Auswahl der wissenschaftlichen Literatur zum Thema „Leukemia Physiopathology“

As childhood cancer treatment has become more effective, survival rates have improved, and a number of complications have been described while many of these patients reach adulthood. Obesity is a well-recognized late effect, and its metabolic effects may lead to cardiovascular disease. Currently, studies concerning overweight have focused on acute lymphocytic leukemia and brain tumors, since they are at risk for hypothalamic-pituitary axis damage secondary to cancer therapies (cranial irradiation, chemotherapy, and brain surgery) or to primary tumor location. Obesity and cancer have metabolic syndrome features in common. Thus, it remains controversial if overweight is a cause or consequence of cancer, and to date additional mechanisms involving adipose tissue and hypothalamic derangements have been considered, comprising premature adiposity rebound, hyperinsulinemia, leptin regulation, and the role of peroxisome proliferator-activated receptor γ. Overall, further research is still necessary to better understand the relationship between adipogenesis and hypothalamic control deregulation following cancer therapy.

Over the past decades, genetic advances have allowed a more precise molecular characterization of AML with the identification of novel oncogenes and tumor suppressors as part of a comprehensive AML molecular landscape. Recent advances in genetic sequencing tools also enabled a better understanding of AML leukemogenesis from the preleukemic state to posttherapy relapse. These advances resulted in direct clinical implications with the definition of molecular prognosis classifications, the development of treatment recommendations based on minimal residual disease (MRD) measurement and the discovery of novel targeted therapies, ultimately improving AML patients’ overall survival. The more recent development of functional genomic studies, pushed by novel molecular biology technologies (short hairpin RNA (shRNA) and CRISPR-Cas9) and bioinformatics tools design on one hand, along with the engineering of humanized physiologically relevant animal models on the other hand, have opened a new genomics era resulting in a greater knowledge of AML physiopathology. Combining descriptive and functional genomics will undoubtedly open the road for an AML cure within the next decades.

Fanconi anemia (FA), the most common inherited bone marrow failure and leukemia predisposition syndrome, is generally attributed to alterations in DNA damage responses due to the loss of function of the DNA repair and replication rescue activities of the FANC pathway. Here, we report that FANCA deficiency, whose inactivation has been identified in two-thirds of FA patients, is associated with nucleolar homeostasis loss, mislocalization of key nucleolar proteins, including nucleolin (NCL) and nucleophosmin 1 (NPM1), as well as alterations in ribosome biogenesis and protein synthesis. FANCA coimmunoprecipitates with NCL and NPM1 in a FANCcore complex–independent manner and, unique among the FANCcore complex proteins, associates with ribosomal subunits, influencing the stoichiometry of the translational machineries. In conclusion, we have identified unexpected nucleolar and translational consequences specifically associated with FANCA deficiency that appears to be involved in both DNA damage and nucleolar stress responses, challenging current hypothesis on FA physiopathology.

Mastocytosis is a rare disease due to the abnormal accumulation of mast cells in various tissues. Its clinical presentation is heterogeneous depending on mast cell infiltration and mediators release. In some cases, it is associated with hematological malignancies. Prognosis varies from very good with a life expectancy similar to the general population in indolent forms of the disease to a survival time of just a few months in mast cell leukemia. Although in most cases a somatic KIT D816V mutation is found in tumor mast cells, the physiopathology of the disease is not yet fully understood. Additional germline and somatic mutations may explain this heterogeneity. Treatments aim at blocking effect of mast cell mediators, reducing mast cell activation and tumor burden. New drugs mainly directed against the tyrosine kinase activity of KIT have dramatically changed the quality of life and prognosis of mast cell diseases. Present and future therapeutic strategies are discussed in this review.

Among oncohematological diseases, acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) are characterized by the uncontrolled production and accumulation of blasts that can lead to death. Although the physiopathology of these diseases is multifactorial, a genetic factor seems to be at play. Several studies worldwide have shown association of ALL and AML with several alleles of the major histocompatibility complex (MHC).Objective. To determine gene frequencies of HLA-B alleles in Mexicans (individuals with Native American genetic background admixed with European descent) with ALL and AML.Methods. We compared the HLA-B alleles in 213 patients with ALL and 85 patients with AML to those present in 731 umbilical cord blood (UCB) samples as a control group; this was done by means of the PCR-SSP technique.Results. We found an increased frequency of the HLA-B*40 allele in ALL patients as compared to the control group (14.5% versus 9.84%,P=0.003, OR = 1.67); this was particularly evident in a subgroup of young (less than 18 years old) ALL patients (P=0.002, OR = 1.76); likewise, a decreased frequency of HLA-B*40 allele in AML patients was observed as compared to the control group (4.70% versus 9.84%,P=0.02, OR = 0.42).Conclusions. These results might suggest opposing effects of the HLA-B*40 in the genetic susceptibility to develop ALL or AML and offer the possibility to study further the molecular mechanisms of cell differentiation within the bone marrow lineage.

Abstract Fanconi anemia (FA) is the most frequent inherited cause of BM failure (BMF). Fifteen FANC genes have been identified to date, the most prevalent being FANCA, FANCC, FANCG, and FANCD2. In addition to classical presentations with progressive BMF during childhood and a positive chromosome breakage test in the blood, atypical clinical and/or biological situations can be seen in which a FA diagnosis has to be confirmed or eliminated. For this, a range of biological tools have been developed, including analysis of skin fibroblasts. FA patients experience a strong selective pressure in the BM that predisposes to clonal evolution and to the emergence in their teens or young adulthood of myelodysplasia syndrome (MDS) and/or acute myeloid leukemia (AML) with a specific pattern of somatic chromosomal lesions. The cellular mechanisms underlying (1) the hematopoietic defect which leads to progressive BMF and (2) somatic clonal evolutions in this background, are still largely elusive. Elucidation of these mechanisms at the molecular and cellular levels should be useful to understand the physiopathology of the disease and to adapt the follow-up and treatment of FA patients. This may also ultimately benefit older, non-FA patients with aplastic anemia, MDS/AML for whom FA represents a model genetic condition.

Abstract Introduction: Primary Plasma Cell Leukemia (pPCL) is a rare form of multiple myeloma (MM) that is characterized by an aggressive disease course with >20% peripherally circulating plasma cells (PCs) and poor clinical outcome. Despite the advances of modern anti-MM therapy, pPCL patients continue to experience low median overall survival (OS) suggesting a distinct biological background. Due to its low incidence of 1-2% of all MM patients, studies on physiopathology remain challenging and are limited. The aim of this study was to elucidate the differences in biology and outcome between non-pPCL MM and pPCL, to determine the genetic landscape of pPCL and to identify distinct signatures and pathways that potentially could be used as therapeutic targets. Methods: We performed gene expression profiling (GEP; Affymetrix U133 Plus 2.0) of matched circulating peripheral PCs and bone marrow (BM) PCs from 13 patients. Whole exome sequencing (WES) was performed on purified CD138+ PCs from BM aspirates from 19 pPCL patients with a median depth of 61x. CD34+ sorted cells, taken at the time of stem cell harvest from the same 19 patients, were used as controls. Translocations and mutations were called using Manta and Strelka and annotated as previously reported. Copy number was determined by Sequenza. Results: GEP from the BM and circulating peripheral PCs showed that the expression patterns of the two samples from each individual clustered together, indicating that circulating PCs and BM PCs in pPCL result from the same clone and are biologically clearly related. The clinical characteristics from the patient cohort used for WES analysis were as follows: median age was 58 years (range 36-77), females accounted for 74% (14/19), an elevated creatinine level was found in 78% (14/18) and an elevated LDH level in 71% (10/14). All patients presented with an ISS stage of III. Median OS of the whole dataset was poor at 22 months, which is consistent with OS from previously reported pPCL cohorts. Primary Immunoglobulin translocations were common and identified in 63% (12/19) of patients, including MAF translocations, which are known to carry high risk in 42% (8/19) of patients [t(14;16), 32% and t(14;20), 10%] followed by t(11;14) (16%) and t(4;14) (10%). Furthermore, 32% (6/19) of patients had at least one MYC translocation, which are known to play a crucial role in disease progression. MYC breakpoints (8q24) were identified in 25% with Ig partner loci including IGH (5%), IGK (10%), and IGL (10%). The remaining samples had partner loci including FAM46C (5%), MYNN (5%), SPARC (5%), QRSL1 (5%), RNF126 (5%), PLXNA4 (5%) and CDH7 (5%). The mutational burden of pPCL consisted of a median of 98 non-silent mutations per sample, suggesting that the mutational landscape of pPCL is highly complex and harbors more coding mutations than non-pPCL MM. Driver mutations, that previously have been described in non-pPCL MM showed a different prevalence and distribution in pPCL, including KRAS and TP53 with 47% (9/19) and 37% (7/19) affected patients respectively compared to 21% and 5% in non-PCL MM. PIK3CA (5%), PRDM1 (10%), EP300 (10%) and NF1 (10%) were also enriched in the pPCL group compared to previously reported cases in non-pPCL MM. Biallelic inactivation of TP53 - a feature of Double Hit myeloma - was found in 6/19 (32%) samples, indicating a predominance of high risk genomic features compared to non-pPCL MM. Furthermore, analysis of mutational signatures in pPCL showed that aberrant APOBEC activity was highly prevalent only in patients with a MAF translocation, but not in other translocation groups. Conclusion: In conclusion we present one of the first WES datasets on pPCL with the largest patient cohort reported to date and show that pPCL is a highly complex disease. The aggressive disease behavior can, at least in part, be explained by a high prevalence of MAF and MYC translocations, TP53 and KRAS mutations as well as bi-allelic inactivation of TP53. It is of interest that only KRAS but not NRAS mutations are highly enriched in pPCL. From all highly prevalent genomic alterations in pPCL, only KRAS mutations offer a potential for already available therapeutically targeting with MEK inhibitors, which should be further explored. Disclosures Davies: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; TRM Oncology: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Abbvie: Consultancy; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria. Barlogie:Multiple Myeloma Research Foundation: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Millenium: Consultancy, Research Funding; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC. Morgan:Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Research Funding.

Abstract Despite the major progress obtained in prognosis with the use of tyrosine kinase inhibitors (TKI), the great majority of patients with CML remain on long-term therapy and progression occurs in patients with either primary or secondary resistance. The mechanisms of progression towards accelerated phase (AP) and blast crisis (BC) have been studied so far only in primary patient samples in BC. Currently, there is no in vitro model to study sequentially the molecular events leading from CP to BC as only some primary sequential samples are amenable to analysis. Using induced Pluripotent Stem Cell (iPSC) technology, it is now possible to reprogram the primary leukemic cells to pluripotency and generate a major source of stem cells. To determine the feasibility of studying progression of CML towards AP and BC, we have used a patient-specific iPSC line that we have generated from the primary leukemic cells of a patient who later showed a TKI-resistance requiring an allogeneic stem cell transplant. These iPSC expressed BCR-ABL, exhibited all pluripotency markers and after injection in NSG mice, generated teratoma with differentiation into three germ layers. In hematopoietic differentiation assays using day 19-embryoid bodies (EB), increased numbers of hematopoietic progenitors were found as compared to control iPSC (5-fold increase n= 3). We have then treated leukemic iPSC with the mutagenic agent N-ethyl-N-nitrosourea (ENU) during regular medium changes. After 61 days in ENU cultures, day-19 derived embryoid bodies generated hematopoietic cells (>90% CD45+, CD43+) which proliferated in liquid cultures with myeloid and some blast cell morphology. Cytogenetic analyses of iPSC revealed chromosomal abnormalities such as loss of Y and loss of der q9+, both alterations known to occur in CML during progression. They exhibited increased numbers of micronuclei (MN) as compared to leukemic iPSC without ENU (X 3 Fold increase) suggesting acquisition of a progressive chromosomal instability. CGH array analyses were performed using ENU-treated iPSC-derived hematopoietic cells in two different timepoints as compared to leukemic iPSC cultured without ENU. Genomic aberrations were analyzed by Agilent Cytogenomics software with Mosaicism workflow on HG19 genome. 249 gene loci alterations were detected after polymorphism filtration on European population. These analyses showed DNA losses and DNA gains in genes implicated in mesoderm development and hematopoietic lineage as well as genes implicated in DNA damage response. Several loci of transcription factors were found to be involved such as IKZF1 described in imatinib-refractory chronic myeloid leukemia (Bolton-Gillespie et al. 2013). The aberrations included SIRT1and BLM which is implicated in DNA repair. Several cancer genes were found to be involved, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Remarkably, transcriptome geodataset GSE4170 (Radich et al. 2006) allowed us to associate 125 of 249 of the aberrations that we detected in CML iPSC, with the CML progression genes already described during progression from chronic and AP to BC (p-value =9.43E-32, after ANOVA with 1000 permutations). 38 most predictive aberrations allowed perfect reclassification of BC and chronic phase samples by unsupervised classification. Among these candidates, eleven of them have been described in CML physiopathology and connected to TKI resistance and genomic instability. Majority of them ( 7/11) are connected to chronic phase (FAS, ACTB, TRIM21, ANPEP, MLK1, CSF2RA, and MAGEC2) whereas a minority of them (4/11) are connected to BC (ACP1, SH3YL1, FHL1, IL3RA). Interestingly, these experiments also allowed us to discover the connection of a new multidrug resistance molecule associated to BC and having the ability to modify interferon pathway connected to the TKI sensitivity. Thus, genomic instability pattern that we have generated using a single leukemic iPSC allowed duplication of genomic abnormalities described in CML progression and allowed identification of some novel genes. Overall, these results demonstrated that we have generated for the first time to our knowledge, an in vitro BC model, reproducing genomic events described in patients with BC. This "blast crisis in a dish" tool using patient-derived iPSC will be of major interest to study CML progression and eventually to test novel therapies. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Addendum attached to back facing leaves. Includes bibliographical references (leaves 172-199). The findings establish leukemia inhibitory factor, and possibly oncostatin M, as new members of a small but growing class of cytokines produced in an intracellularly active form and also suggest that the production of alternate transcripts and intercellularly-retained proteins may be a common and important feature of cytokines of the IL-6 and other families.

O STI (imatinib, Glivec) é um inibidor da tirosina quinase BCR-ABL, responsável pela patogênese da leucemia mielóide crônica (LMC). Um total de 110 pacientes com LMC na fase crônica (FC) que falharam ou foram intolerantes ao tratamento com interferon, fase acelerada (FA) e crise blástica (CB) foram tratados com imatinibe entre dezembro de 2000 e setembro de 2003. Resposta hematológica completa e resposta citogenética maior foram observadas em 95,9% e 69,4% respectivamente em pacientes em FC e 93,2% e 36,4% em FA. Apenas 2 pacientes na CB estão vivos. O imatinib foi bem tolerado com altas taxas de resposta.
STI571 (Imatinib, Glevec) is an inhibitor of the Bcr-Abl tyrosine kinase that is central to the pathogenesis of chronic myelogenous leukemia (CML). A total of 110 patients with CML chronic phase (CP) who failed or were intolerant to interferon, accelerated phase (AP) and blastic crisis (BC) were treated with imatinib from December 2000 until September 2003. Complete hematologic response and major cytogenetic response were observed in 95,9% and 69,4% respectively of patients in CP and 93,2% and 36,4% in AP. Only 2 patients are alive in BC. Imatinib is well tolerated with high rates of response

L’objectif de notre travail était de déterminer le rôle joué par l’érythropoïétine et la

thrombopoïétine, respectivement, dans l’anémie et la thrombocytopénie observées

chez des enfants souffrant d’une hémopathie maligne.

Par le dosage simultané de la forme soluble du récepteur de la transferrine et de

l’érythropoïétine dans le sérum nous avons montré que l’anémie observée chez ces

patients est bien la conséquence d’une réduction du nombre de progéniteurs

érythropoïétiques (atteinte médullaire centrale) mais que celle-ci n’est pas la

conséquence d’une production insuffisante d’érythropoïétine. Nous avons fait la

même observation chez des enfants souffrant d’une tumeur solide non

hématologique et chez des patients en cours de traitement par chimiothérapie.

Chez ces derniers patients, en appliquant un modèle de culture de moelle à long

terme, nous avons pu démontrer l’existence d’une altération du microenvironnement

médullaire, probablement induite par la chimiothérapie, se

traduisant par une réduction de son aptitude à supporter le développement de la

lignée érythroïde. Ceci expliquant au moins partiellement l’inadéquation de la

réponse érythropoïétique observée chez ces patients en réponse à l’anémie.

Dans la dernière partie du travail, nous avons montré que la thrombocytopénie très

fréquemment observée chez les patients leucémiques s’accompagne dans la

majorité des cas d’une élévation exponentielle de la concentration de

thrombopoïétine, excepté dans les cas de leucémies de la lignée myéloïde. Chez ces

derniers la concentration de thrombopoïétine est proche des valeurs observées chez

des sujets normaux alors qu’elle devrait être 10 à 100 fois plus élevée compte tenu

du nombre de plaquettes extrêmement bas. Nous avons pu montrer que ces taux

très bas sont la conséquence de la liaison de la thrombopoïétine à un récepteur

spécifique et fonctionnel présent à la surface des cellules leucémiques myéloïdes

qui, en l’utilisant comme facteur de croissance, (stimulant leur prolifération et

retardant leur mort cellulaire) « consomment » la thrombopoïétine présente dans le

sérum.
Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

Malgré l'amélioration des traitements, environ 20% des patients atteints de leucémie aigue lymphoblastiques (LAL) rechutent dans la moelle osseuse ou dans des sites extra-médullaires tels que les ovaires et les testicules, ce qui est particulièrement fréquent dans les rechutes tardives de LAL-B présentant un remaniement ETV6/RUNX1. Les travaux réalisés par Virginie Gandemer en 2007, ont montré que l'expression de CD9 permettait de distinguer les leucémies ETV6/RUNX1 des autres types de leucémie. Le gène CD9 code pour une protéine de la famille des tétraspanines dont l'expression a été corrélée avec le risque métastatique et la survie des patients. Par ailleurs il a été démontré que la protéine CD9 était impliquée dans le homing et la prise de greffe des cellules souches hématopoïétiques et leucémiques. Nous avons donc émis l'hypothèse qu'à travers ses propriétés fonctionnelles sur la migration et le homing, CD9 pourrait être un acteur clé des rechutes de LAL-B. Le but de ce travail de thèse était donc premièrement de déterminer le mode de régulation de CD9 dans les LAL-B ETV6/RUNX1 et deuxièmement de déterminer les effets de l'expression de CD9 sur la motilité et la prise de greffe des LAL-B. Les analyses préalablement réalisées au laboratoire avaient suggéré que CD9 pouvait être régulé par des miARNs. Nous avons identifié un cluster de 3 miARNs potentiellement impliqués dans la régulation de CD9 dans les LAL-B ETV6/RUNX1. Ces résultats doivent cependant être complétés par d'autres analyses fonctionnellles afin d'être confirmés. Nous avons étudié le rôle de la protéine CD9 dans la dissémination des cellules de LAL-B. Nous avons démontré que CD9 était un régulateur potentiel de l'adhésion et un nouveau facteur impliqué dans la migration et le homing dépendants de CXCR4 en favorisant l'activation de RAC1 et les réarrangements de l'actine en réponse au CXCL12. Enfin, nous avons décrit pour la première fois l'influence de CD9 sur la migration et le homing dans les testicules via RAC1. Nos résultats montrent donc que CD9 favorise la dissémination des cellules de LAL-B dans les testicules et suggèrent que cette protéine pourrait constituer un acteur majeur des rechutes tardives de LAL-B dont les mécanismes d'apparitions sont peu connus
Despite improvements in survival rates, approximately 20% of children suffering from acute lymphoblastic leukemia (B-ALL) present relapses from bone marrow or from B-extramedullary sites, such as the testes or ovaries, particularly in cases of late relapse of ETV6/RUNX1-ALL. Virgine Gandemer showed in 2007, that the expression of CD9, a protein from the tetraspanin superfamily, can be used to distinguish ETV6/RUNX1 lymphoblastic leukemia from other types of ALL. CD9 expression has been correlated with the risk of metastasis and is associated with a poor clinical outcome in various types of cancer. Moreover CD9 has been implicated in hematopoietic and leukemic stem cell homing. We hypothesized, that CD9 protein, through its functional properties on migration and homing, could be a key actor of B-ALL relapses. The purpose of our study was then to investigate, first the transcriptional regulation of CD9 in ETV6/RUNX1 B-ALL and secondly, the effect of CD9 expression on motility and engrafment of B lymphoblasts. The analysis of CD9 transcriptional regulation previously made in the team, suggested that it could be regulated by miRNAs. We identified a cluster of 3 miRNAs potentially implicated in the regulation of CD9 expression in ETV6/RUNX1 B-ALL. This result has to be confirmd by more functional analysis. We investigated the role of CD9 in the dissemination of B-ALL. We identified CD9 as a potential regulator of B-ALL cell adhesion and a new factor involved in CXCR4-mediated migration and homing, through the promotion of actin rearrangement in response to CXCL12. We also characterized the effect of CD9 protein expression on RAC1 activation, which had an impact on blast migration and engraftment. Finally, we described, for the first time, the influence of CD9, mediated by RAC1 signaling, on B-cell chemotactic migration and homing in the testis. Our work provides evidence for an impact of CD9 on the ability of pre-B leukemic cells to disseminate to testes, through its effects on migration and homing, and suggests that CD9 may be a key player in late relapses of B-ALL, which are currently poorly understood

L’objectif général de cette thèse a été de mettre en évidence la contribution de l’approche transcriptomique dans la physiopathologie et le traitement des hémopathies malignes. En particulier, comment la technologie des microarrays nous a aidée à étudier diverses problématiques en onco-hématologie.Dans la première partie, notre objectif était d’étudier les cellules Natural killer (Nk) chez les patients atteints de leucémie aiguë myéloïde (LAM). Nous avons comparé la signature transcriptomique des cellules Nk de patients LAM à celle des cellules Nk de sujet sains et suggéré que le facteur de transcription ETS-1 est un bon candidat capable de réguler les récepteurs activateurs NCR (natural cytotoxicity receptors) dont les gènes sont situés sur deux chromosomes différents, même si leur expression reste fortement cordonnée.Dans la seconde partie, nous nous sommes intéressés à la prédiction du sepsis en utilisant une approche transcriptomique dans le cadre de l’autogreffe de cellules souches hématopoïétiques (auto-CSH). En utilisant le même modèle, dans la troisième partie, nous avons mis en évidence l’effet du melphalan en tant que chimiothérapie de conditionnement sur les cellules mononuclées du sang périphérique et identifié un marqueur potentiel de rechute précoce chez les patients atteints de myélome dans le cas de l’auto-CSH. Enfin, dans la dernière partie, notre objectif a été d’analyser le profil d’expression génique des lymphomes B diffus à grandes cellules liés à l’infection par le VIH afin de vérifier ou pas l’existence des sous-types décrits chez les patients immunocompétents
The aim of this research is to demonstrate transcriptomic approach contribution in the physiopathology and treatment of hematological malignancies. In particular, how microarrays technology is used to study several oncohematology difficulties; which remain deaths-related infection, as well as the failure to obtain remission and death related relapse. In the first part, our focus was to study natural killer cells (Nks) in patients affected with acute myeloid leukemia (AML). We compared transcriptomic AML-NKs signature with healthy donors-NKs signature and suggested that ETS-1 transcription factor is a good candidate able to regulate the natural cytotoxicity receptors (NCRs), whose coding genes, are located on two different chromosomes even if their expression remain strongly coordinated.Our second part, aimed to predict sepsis using a transcriptomic approach in the case of autologous stem cell transplantation (auto-HSCT). Using the same model, in the third part, we highlighted the melphalan high-dose chemotherapy effect on peripheral blood mononuclear cells and identified a potential good biomarker of early relapse in patients affected by myeloma in the case of auto-HSCT.Our final focus was to analyze gene expression profile of HIV-related large diffuse B-cell lymphoma type in order to verify the existence of subgroups described in immune-competent patients

Apart from CAD, the transient liberation of AIF during day 6 of TF-1 differentiation could pose another threat to the genomic DNA in cells. We have demonstrated the absence of AIF in the nucleus of TF-1 cells despite its release from the mitochondria by using confocal studies. Moreover, the expression of heat shock protein 70 kDa (Hsp70), a well-known antagonist of AIF, was found to be temporarily increased at day 6. Taken together, our results implied a plausible retention of AIF in the cytoplasm by Hsp70. Although Hsp70 is commonly utilized by many cancer cells to counteract AIF and avoid DNA fragmentation, we are the first to demonstrate its role in suppressing AIF during normal erythroid maturation.
As a whole, we have illustrated that the activated caspase-3, mediated most likely by the mitochondrial pathway, is an essential component in the differentiation of TF-1 cells. Its activation was nevertheless not coupled with DNA fragmentation due to some protective mechanisms such as CAD downregulation, Hsp70 upregulation and overexpression of Bcl-XL. Our study therefore provides some insights in the understanding of the relationship between human erythropoiesis and apoptosis and a better understanding in this regard will undoubtedly facilitate the development of new drugs in the treatment of different hematopoietic diseases.
Caspases play a central role in apoptosis. Their activations during the process are accounted for different biochemical and morphological changes in apoptotic cells. Yet in recent years, increasing studies had shown that caspases were also involved in some non-apoptotic cellular events, including T and B-lymphocytes activation, as well as the terminal differentiation of lens cells, megakaryocytes and erythrocytes.
In order to find out other unknown cellular mechanisms in erythropoiesis, mRNA differential display was employed to compare the gene expression pattern of TF-1 cells at different stages of differentiation. Several differentially expressed genes were identified and subsequently confirmed by RT PCR. These genes include formin binding protein 3, destrin and T-complex protein-1 (TCP-1). Their involvement in erythroid differentiation was still not clear at the moment but would be investigated in the near future. Furthermore, aiming at identifying the interacting proteins or inhibitors of caspase-3 in the system, a pull down assay was developed by means of the bacterial expression of a recombinant human caspase-3 mutant protein. With the mutation in the active site, the binding of our recombinant caspase-3 mutant with two known partners ICAD and BIRII (Baculovirus Inhibitor of apoptosis protein Repeat II) domain has been demonstrated. We hope in the near future that it can be employed to fish out some novel caspase-3 substrates from the differentiating TF-1 cell lysate.
In the present study, the participation of caspase in in vitro erythropoiesis was investigated using a human erythroleukemia cell line TF-1. Erythropoietin (EPO) induced erythroid maturation of TF-1 as indicated by the expression of erythroid-lineage markers like glycophorin A (GPA), transferrin receptors (CD71) and synthesis of hemoglobin (Hb). Activation of caspase-3 was observed from day 6 to day 12 during TF-1 differentiation after EPO treatment. With the administration of caspase-3 specific inhibitor, expressions of GPA and CD71 were partially blocked, suggesting that caspase-3 activation is essential in erythropoiesis in our TF-1 model.
Possible involvement of the intrinsic and extrinsic apoptotic pathways was studied by investigating respectively the activation of pro-caspase-9 and -8. It was found that caspase-9, but not -8, was activated at the corresponding time point when caspase-3 was activated. Besides, a transient mitochondrial depolarization coupled with the release of cytochrome c and apoptosis inducing factor (AIF) were detected on day 6, strongly implying a role of mitochondria in triggering the activation of executioner caspase-3. On the other hand, GPA and CD71 expressions were blocked by the application of mitochondrial depolarization inhibitor cyclosporin A (CyA). Also, the recovery of mitochondrial membrane potential was found to be correlated with an overexpression of Bcl-XL at a late stage of TF-1 differentiation, and the role of Bcl-XL was subsequently manifested further by a significant retardation of erythroid differentiation in the siRNA Bcl-XL knocked down TF-1 cells.
The exact role of caspase-3 in erythroid differentiation is far from clear at this moment. Yet, its regulation in the process is equally intriguing. On the course of TF-1 maturation, activated caspase-3 was able to cleave and de-localize the Inhibitor of Caspase-activated DNase (ICAD) from the nucleus, but at the same time DNA fragmentation was not detected by TUNEL assay nor agarose electrophoresis. Furthermore, protection against DNA fragmentation was observed in the EPO-treated TF-1 cells when challenged with a potent apoptotic inducer staurosporine (STS). These observations are in contrast to our understanding that DNA is fragmented by CAD (Caspase-activated DNase) when ICAD in the ICAD-CAD complex is cleaved by caspase-3. For these apparently contradictory observations, we demonstrated that downregulation of CAD occurred at the mRNA and protein levels during the erythroid differentiation in TF-1. This provides a cell rescuing mechanism in non-apoptotic cells with activated caspases.
Lui Chun Kin Julian.
"September 2006."
Adviser: Siu Kai Kong.
Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1620.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (p. 239-253).
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Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.