Abstract Epidemiological and experimental studies indicate a causative role of viruses in malignancies. Recently, a link between bacterial infections and the development of cancer has been suggested. The purpose of this study was to evaluate the association between chlamydial infection and cancer. The association between C. trachomatis infection and cervix cancer was analysed in a prospective study. The presence of IgG antibodies to C. trachomatis and C. pneumoniae was determined from the serum samples of 182 Nordic women with invasive cervical carcinoma and 538 matched cancer-free controls by the microimmunofluorescence (MIF) method. Serum antibodies to C. trachomatis were associated with an increased risk for cervical squamous cell carcinoma (SCC) (OR 2.2, 95% CI 1.3-3.5), but not for cervical adenocarcinoma (OR 0.4, 95% CI 0.1-1.7). C. trachomatis serotype G was highly significantly associated with an increased risk for SCC (adjusted OR 6.6, 95% CI 1.6-27). The presence of serum IgG antibodies to more than one serotype of C. trachomatis, on the other hand, also increased the risk of SCC. The association between C. pneumoniae infection and lung cancer was analysed separately in men and women. C. pneumoniae-specific antibodies and immune complexes (IC) were analysed from 230 Finnish smoking males with lung cancer and their matched controls using serum samples collected before the lung cancer diagnosis. Suggestive chronic C. pneumoniae infection was associated with an increased risk for lung cancer (OR 1.6; 95% CI 1.0-2.3). The risk was increased especially in men younger than 60 years (OR 2.9; 95% CI 1.5-5.4), but not in the older age group (OR 0.9; 95% CI 0.5-1.6). Chlamydial antibodies and chlamydia-specific ICs were analysed from serum samples of 29 Finnish women with lung cancer and 87 matched cancer-free controls by MIF. The mean follow-up from serum sampling to cancer diagnosis was 6.7 years. IgG class antibodies to C. pneumoniae were common in pregnant Finnish women (66% among cases, 62% among controls), whereas IC-bound C. pneumoniae IgG antibodies were rare. No additional risk for lung cancer in association with chlamydial antibodies was found among women. The association between chlamydial infections and lymphomas was evaluated in a cross-sectional study. Seventy-two lymphoma patients from Tampere University Hospital and 72 matched controls were selected, and IgG antibodies and ICs to C. pneumoniae and C. trachomatis were analysed from their serum samples by MIF and enzyme immunoassay (EIA). The serological markers suggesting chronic chlamydial infection were associated with an increased risk for malignant lymphoma. The association was most evident for the presence of C. pneumoniae-specific ICs in non-Hodgkin's lymphoma (OR = 7.3, 95% CI 2.2-25) and appeared to be limited to men. Infection with C. trachomatis was found to increase the risk of subsequent development of invasive cervical SCC. Chronic C. pneumoniae infection was also found to be a new independent risk factor for lung cancer in males. Serological markers suggestive of chronic chlamydial infection were associated with lymphomas, proposing that chlamydial infection may have a similar role as H. pylori in the pathogenesis of lymphomas.

Le lymphome des cellules du manteau (LCM) est un lymphome d’une rare agressivité causée par la translocation chromosomique t(11; 14)(q13; q32) juxtaposant le locus de la cycline D1 (CCND1) sur le chr 11 avec le locus de la chaîne lourde de l'immunoglobuline (IgH) sur le chr 14. En conséquence, une cycline D1 proto-oncogène devient active alors qu’elle n’est pas exprimée dans les cellules-B normales. L’hypothèse initiale semble indiquer une influence directe du fort enhancer IgH sur le promoteur du gène CCND1 afin de surexprimer sa transcription. Quoi qu’il en soit, le locus CCND1 peut être éloigné jusqu'à 200kb du point de cassure du chromosome. Nous avons montré que le locus 11q13 relocalise depuis la périphérie du noyau jusque au centre actif de transcription et au nucléole (Allinne et al., 2014). Ce phénomène qui mène à l’activation du locus entier, suggère un mécanisme epigénétique de régulation des gènes dans les LCM plutôt que simplement un simple effet enhancer-promoteur. Plusieurs nouveaux traitements contre le MCL ont été proposés, y compris les inhibiteurs d’histone deacetylase (HDACis) qui impliquent des mécanismes épigénétiques. Dans les lignées cellulaires LMC, les HDACis sont décrites comme aillant des effets antiprolifératifs, et paradoxalement, le niveau d’expression protéique de la cycline D1 dans la cellule diminue. Jusqu'à présent, il n'y a pas une compréhension claire de ce phénomène, de même que les mécanismes d’action des HDACis reste inconnus. Pour ces raisons, une étude du « paysage épigénétique » sur les loci 11q13 et 14q32 devrait fortement améliorer notre connaissance sur les mécanismes de surexpression de la cycline D1 dans les LMC. L’objectif de ce travail est d'étudier la structure de la chromatine dans le locus réarrangé (11; 14)(q13; q32) dans des cellules LMC par rapport au locus 11q13 et 14q32 dans les lymphocytes humains normaux. Nous avons ensuite étudié l'effet de différentes HDACis sur le locus réarrangé (11; 14)(q13; q32) à plusieurs niveaux: l'acétylation des histones / la méthylation de la chromatine ainsi que sa conformation et l'expression des gènes. Nous avons montré que t(11:14)(q13; q32) conduit à la surexpression de CCND1 avec un groupe de gènes couvrant plus de 15 Mb autour du point de translocation. Les mêmes gènes, sensibles à la dérégulation par la translocation t(11; 14, réagissent au traitement HDACi en augmentant leur expression. Nos résultats indiquent que bien que HDACi stimule la désagrégation de l'hétérochromatine sur l'ensemble du génome, les promoteurs de gènes restent à l'abri de ces effets. / Mantle cell lymphoma (MCL) is a rare aggressive lymphoma caused by the chromosome translocation t(11;14)(q13;q32) juxtaposing the cyclin D1 (CCND1) locus on chr 11 with the immunoglobulin heavy chain (IgH) locus on chr 14. As a result, a proto-oncogene cyclin D1 which is not expressed in normal B-cells, becomes active. The initial hypothesis favored direct influence of the strong IgH enhancer on CCND1 gene promoter to upregulate its transcription. However, the CCND1 locus may be as far as 200 kb from the chromosome breakpoint. We have shown that 11q13 locus relocalizes from the nucleus periphery towards the transcriptionally active center and nucleolus (Allinne et al., 2014). This may lead to activation of the entire locus, suggesting an epigenetic mechanism of gene regulation in MCL, rather than just simple enhancer-promoter effect.Several new treatments are proposed for MCL, including histone deacetylase inhibitors (HDACis) with epigenetic mechanism of action. In MCL cell lines, HDACis were shown to have antiproliferative effects, and paradoxically, they decreased the cyclin D1 protein level in the cells. Until now, there is no clear understanding of this phenomenon, nor of HDACis mechanism of action. Therefore, a study of «epigenetic landscape» in 11q13 and 14q32 loci should significantly advance our knowledge about the mechanisms of cyclin D1 upregulation in MCL.The purpose of the present work was to study chromatin structure in the rearranged (11;14)(q13;q32) locus in MCL cells as compared to the 11q13 and 14q32 loci in normal human lymphocytes. We then studied the effect of different HDACis on the rearranged (11;14)(q13;q32) locus at several levels: histone acetylation / methylation, chromatin conformation and gene expression.We have shown that t(11:14)(q13;q32) translocation leads to overexpression of CCND1 along with a group of genes spanning over 15 Mb around the translocation point. The same genes, sensitive to deregulation by t(11;14) translocation, react to the HDACi treatment by increasing their expression. Importantly, while HDACi stimulates genome-wide disaggregation of heterochromatin, genes’ promoters stay shielded from its effect.

Epstein-Barr Virus (EBV) is well known to manipulate the host ubiquitin machinery to facilitate its latent persistence and oncogenesis, exemplified by LMP1 signal transduction that activates multiple transcription factors, including NFκB, AP1, and IRF7/IRF4, which promote cell survival and outgrowth, and control immune response and inflammation. It is therefore vital to delineate the detailed mechanisms underlying LMP1 signal transduction for understanding EBV-mediated oncogenesis. p62 (also called SQSTM1, Sequestosome 1) is a ubiquitin sensor and a signal transducing adaptor that interacts with TRAF6 and facilitates the recruitment of ubiquitinated signal intermediators for the activation of NFκB and AP1 in diverse contexts. In turn, p62 is induced by NFκB. However, the interaction between p62 and EBV latency has never been studied. We have recently published interesting and important results, which imply a crucial role for p62 in EBV-mediated oxidative stress. In this study, we further show that p62 is upregulated in EBV latency, with the contribution of LMP1-mediated NFκB and AP1 activities. In turn, p62 participates in LMP1 signal transduction through its interaction with TRAF6, promoting TRAF6 ubiquitination. shRNA-mediated p62 depletion downregulates LMP1-TRAF6 interaction and TRAF6 ubiquitination, and significantly impairs AP1 activity; however, with no detectable effects on NFκB activity. These observations imply that TRAF6-p62 interaction differentiates LMP1 signaling to NFκB and AP1 activation. As a consequence, p62 depletion promotes etoposide-induced apoptosis. These findings identify p62 as a novel player in EBV LMP1 signaling to AP1 activation that is crucial for LMP1-mediated ROS production.

Chromosomal translocation is a characteristic feature of human lymphoid malignancies and a driver of the initiation and progression of the disease. They arise from the mis-repair of physiological DNA double-strand breaks (DSBs) generated during the assembly and subsequent modifications of the antigen receptor gene loci, namely V(D)J recombination and class switch recombination (CSR). Mammalian cells have three DSB repair pathways –classical non-homologous end-joining (cNHEJ), alternative end-joining (A-EJ), and homologous recombination. DNA end-resection that generates a single-strand 3’ overhang is a critical regulator for the repair pathway choice. Specifically, localized end-resection prevents cNHEJ and exposes flanking microhomology (MH) to promote error-prone A-EJ. In addition to DNA repair, DNA end-resection generates extended single-strand DNA, which activates the ATR-mediated cell cycle checkpoint and indirectly contributes to genomic integrity. The central goal of my thesis research is to investigate the physiological role of DNA end-resection initiation in lymphocyte development and lymphomagenesis. DNA end-resection in mammalian cells is mostly initiated by the endonuclease activity of MRE11-RAD50-NBS1 (MRN) complex aided by CtIP. In addition, MRN protein also recruits EXO1 and DNA2 nucleases in combination with Top3 helicase complex for more extensive resection. The CtIP protein is essential for the endonuclease activity of the MRN complex that initiates DNA end-resection. CtIP is essential for embryonic development. Here I utilized B cell-specific conditional deletion models and loss-of-function mutations to investigate the role and regulation of CtIP and CtIP-mediated DNA end-resection in lymphocyte development and tumorigenesis. The level and extent of CtIP-mediated resection are tightly regulated. For the first aim, we applied the ATAC-Seq and EndSeq methods to test whether chromatin accessibility determines the level of DNA end-resection. Specially, we found that chromatin-bound DNA damage response factors – H2AX and 53BP1- reduced the accessibility of the DNA around the DSBs and antagonized end-resection. Our data also suggest that during DNA damage response, the nucleosome-free or accessible regions are more prone to secondary DNA breakages. Mechanistically, the preferential vulnerability is correlated with the availability of chromatin-bound DNA damage response factor 53BP1, which protects the nucleosome covered region at the price of the nucleosome-free regions. The work provides one explanation for tissue and cell type-specific translocations in transcriptionally active regions and super-enhancers. For the second and third aims, I investigated the role of CtIP and CtIP-mediated end-resection in lymphocyte development and lymphomagenesis in vivo using the conditional deletional CtIP allele and a phosphorylation-deficient CtIP-T855A mutant. T855 phosphorylation promotes end-resection but is not essential for cellular viability. I identified a sequence-context-dependent role of CtIP and end-resection in A-EJ mediated repair. We found that the reduced level of end-resection did not alter the frequency of the A-EJ mediated joining during B cell CSR, nor the levels of micro-homology at the junction, a defining feature of A-EJ mediated repair. These findings, for the first time, showed that DNA end-resection is not essential for A-EJ-mediated chromosomal DSBs repair nor for the generation of MH at the junction in vivo. This unexpected observation also highlights a tissue- and cell type-specific regulation of A-EJ and the importance of sequence context for A-EJ. Moreover, we found that ATM kinase suppresses A-EJ mediated translocation and reported the very first cell cycle-dependent analyses of CSR junctions. In cNHEJ-deficient B cells (e.g., Xrcc4- or DNA-PKcs- deficient), the A-EJ pathway is responsible for both the residual CSR events and the generation of the oncogenic IgH-Myc chromosomal translocations. In the last chapter, I determined how CtIP contributes to oncogenesis using the CtIP-T855A phospho-deficient mouse model. The result showed that CtIP T855 phosphorylation is critical for the neonatal development of Xrcc4-/-p53-/- mice and IgH-Myc translocation driven lymphomagenesis in DNA-PKcs-/-Tp53-/- mice. Mechanistically, phospho-deficient CtIP compromises the extent of end-resection without affecting the initiation. Reduced end-resection in CtIP-T855A mice and cells attenuated G2/M checkpoints and reduced the tolerance to the oncogene-induced replication stress, thereby limit lymphomagenesis. Collectively, our data provided the first in vivo characterization for the role of CtIP and its related end-resection pathway in lymphocyte development and lymphomagenesis. The results highlight the importance of end-resection for checkpoint maintenance (§ 4) and the context-dependent regulation of A-EJ and DNA repair pathway choice in vivo (§ 3), explaining why A-EJ is more robust at the repetitive switch regions. Finally, the application of HTGTS, EndSeq, and ATAC-Seq technologies in lymphocyte-specific gene rearrangements markedly improved the analysis depth and sensitivity while reducing the cost of repair-junction sequencing, allowing the quantitative detection of subtle changes and additional mechanistic insights. Specifically, we showed that end-resection could be regulated at the level of chromatin accessibility, which is determined by both baseline chromatin occupancy and DNA damage-induced changes (§ 2). These findings provide one explanation for the tissue and cell type-specificity of translocation targeting. These techniques can be used to analyze the impact of other DNA repair factors during lymphocyte development and lymphomagenesis and in translocation in general.

Marginal zone B-cell lymphomas (MZBL) constitute 7% of all non-Hodgkin lymphomas, being the third most common subtype after diffuse large B-cell lymphoma(DLBCL) and follicular lymphoma. Extranodal MZBL (ENMZBL) most commonly arise from the mucosa-associated lymphoid tissue (MALT lymphoma), with stomach being the most common site. ENMZBL involving the dura is anatomically unusual and very rare. A 54 year old female presented to the hospital with worsening headaches and new onset generalized tonic clonic seizures. Complete blood counts and chemistry were unremarkable. No constitutional symptoms, new / progressive lymphadenopathy reported. Magnetic resonance Imaging( MRI) brain showed an enhancing right subdural soft tissue lesion overlying the right frontotemporal lobe suggestive of meningioma versus metastasis. Computed Tomography (CT) chest/abdomen/pelvis revealed no mass or lymphadenopathy. Lumbar Puncture cerebrospinal fluid cytology was negative for malignancy. She underwent brain biopsy. Pathology revealed diffuse infiltrate of small to medium-sized lymphoid cells, Immunohistochemical stains positive for CD 20, PAX5, CD 79a, Ki-67 at 5-10%, weakly positive for MUM1 and BCL2, negative for CD3, CD5, CD10, BCL6, cyclin D1, consistent with ENMZBL. Bone marrow biopsy and aspiration negative for involvement with lymphoma. Patient received local/regional therapy with radiation (XRT), total dose 24 Gy in 12 fractions. She presented six months later with worsening neck pain. MRI cervical spine revealed diffuse thick dural based enhancement within the spinal canal at C1-C4 levels leading to moderate-to-severe spinal canal stenosis at C2-C3 level with significant soft tissue extension. Repeat labs, systemic imaging, and bone marrow biopsy continued to show no evidence of systemic disease. She received low dose XRT to the entire craniospinal axis (dose-4Gy). Patient developed profound pancytopenia secondary to craniospinal XRT. After count recovery she initiated daily oral Ibrutinib (560 mg) with plans for a treatment duration of one year. Dural based ENMZL usually present as solitary masses, mimicking meningioma's. Marked female predilection is seen with median age of onset 50 years. Very few cases have been reported in literature with no standard therapy being described. ENMZL are usually indolent requiring less aggressive therapy. In contrast, primary CNS lymphoma (PCNSL) of diffuse large B cell histology is usually aggressive with high tendency to relapse, requiring treatment with high dose methotrexate based regimes. Dural based ENMZL therapy entails local treatments such as surgery and radiation therapy (relatively low dose radiation usually effective with prolonged durable responses). Systemic treatment with single agent Rituximab or with Tyrosine Kinase inhibitors like Ibrutinib with CNS penetration can also be considered. Long-term follow-up is recommended even in those patients who achieved complete remission as relapses may occur.

Inconsistent use of nomenclature in different canine malignant lymphoma (CML)classification systems, which lead to incorrect diagnosis and prognosis, necessitated a retrospective study of 103 cases of CML. Histological classification was done according to the Working Formulation, on H&E sections, after standard processing. Immunophenotyping, using CD3 (T cell) and CD79a (8cell) markers, was carried out on the same sections. Intermediate grade lymphomas were the largest category (49.5%), with 16.5%high grade lymphomas. More than half (53.3%) of the lymphomas were of 8 cell immuno phenotype, and 36.5% were T cell lymphomas. Only 9.7% of the total number of lymphomas exhibited double negative staining. Only two categories, the immunoblastic and medium-sized macro nucleolated (MMC) category (Fournel-Fleury et al.). exhibited constant (8 cell) immunophenotype. All the other categories exhibited mixed immunophenotype. The Working Formulation, with omission of the follicular tyoes (due to the rarity thereof in CML) and addition of the MMC category and immunophenotyping, is best for classifying CML. / Dissertation (MMed Vet (Pathology))--University of Pretoria, 1999. / Anatomy and Physiology / Unrestricted

Activated B-cell Factor-I (ABF-1) is a class II basic helix-loop-helix protein expressed in activated B-cells, EBV -immortalized lymphoblastoid cell lines and embryonic skeletal muscle in mice. ABF -1 dimerizes with another bHLH protein E4 7 to form a transcriptional repressor ofE2A, which is a gene essential to the proper development of B- and T -cells. In an effort to study genes in B-cells regulated by ABF- 1, I have attempted to construct a Burkitt Lymphoma cell line allowing tetracycline regulated expression of ABF-1. The tetracycline repressor gene was first added to these cells, creating a parental line oftet repression. Next, both a full-length and a truncated version of ABF -1 were added to the cell line. The truncated version of the protein is expressed in the presence of tetracycline, but completely repressed in its absence, demonstrating a tightly-regulated system of inducible expression. The full-length version of ABF-1 has yet to be expressed in this cell line; however, it has been expressed in a HeLa cell line, demonstrating that the construct has been properly made.

Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 4 (STAT4) is an important transcription factor activated by IL-12 signaling. Activated STAT4 is essential for Th1 cell differentiation, a process characterized by increased potential for interferon (IFN)-γ production. Defective IFN-γ production due to STAT4 deficiency occurs after autologous stem cell transplantation for lymphoma. We have investigated the mechanisms of post-transplant STAT4 deficiency. The tumor-bearing state is ruled out to be the cause because STAT4 levels were not significantly different in peripheral blood mononuclear cells (PBMCs) obtained from lymphoma patients prior to treatment and healthy control subjects. The magnitude of the decrease in STAT4 levels corresponded with increasing intensity of chemotherapeutic treatment in vivo. Furthermore, treatment of normal PBMC cultures or a natural killer (NK) cell line with chemotherapy drugs in vitro also resulted in reduced STAT4 protein and reduced IL-12-induced IFN-γ production. Chemotherapy drugs are shown to have no impact on the stability of STAT4 mRNA, while steady-state levels of STAT4 transcripts are decreased in lymphoma patients. Our findings demonstrated that chemotherapeutic drugs up-regulate the ubiquitination rates of the STAT4 protein, which in turn promotes its degradation via the proteasome-mediated pathway. Treatment with the proteasome inhibitor bortezomib largely reversed the chemotherapy-induced STAT4 deficiency. Thus, acquired STAT4 deficiency in lymphoma patients is a consequence of treatment with chemotherapy. These results have important implications for design of optimal immunotherapy for lymphoma.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy