Previous studies of genes associated with retroviral-induced neoplasia have provided the foundation for much of our current knowledge of both tumor suppressor and oncogenes, and have contributed to our understanding of both gene function and malignant transformation. The study of Friend virus-induced erythroleukemia, a well-studied example of multistage malignancy, has led to the identification of several oncogenes, including the Ets transcription factor, fli-1. Fli-1 plays a vital role in hematopoiesis, and vasculogenesis through the transcriptional regulation of its target genes, some of which are critical for the control of cellular proliferation, differentiation, and survival. The aberrant regulation of Fli-1 is associated with a number of cancers and human diseases, including erythroleukemia, Ewing’s sarcoma, lupus, and Jacobsen or Paris Trousseau syndrome. The essential goal set out to be achieved by the research presented herein is to establish a better understanding of both the oncogenic and developmental roles of Fli-1 by investigating the molecular basis by which its deregulated expression leads to fundamental aberration in the fine balance between proliferation and differentiation.

Previous studies of genes associated with retroviral-induced neoplasia have provided the foundation for much of our current knowledge of both tumor suppressor and oncogenes, and have contributed to our understanding of both gene function and malignant transformation. The study of Friend virus-induced erythroleukemia, a well-studied example of multistage malignancy, has led to the identification of several oncogenes, including the Ets transcription factor, fli-1. Fli-1 plays a vital role in hematopoiesis, and vasculogenesis through the transcriptional regulation of its target genes, some of which are critical for the control of cellular proliferation, differentiation, and survival. The aberrant regulation of Fli-1 is associated with a number of cancers and human diseases, including erythroleukemia, Ewing’s sarcoma, lupus, and Jacobsen or Paris Trousseau syndrome. The essential goal set out to be achieved by the research presented herein is to establish a better understanding of both the oncogenic and developmental roles of Fli-1 by investigating the molecular basis by which its deregulated expression leads to fundamental aberration in the fine balance between proliferation and differentiation.

Both tissue microenvironment and genetic changes are involved in development of cancer. We employed the Friend murine leukemia virus (F-MuLV)- induced erythroleukemia model to study the role of these parameters in induction of malignancy. The tissue microenvironment is composed of non-cellular and cellular components. In regards to the non-cellular part, we previously reported that vascular endothelial growth factor (VEGF), in combination with macrophage chemoattractant protein-5, contributes to leukemia progression in F-MuLV- infected mice. To study the influence of constitutively elevated VEGF levels on the progression of erythroleukemia, we inoculated VEGF hi/+ mice, which are heterozygous for a VEGF “hypermorphic” allele, with F-MuLV. Unexpectedly, a significant delay in erythroleukemia was observed in these mice when compared with wild-type controls. The VEGF hi/+ mice exhibited a higher natural killer (NK) cell activity, elevated B cells, and a decrease in T-cell number. Furthermore, higher erythroid progenitors (i.e. CD34+, CD36+, and TER119+ cells) were evident in the bone marrow, spleen, and peripheral blood of these mice. Also, the CFU-E levels were significantly elevated in VEGF hi/+ bone marrow cultures. We propose that a compensatory erythropoietic response combined with increased NK cell activity account for the extended survival of erythroleukemic, VEGF hi/+mice. In regards to the cellular component of tissue microenvironment we studied the role of B cells in response to F-MuLV. To test the hypothesis that virus- neutralizing antibodies are involved in providing sterilizing immunity to F-MuLV we inoculated adult female mice with F-MuLV so that their newborns are provided with anti-viral antibodies. F-MuLV challenge of these newborns did not lead to induction of erythroleukemia. Conversely, mice from a control group (newborns whose mother had not received viral inoculation) contracted erythroleukemia upon F-MuLV challenge, as shown by hepatosplenomegaly, anemia, and emergence of leukemic cells in the spleen. These results indicated the importance of anti-viral antibodies in immunity to F-MuLV and suggested that anti-F-MuLV antibodies were generated in mothers, transferred to their offspring and protected them from viral challenge. The key genetic event upon F-MuLV infection is viral integration at the Fli-1 locus. We set to identify F-MuLV integration sites in SCID mice following two observations that these mice show a delay in induction of leukemia and also they do not exhibit viral integration at the Fli-1 locus. We hypothesized that development of leukemia in these mice is due to F-MuLV integration at a region other than the Fli-1 locus. Using a GenomeWalking approach we identified a total of 15 viral integration sites in F-MuLV-infected SCID mice, with eight of them interrupting the following genes: Mex3d, Fam125b, Prdm16, Rhoq, Ahdc1, Zc3h4, Msh3, and Hcls1. Using PCR to amplify the virus- host DNA junction fragment we found that one of the viral insertion sites (chromosome 10; position 20,942,825) occurs with a frequency of 35 % and therefore is considered as a common integration site.

Both tissue microenvironment and genetic changes are involved in development of cancer. We employed the Friend murine leukemia virus (F-MuLV)- induced erythroleukemia model to study the role of these parameters in induction of malignancy. The tissue microenvironment is composed of non-cellular and cellular components. In regards to the non-cellular part, we previously reported that vascular endothelial growth factor (VEGF), in combination with macrophage chemoattractant protein-5, contributes to leukemia progression in F-MuLV- infected mice. To study the influence of constitutively elevated VEGF levels on the progression of erythroleukemia, we inoculated VEGF hi/+ mice, which are heterozygous for a VEGF “hypermorphic” allele, with F-MuLV. Unexpectedly, a significant delay in erythroleukemia was observed in these mice when compared with wild-type controls. The VEGF hi/+ mice exhibited a higher natural killer (NK) cell activity, elevated B cells, and a decrease in T-cell number. Furthermore, higher erythroid progenitors (i.e. CD34+, CD36+, and TER119+ cells) were evident in the bone marrow, spleen, and peripheral blood of these mice. Also, the CFU-E levels were significantly elevated in VEGF hi/+ bone marrow cultures. We propose that a compensatory erythropoietic response combined with increased NK cell activity account for the extended survival of erythroleukemic, VEGF hi/+mice. In regards to the cellular component of tissue microenvironment we studied the role of B cells in response to F-MuLV. To test the hypothesis that virus- neutralizing antibodies are involved in providing sterilizing immunity to F-MuLV we inoculated adult female mice with F-MuLV so that their newborns are provided with anti-viral antibodies. F-MuLV challenge of these newborns did not lead to induction of erythroleukemia. Conversely, mice from a control group (newborns whose mother had not received viral inoculation) contracted erythroleukemia upon F-MuLV challenge, as shown by hepatosplenomegaly, anemia, and emergence of leukemic cells in the spleen. These results indicated the importance of anti-viral antibodies in immunity to F-MuLV and suggested that anti-F-MuLV antibodies were generated in mothers, transferred to their offspring and protected them from viral challenge. The key genetic event upon F-MuLV infection is viral integration at the Fli-1 locus. We set to identify F-MuLV integration sites in SCID mice following two observations that these mice show a delay in induction of leukemia and also they do not exhibit viral integration at the Fli-1 locus. We hypothesized that development of leukemia in these mice is due to F-MuLV integration at a region other than the Fli-1 locus. Using a GenomeWalking approach we identified a total of 15 viral integration sites in F-MuLV-infected SCID mice, with eight of them interrupting the following genes: Mex3d, Fam125b, Prdm16, Rhoq, Ahdc1, Zc3h4, Msh3, and Hcls1. Using PCR to amplify the virus- host DNA junction fragment we found that one of the viral insertion sites (chromosome 10; position 20,942,825) occurs with a frequency of 35 % and therefore is considered as a common integration site.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22326号 / 医博第4567号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 小川 誠司, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This investigation focused on four aspects of hexamethylene bisacetamide's ( HMBA ) involvement in induction of differentiation in murine erythroleukemia (MEL) cells: (a) Effects of APAH , a N 8 -acetylspermidine deacetylase inhibitor, on differentiation induced by HMBA and its two deacetylated metabolites, NADAH and DAH , (b) influence of APAH on intracellular levels of HMBA and its deacetylated metabolites in HMBA treated MEL cells, (c) Ca 2+ mobilizing effects of HMBA, NADAH and DAH and (d) effect of APAH on HMBA induced changes in c- myc gene expression during differentiation. HMBA (5 mM) and DAH (2 mM) were equally effective in inducing MEL cell differentiation as measured by the amount of hemoglobin (Hb) produced, while NADAH (5 mM) was least effective. APAH (10–500 0μM) inhibited HMBA and NADAH induced differentiation without affecting DAH induced differentiation. APAH (500 μM) was shown to affect the deacetylation pathway for HMBA. There was a significant increase in intracellular NADAH levels and a decrease in DAH levels in MEL cells treated with both HMBA and APAH compared to HMBA alone (measured by LC/MS). This indicated that APAH inhibited the second deacetylation step, the conversion of NADAH to DAH but not the first, the conversion of HMBA to NADAH. Ca 2+ influx is necessary for HMBA induced MEL cell differentiation. BAPTA-AM (10 μM), a calcium chelator, inhibited HMBA induced Hb production while Tg (0.5 nM), the SERCA pump blocker, potentiated Hb production. 2-APB, a store operated channel (SOC) regulator, at higher concentrations (50,75 μM) prevented HMBA induced differentiation while at lower concentrations (5,10 μM) potentiated induced differentiation. DAH (0.5 mM), caused an immediate increase in [Ca 2+ ] i in MEL cells, while a slower response was seen with NADAH (3 mM). HMBA (5 mM) had the longest lag period (∼6 min) before it elevated [Ca 2+ ] i . APAH effectively prevented [Ca 2+ ] i increase caused by HMBA and NADAH but failed to alter DAH induced increase suggesting that DAH was the metabolite that raised [Ca 2+ ] i levels. Permeabilized cell assays demonstrated that DAH mobilized Ca 2+ from intracellular IP 3 sensitive stores in the ER. The identity of SOC for DAH induced Ca 2+ influx was inconclusive since 2-APB was not able to alter DAH induced Ca 2+ mobilizing responses. In addition to preventing HMBA induced MEL cell differentiation, APAH also inhibited the second phase of repression of c- myc gene expression, a hallmark of induced differentiation. In summary, the present study suggests the mechanism of action of HMBA requires the active involvement of a metabolite, DAH, in differentiation of hematopoietic cells.

Acute erythroleukemia (AEL) is a rare disease typically associated with a poor prognosis. Themedian survival ranges between 3–9months frominitial diagnosis. Hypomethylating agents (HMAs) have been shown to prolong survival in patients with myelodysplastic syndromes (MDS) and AML, but there is limited data of their efficacy in AEL. We collected data from 210 AEL patients treated at 28 international sites. Overall survival (OS) and PFS were estimated using the Kaplan-Meier method and the log-rank test was used for subgroup comparisons. Survival between treatment groups was compared using the Cox proportional hazards regression model. Eighty-eight patients were treated with HMAs, 44 front line, and 122 with intensive chemotherapy (ICT). ICT led to a higher overall response rate (complete or partial) compared to first-line HMA (72% vs. 46.2%, respectively; p 0.001), but similar progression-free survival (8.0 vs. 9.4 months; p = 0.342). Overall survival was similar for ICT vs. HMAs (10.5 vs. 13.7months; p = 0.564), but patients with high-risk cytogenetics treated with HMA first-line lived longer (7.5 for ICT vs. 13.3 months; p = 0.039). Our results support the therapeutic value of HMA in AEL.

Les facteurs de transcription FLI-1 et SPI-1/PU.1 appartiennent à la famille ETS et reconnaissent le même motif sur l’ADN GGAA. Leur activation est observée de manière récurrente dans les érythroleucémies murines induites par le virus de Friend. Ces observations suggèrent un rôle crucial de ces deux facteurs dans la transformation de la lignée érythrocytaire potentiellement par la dérégulation de gènes cibles communs. Mon travail de thèse a consisté à tester la contribution de ces deux facteurs au phénotype des cellules érythroleucémiques et à rechercher les gènes cibles directs communs.Nous avons pu montrer que FLI-1 et SPI-1/PU.1 ont des contributions additives au phénotype des cellules érythroleucémiques surexprimant les deux facteurs. Par une approche transcriptomique, nous avons identifié une grande proportion de gènes cibles directs communs à FLI-1 et à SPI-1/PU.1 impliqués dans différentes étapes de la biogenèse des ribosomes. La déplétion de ces facteurs induit une réduction de la biogenèse des ribosomes qui n’induit pas de stress ribosomique stabilisant p53. Néanmoins, nous avons mis en évidence une contribution spécifique de RPL11, un médiateur essentiel du stress ribosomique, à la différenciation des cellules érythroleucémiques induites par l’absence de ces facteurs.Nous avons mené en parallèle l’inventaire par ChIP-Seq des sites de recrutement de FLI-1 et de SPI-1/PU.1 sur le génome entier de 3 lignées érythroleucémiques indépendantes. Cette stratégie nous a permis de montrer que les régions de recrutement communes sont la conséquence de la proximité de consensus spécifiques et distincts et du recrutement de FLI-1 et de SPI-1/PU.1 sur leur propre consensus. / The transcription factors FLI-1 and SPI-1/PU.1 belong to the ETS family and recognize the same DNA motif GGAA. Their activation is recurrently observed in murine erythroleukemia induced by Friend virus. These observations suggest a crucial role of these two factors in erythroid lineage transformation potentielly by deregulating common target genes. My thesis work consisted of testing both factors contribution to the phenotype of erythroleukemia cells and of searching for common direct target genes.We showed that FLI-1 and SPI-1/PU.1 have additive contributions to the phenotype of erythroleukemia cells overexpressing both factors. By a transcriptomic approach, we identified a high proportion of common direct target genes of FLI-1 and SPI-1/PU.1 involved in ribosome biogenesis at different levels. The déplétion of these factors induced a decrease of ribosome number which doesn’t induce a ribosomal stress leading to the p53 stabilization. However, we highlighted a specific contribution of RPL11, an essential ribosomal stress médiator, in erythroleukemia cell differentiation induced by depletion of both factors. In parallel, we mapped at whole génome scale by ChIP-Seq the recruitment site of FLI-1 and SPI-1/PU.1 in 3 independent erythroleukemia cell lines. This strategy allowed us to show that the common recruitment régions are the conséquence of a very close association of clearly distinct and specific consensus binding sites for FLI-1 and SPI-1/PU.1 and that each of those factor sis recruited to its own consensus.

Les stratégies actuelles de lutte contre le cancer consistent à développer de nouveaux traitements à base de molécules d’origine naturelle susceptibles de déclencher l’apoptose de cellules malignes et d’inhiber les principales voies de survie cellulaire. Le premier objectif de ces travaux porte sur les effets anti-prolifératifs et apoptotiques de l’extrait éthanolique de la plante Berberis libanotica sur les lignées érythroleucémiques humaines HEL, K562 et K562 (COX-2+) et sur son effet sur l’expression de la COX-2 dans ces lignées. Nos résultats montrent que l’extrait induit l’apoptose dans les lignées étudiées et ceci par activation de la caspase-3, le clivage de la PARP-1 et la fragmentation de l’ADN. De même, il induit la diminution de l’expression de COX-2. Nous avons démontré que les voies survie de NF-ĸB et p-AKT sont inhibées. Le deuxième objectif de ces travaux consistent à identifier la molécule présente dans cet extrait et qui est capable de déclencher ces effets anticancéreux. Nous avons démontré que la berbérine est la molécule majoritaire dans cet extrait et possède des effets apoptotiques et des effets inhibiteurs des voies de survie, ce qui est similaire aux effets de l’extrait brut.Mots clés :érythroleucémie, apoptose, berberis libanotica, berbérine, COX-2, NF-kB, p-AKT. / The first aim of this study focuses on the apoptotic effect of Berberis Libanotica on human erythroleukemia cell lines HEL, K562, and K562(COX-2+) and it is effect on the expression of COX-2. In light of the reported chemopreventive and chemosensitive effects of natural products on various tumor cells and animal models, we postulated that our Bl extract may mediate their effects through apoptosis induction with suppression of cell survival pathways. We showed that this extract induces apoptosis in eryrhtroleukemia cells by activation of the late markers of caspase-3 activation, PARP cleavage and DNA fragmentation. In the other hand, we showed that Bl extract reduced significantly expression of COX-2 by a dose-dependent manner. In regard to our results, it is clear that the simultaneous inhibition of Akt and NF-κB signalling can significantly contribute to the anticancer effects of Bl extract in human erythroleukemia cells.The second objective of this report is to elucidate wich molecule present in our extract can exert this effects. We found that berberine, the majoritory compound, can induce an apoptotic effect and inhibits the survival pathway of NF-ĸB and p-AKT similarly to the extract.Key words: erythroleukemia, apoptosis, Berberis libanotica ,berberine, COX-2, NF-ĸB, p-AKT

Polyamines are naturally occurring cellular polycations essential for cell growth and differentiation. This investigation focused on the quantitative analysis of polyamines and metabolites in mouse erythroleukemia (MEL) cells by mass spectrometry. Hexamethylene bisacetamide (HMBA) is a synthetic polyamine derivative known to induce differentiation of a variety of transformed cells such as MEL cells. A fast and sensitive quantitative method for HMBA and metabolites NADAH, DAH and AcHA was developed using atmosphere pressure chemical ionization mass spectrometry (APCI/MS) by flow injection analysis. Selected ion monitoring (SIM) mode was employed for the mass spectrometric detection and d 4 -DAH was used as the internal standard for quantitation. The intracellular concentrations of HMBA and metabolites were obtained in MEL cells treated with 5mM HMBA in the presence or absence of 500μM APAH, a potent N 8 -acetylspermidine deacetylase inhibitor. A significant increase in intracellular NADAH and decrease in DAH levels in MEL cells were observed in HMBA treatment in the presence of APAH. This result indicates that APAH inhibits the second deacetylation step in HMBA metabolism, the conversion of NADAH to DAH, but not the first deacetylation of HMBA to NADAH. Two histone deacetylase (HDAC) inhibitors including sodium butyrate (NaB) and m -carboxycinnamic acid bis-hydroxamide (CBHA) were also used as inducing agents for MEL cell differentiation. Both agents caused accumulation of hyperacetylated histone H4 and H2B in MEL cells at concentrations optimal for inducing differentiation, while neither HMBA nor APAH had detectable effect on the acetylation level of histones. A fast and sensitive method for five important polyamines including putrescine (PU), spermidine (SPD), spermine (SPM), N 1 -acetylspermidine (N 1 -AcSPD) and N 8 -acetylspermidine (N 8 -AcSPD) was also developed using APCI/MS by flow injection analysis. Selected reaction monitoring (SRM) mode was employed for the mass spectrometric detection and 1,7-diaminoheptane was used as the internal standard for quantitation. The intracellular polyamine concentrations was obtained in MEL cells treated with 5mM HMBA, 2mM NaB, 3μM CBHA and 500μM APAH respectively. A significant increase in N 8 -acetylspermidine levels was observed during 3hr to 4 days treatment with APAH. There was no change in N 8 -acetylspermidine levels in MEL cells treated either with NaB or CBHA. The results from the present study suggest APAH has a selective inhibitory effect on N 8 -acetylspermidine but not histone deacetylation. While HDAC inhibitors inhibit histone deacetylase but have no effect on N 8 -acetylspermidine deacetylation. In conclusion, despite the known similarities they share, the enzymes involved in the deacetylation process of N 8 -acetylspermidine and histones in MEL cells are different.