published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Η διάγνωση και τυποποίηση της οξείας μυελογενούς λευχαιμίας στηρίζεται στο συνδυασμό των μορφολογικών ευρημάτων του επιχρίσματος υλικού αναρρόφησης μυελού των οστών ή περιφερικού αίματος, και των ευρημάτων της κυτταροχημείας, της ανοσοκυτταροχημείας, ή της ανοσοφαινοτυπικής εξέτασης με τη μέθοδο της κυτταρομετρίας ροής. Ωστόσο, σε ορισμένες περιπτώσεις το υλικό αναρρόφησης του μυελού των οστών ή του περιφερικού αίματος δεν επαρκεί για να στηρίξει διάγνωση, με συνέπεια η οστεομυελική βιοψία να αποτελεί το μοναδικό διαθέσιμο υλικό για να τεθεί η διάγνωση. Με σκοπό την εκτίμηση της ακρίβειας της ανοσοϊστοχημικής τυποποίησης της οξείας μυελογενούς λευχαιμίας σε υλικό οστεομυελικής βιοψίας, μελετήσαμε 50 περιπτώσεις de novo οξείας μυελογενούς λευχαιμίας, οι οποίες είχαν διαγνωσθεί κλινικά σε υλικό αναρρόφησης μυελού των οστών με κυτταροχημική και/ή ανοσοφαινοτυπική εξέταση, και είχαν ταξινομηθεί σύμφωνα με τα κριτήρια της FAB-Ταξινόμησης. Τομές παραφίνης των οστεομυελικών βιοψιών χρώσθηκαν ανοσοϊστοχημικά με αντισώματα εναντίον της μυελοπεροξειδάσης (αντι-ΜΡΟ), δύο επιτόπων του CD68 (KP-1 και PGM-1), της γλυκοφορίνης Α (αντι-GlycA), του παράγοντα von Willebrand (αντι-vW), του CD34 και της TdT. Τα αποτελέσματά μας εισηγούνται ότι η ανοσοϊστοχημική τυποποίηση της οξείας μυελογενούς λευχαιμίας είναι εφικτή με τα αντισώματα αντι-ΜΡΟ, PGM-1, αντι-GlycA, αντι-vW και αντι- CD34. Συμφωνία μεταξύ της κλινικής και παθολογοανατομικής τυποποίησης της οξείας μυελογενούς λευχαιμίας παρατηρήθηκε σε 47 (94%) περιπτώσεις. / The diagnosis and subtyping of acute myeloid leukemia are based on the morphologic examination of bone marrow aspirate and peripheral blood smears in conjunction with cytochemistry, immunocytochemistry, or immunophenotyping by flow cytometry. However, there are situations in which the smears of bone marrow aspirate or peripheral blood are inadequate for study and the bone marrow biopsy specimens are the only available source for the diagnosis and classification of acute myeloid leukemia. To evaluate the accurate subtyping of acute myeloid leukemia based on the immunohistochemical examination of routinely processed bone marrow biopsy specimens, we studied 50 de novo cases of acute myeloid leukemia that where clinically diagnosed on the basis of cytochemical findings and/or immunophenotyping by flow cytometry of bone marrow aspirate smears and classified according to FAB criteria. Paraffin-embedded bone marrow specimens were stained using a panel of antibodies that included antimyloperoxidase (anti-MPO), two epitopes of CD68 (KP-1 and PGM-1), antiglycoforin A (anti-GlycA), anti-von Willebrand factor (anti-vW), anti-CD34 and anti-TdT. Our results suggest that the immunohistochemical subtyping of acute myeloid leukemia is possible using the paraffin reactive antibodies anti-MPO, PGM-1, anti-GlycA, anti-vW and anti-CD34. Concordance between clinical and pathological subtyping of acute myeloid leukemia was observed in 47 (94%) cases.

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Benzene is an ubiquitous pollutant and industrial solvent that has been identified as a human leukemogen. Early exposure to environmental carcinogens such as benzene has been postulated to play a role in the etiology of childhood leukemia, however the association remains controversial. Genotoxic agents such as benzene can cause an increase in the frequency of DNA double-strand breaks, which may remain unrepaired or result in the initiation of DNA recombinational repair mechanisms. The first objective was to investigate the induction of DNA double-strand breaks following in utero treatment to 200 mg/kg and 400 mg/kg benzene i.p. using the phosphorylated histone γ-H2A.X as a marker. Using immunoblotting, treatment with benzene did not increase the formation of γ-H2A.X in bone marrow cells of adult C57Bl/6N male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells following in utero exposure to 200 mg/kg or 400 mg/kg benzene throughout gestational days 7 to 15. Secondly, the study investigated the induction of micronuclei following in utero exposure to benzene. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells. In utero exposure to 400 mg/kg benzene throughout gestational days 7 to 15 also caused a statistically significant increase in the percentage of micronucleated cells in maternal bone marrow and post-natal bone marrow cells. Fetal liver cells also demonstrated a statistically significant increase in the percentage of micronucleated cells following 200 mg/kg and 400 mg/kg benzene. The third objective was to investigate the initiation of DNA recombination following in utero exposure to benzene using the pKZ1 mutagenesis mouse model as a surrogate marker for non-homologous end joining activity. Adult pKZ1 mouse tissue yielded no recombination events; however, post-natal bone marrow cells did contain detectable recombination frequencies. iii In utero benzene exposure did cause an increasing trend in recombination events, and upon analysis of only the samples containing detectable levels of recombination, in utero exposure to 400 mg/kg of benzene caused a statistically significant increase in recombination frequency within this group. These results demonstrate that benzene does not increase the formation of γ-H2A.X after acute and in utero exposure, however, the induction of micronuclei following acute and in utero benzene exposure confirmed that benzene is a genotoxic agent causing chromosomal breaks. In utero benzene exposure increased the frequency of DNA recombination in bone marrow from post-natal day 9 pups exhibiting detectable levels of recombination. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-08-22 11:07:49.162

The E2A-PBX1 fusion gene results from the t(1;19) chromosomal translocation that is found in 25% of pre-B-cell cases of acute lymphoblastic leukemia (ALL). The resulting encoded product contains the transactivation domains of E2A, a Class I basic helix-loop-helix transcription factor, and most of PBX1. PBX1 is a major cofactor for most members of the HOX family of homeodomain proteins and is necessary for regulating the essential role that HOX proteins play in development and tissue homeostasis. We have identified an interaction between the E2A-encoded portion of E2A-PBX1 and the CREB-binding domain (KIX) of the transcriptional coactivator CBP and demonstrated a requirement for this interaction in leukemia induction. Others have shown that HOX proteins and CBP also interact directly, with resulting inhibitory effects on the DNA-binding ability of HOX proteins and on the acetylation of substrate proteins by CBP. Several publications have also identified the interaction of HOX proteins with the PBX1 portion of E2A-PBX1 and the result is a potent transcriptional activator at PBX1/HOX target sequences. In an attempt to develop a molecular model for the induction of ALL by E2A-PBX1, we hypothesize that the addition of CBP interactive peptide elements encoded by E2A to PBX1 allows E2A-PBX1 to stabilize a ternary complex involving E2A-PBX1, HOX, and CBP resulting in the deregulated expression of critical PBX1 or HOX target genes. I demonstrate using in vitro protein-protein interactions that this ternary complex involving E2A-PBX1, HOXB4 (chosen as a representative member of the HOX family), and CBP does form. This direct interaction appears to reduce transcriptional activation by E2A-PBX1/HOXB4 heterodimers from PBX1/HOX enhancer elements. I also show that this suppression of transactivation appears to involve CBP antagonism of DNA binding by E2A-PBX1/HOXB4 heterodimers. My results are consistent with the idea that E2A-PBX1 contributes to ALL induction by promoting the redistribution of CBP away from DNA sites bound by E2A-PBX1/HOXB4 heterodimers and in favour of those sites bound by E2A-PBX1 homodimers. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-09-29 13:57:25.324

E2A-PBX1 is expressed as a consequence of a recurring chromosomal translocation seen in 5% of acute lymphoblastic leukemia cases. We recently reported that substitution of a leucine residue (L20A) within the N-terminal transcriptional activation domain (AD1) of E2A-PBX1 markedly impairs binding to the KIX domain of CBP/p300 and, importantly, leukemia induction in a mouse bone marrow transplantation model. Since both the protein-protein interaction and consequent leukemogenesis rely on a focal contact point and might therefore be susceptible to antagonism by small molecules, we devised a cell-free assay based on fluorescence anisotropy (FA) to detect binding of a fluorescently labeled peptide derived from AD1 of E2A-PBX1 (FITC-E2A) with recombinantly expressed KIX domain. The optimized FA assay reveals a dissociation constant of 2 µM for the wild-type interaction and correctly detects disruption of the complex by naphthol AS-E phosphate, a compound previously shown to antagonize KIX binding. The optimized FA assay was used to screen the Prestwick, Spectrum and Chembridge libraries containing 12400 compounds in total. Of the initial 43 positive hits from the libraries, 10 caused a reproducible decrease in FA. Since intrinsic small molecule fluorescence can produce false positive results in the FA-based screen, intrinsically fluorescent compounds were excluded from further analysis unless they could be shown to bind to KIX. Two hits, L1 and C2, were intrinsically fluorescent but demonstrated KIX interactions and one hit, P9, was not intrinsically fluorescent. These three compounds were tested for their ability to inhibit binding of a larger portion of E2A (residues 1 to 483) to full length CBP in a pull down assay with only compound P9 demonstrating efficacy. Further characterization of P9 by NMR showed no binding to KIX, however evaluation by FA showed binding to FITC-E2A with a 20 µM affinity. A cell-based cytotoxicity assay demonstrated that compound P9 was slightly more toxic on leukemic cells that express E2A-PBX1, compared to leukemic cells lacking E2A-PBX1 expression. Mammalian two-hybrid analysis did not provide details of the effects of P9 on the E2A:KIX interaction. We expect the identification of a novel compound, P9, capable of disrupting the oncogenic E2A-PBX1:CBP interaction, to guide the development of effective, less toxic leukemia drugs and provide new tools for elucidating the molecular mechanisms of leukemia induction by E2A-PBX1. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-08-31 11:13:19.517

The E2A proteins are transcription factors critical for B-lymphopoiesis. A chromosomal translocation involving the E2A gene promotes acute lymphoblastic leukemia (ALL) through expression of the oncoprotein E2A-PBX1. Two activation domains of E2A-PBX1, AD1 and AD2, have been implicated in transcription mediated by recruitment of the transcriptional co-activator CBP/p300. A motif has been identified within AD1 that is important for recruiting CBP/p300, known as PCET. This recruitment requires an interaction between the activation domains of E2A-PBX1 and the KIX domain of CBP/p300. The KIX domain recognizes a generic ΦXXΦΦ sequence (Φ corresponds to a hydrophobic residue) found in the activation domains of numerous transcription factors. Mutation of leucine 20 in PCET has been shown to abrogate ex vivo immortalization of murine bone marrow and oncogenesis in a murine bone marrow transplantation model. A similar sequence is also found in AD2 and is implicated in E2A transcriptional activity and recruitment of CBP/p300. The structural details of these interactions remain largely unknown. NMR spectroscopy was used to determine the solution structure of the PCET:KIX complex, and the functional consequences of the Leu20Ala mutation were structurally rationalized. Other PCET mutations informed by this structure were tested and correlations were found between in vitro binding affinities and both transcriptional activation and immortalization. The binding site of the ΦXXΦΦ-containing E2A AD2 peptide was mapped to the same site on the KIX domain used by the PCET motif. A model of this complex was generated and mutations were tested using a similar approach as was used for PCET. E2A AD2 binds the KIX domain with lower affinity than the PCET motif and is not required for immortalizing bone marrow. A mutation that increases the affinity of E2A AD2 for the KIX domain to levels approaching that seen for the PCET:KIX interaction restores transcriptional activation and immortalization, demonstrating that immortalization by E2A-PBX1 is an affinity dependent process involving the KIX domain of CBP/p300. These studies indicate that the activation domains of E2A-PBX1 serve to support the in vivo function of the oncoprotein and that the PCET:KIX complex is a potential target for novel therapeutics in E2A-PBX1+ leukemia. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-09-13 13:30:48.848

T-cell acute lymphoblastic leukemia (T-ALL) is a lymphoid disorder that results from an over proliferation of immature lymphocytes in the blood and bone marrow. It has been determined that 60% of patients stricken with T-ALL aberrantly express TAL-1 and have been shown to respond poorly to chemotherapy. This research sought to determine if TAL-1 influences the expression of the Bcl-2 family members Bcl-2 (anti-apoptotic), Bad and Bax (pro-apoptotic). TAL-1 and Bcl-2 levels were elevated while Bad and Bax levels were lower in etoposide-treated Jurkat cells as compared to TRAIL-treated and dual-treated Jurkat cells in which TAL-1 and Bcl-2 levels were lower while Bad and Bax levels were elevated. These results suggest TAL-1 up-regulates Bcl-2 and suppress Bad and Bax expression in response to etoposide treatment, thus inducing an anti-apoptotic response in the cell. These results also suggest that TRAIL and the dual treatment of etoposide and TRAIL down-regulates TAL-1 and Bcl-2 expression while up-regulating Bad and Bax, thus inducing a pro-apoptotic response in the cell. / Department of Biology