was to evaluate the effect of RFA on frequency and function of regulatory T cells and NK cells. Methods: Peripheral blood mononuclear cells were derived from 1 I subjects undergoing RFA for HCC the day before, 1 and 4 weeks after treatment. Treg and NK cells were quantified by flow-cytometry. In order to test T-cell responses directed to HCC and to evaluate the in-vitro effect of CTLA-4 blocking, 20-mer peptides overlapping by I0 residues representing MAGE-I and MAGE-3 tumor-associated antigens, known to be widely expressed in HCC, were pooled in 4 mixtures and used to generate short term T-cell lines. T-cell lines specificity was tested by intracellular cytokine staining for IFN-gamma. Results: A significant increase of the frequency of NK cells (CDl6+CD56+) was observed after RFA: 10+3.4% before treatment, 15.4+5.4% 1 week after treatment (p <0.005) and 16.9+7.9% 4 weeks 0.01). Frequency of Treg (CD4+CD25+FoxP3+) was after treatment (p i quite stable during treatment, showing however an increase of the mean fluorescence intenstity (MFI) of both FoxP3 (mean MFI on CD4+CD25+ cells 35.2+10% before treatment vs 41.6+22.5% 4 weeks after treatment) and CTLA-4 (mean MFI on CD4+CD25+FoxP3+ cells 36.1+15% before treatment vs 38.1+1 1.1% 4 weeks after treatment). RFA treatment enhanced MAGE-specific T-cell response: 4 MAGE-specific T cell lines could be generated before treatment compared to 1 I T-cell lines 4 weeks after treatment. lOil5 MACE-specific T-cell lines were expanded by blocking CTLA-4 in-vitro.
~
D ) LIVER REGENERATION AND EXPERIMENTAL ONCOLOGY
Methods: gene expression was analyzed in 2 independent lines of SV40 immortalized mouse hepatocytes untreated, treated with desferioxamine l00mcM and FeAmCit 150mcM (4 replicates x 3 conditions x 2 lines) by high-density oligonucleotide microarrays (Affymetrix), qRT-PCR and Western blotting; cell proliferation was evaluated by 3H-thymidine and growth curves; primary human monocytes were isolated by HFE wt/wt and C282Y/C282Y subjects (n= 10) by aCD14-microbeads.
(:-act+
H M CESZY/C28iV 1'1.3;
',,t;W'
:n-?!
HFE status affects Mdm2 and p53 expression in primary human monocytes. Results: FeAmCit downregulated and Dfo upregulated the expression of Mdm-2, the ubiquitin-ligase involved in p53 transcriptional inactivation and degradation, of its coactivator cyclin-G1, in both cell lines. Downregulation of Mdm-2 in the presence of iron was confirmed by Western blotting, and was associated with upregulation of p53 protein levels, and of the p53 target antioxidant genes aldehyde dehydrogenase4al and Sod2. Conversely, antioxidants (NAcCys and Vitamin E) inhibited the iron dependent increase in p53. FeAmCit induced hepatocyte proliferation independently of growth factors, suggesting that iron did not induce apoptosis in immortalized cells. Mdm-2 expression was higher and p53 expression lower in C282Y-'-, characterized by lower intracellular free iron vs. wt/wt primary human monocytes. Conclusions: iron status regulates Mdm-2, p53 and target antioxidant genes in hepatocytes and human monocytes. Mdm-2 down-regulation may be involved in the management of iron-dependent oxidative stress in normal and neoplastic cells.
13791 EVOLVING CYTOGENETIC ANOMALIES IN LONG-TERM CULTURES OF TELOMERASE-IMMORTALIZED HUMAN FETAL HEPATOCYTES B. Haker', S. Fuchs2, J. Dierlamm3, T.H. B r ~em m en d o r f H. ~, Were'. 'Department of Gastrorntrrologv and Hrpatology; 'Drpurtment of Hunzun Genetics; 3Departnzent q j Oncology and Hematologv, linioersity Medical Center Hum burg-E p m i o y f , ; Hum burg, Germany E-mail: [email protected]
Background and Aims: Expansion of human fetal liver cells is limited by replication-associated telomere erosion. We demonstrated that human fetal hepatocytes are amenable to immortalization by ectopic expression of telomerase reverse transcriptase, the catalytic and rate-limiting subunit of the telomerase holoenzyme. Since high levels of telomerase activity are a hallmark of many cancer entities, including hepatocellular carcinoma, and telomerase activation has been described as early step in carcinogenesis, we evaluated permissiveness of telomerase-immortalized human fetal hepatocytes for oncogenic lcaryotype changes. Methods: Telomere length was measured by telomere flow-FISH, a combination of telomere-specific FISH and flow cytometry. Conventional G-banding and multicolor FISH were performed to monitor cytogenetic integrity in two independently immortalized and expanded populations. Oncogenic transformation was determined by soft-agar assays, an established in vitro indicator for malignant growth, and assessment of functional growth control via p53.
S147
Results: Characterization of telomerase-immortalized human fetal hepatocytes revealed maintenance of robust levels of telomerase activity with telomere stabilization above 8 kb despite extended proliferation. Cytogenetic investigations confirmed a stable diploid karyotype beyond the expected stage of replicative senescence. However, following 80 to 100 population doublings (PD), we detected clones with aneuploidy, for example, trisomy 7, 8, and 14. Interestingly, trisomy 7 evolved as characteristic karyotype aberration in two separately cultured populations. Furthermore, cells with trisomy 7 dominated our cultures at later time points (40% of cells at PD 100 and 90% of cells at PD 150), thus, demonstrating a possible selective advantage for cells with trisomy 7. Multicolor FISH confirmed our G-banding results, but did not show additional structural lcaryotype changes. The detected karyotype changes did not result in oncogenic transformation reflected by anchorage-independent growth andor loss of effective p53-mediated DNA damage response. Conclusions: While telomerase reconstitution immortalizes human fetal hepatocytes, these cells do per se not display characteristics of transformed cells in long-term culture. However, telomere maintenance is not sufficient to prevent cytogenetic changes. Moreover, karyotype abnormalities arising in long-term culture resemble some of the cytogenetic events observed in clinical samples. Therefore, telomerase-immortalized human liver cells might serve as culture model to examine cytogenetic changes driving hepatocarcinogenesis.
13801 RADIOFREQUENCY THERMAL ABLATION OF HEPATOCELLULAR CARCINOMA MAY ACTIVATE REGULATORY T CELLS BLOCKING ANTI-TUMOR T-CELL RESPONSES A . Zerbini' , M . Pilli' , G. Pelosi', G. Menozzi' , D. Laccabue' , S. Cerioni', M.G. Pizzi', S. Schivazappa', F. Fagnoni2, C. Ferrari', G. Missale' . 'Lahorutory of Viral lmmunopathology,Azirndu Ospedaliero, Uniurr~xituriadi Purma, P u m a ; 2Luhoratory of Exprrimrntal Oncology, Fonduzionr Saluutorr Muugeri, Clinicu Del Luuoro e Della Riahilituzione, IRCCS, Paoiu, Ituly E-mail: [email protected]
Background and Aim: Radiofrequency thermal ablation (RFA) of HCC liver nodules has been shown to enhance tumor-specific T cell responses in man. Similar studies in animal models have shown that combination of RFA with intralesional injection of DC or with blocking of regulatory T-cells (Treg) by anti-CTLA-4 antibodies may protect mice from tumor rechallenge. Aim of our study was to evaluate the effect of RFA on frequency and function of regulatory T cells and NK cells. Methods: Peripheral blood mononuclear cells were derived from 1 I subjects undergoing RFA for HCC the day before, 1 and 4 weeks after treatment. Treg and NK cells were quantified by flow-cytometry. In order to test T-cell responses directed to HCC and to evaluate the in-vitro effect of CTLA-4 blocking, 20-mer peptides overlapping by I0 residues representing MAGE-I and MAGE-3 tumor-associated antigens, known to be widely expressed in HCC, were pooled in 4 mixtures and used to generate short term T-cell lines. T-cell lines specificity was tested by intracellular cytokine staining for IFN-gamma. Results: A significant increase of the frequency of NK cells (CDl6+CD56+) was observed after RFA: 10+3.4% before treatment, 15.4+5.4% 1 week after treatment (p <0.005) and 16.9+7.9% 4 weeks 0.01). Frequency of Treg (CD4+CD25+FoxP3+) was after treatment (p i quite stable during treatment, showing however an increase of the mean fluorescence intenstity (MFI) of both FoxP3 (mean MFI on CD4+CD25+ cells 35.2+10% before treatment vs 41.6+22.5% 4 weeks after treatment) and CTLA-4 (mean MFI on CD4+CD25+FoxP3+ cells 36.1+15% before treatment vs 38.1+1 1.1% 4 weeks after treatment). RFA treatment enhanced MAGE-specific T-cell response: 4 MAGE-specific T cell lines could be generated before treatment compared to 1 I T-cell lines 4 weeks after treatment. lOil5 MACE-specific T-cell lines were expanded by blocking CTLA-4 in-vitro.