Urinary bladder urothelial carcinoma is the most common cancer of the urinary tract. About 70% of the diagnosed tumors classified as Non-invasive tumor, which is usually multiple. Despite surgical removal and perioperative chemotherapy, tumor recurrence is not uncommon. However, the chance for such non-invasive tumors to advance to the muscle-invasive stage is relatively small and the 5-year survival rate approaches 95%. The rest 30% are classified as invasive tumors which usually pursue aggressive clinical course. In spite of radical cystectomy in conjunction with debilitating chemotherapy and/or radiotherapy, more than 50% of invasive tumors eventually spread to distant organs. The 5-year survival rate for patients with distant metastasis is only about 6%. The current challenge in the management of urinary bladder carcinoma is the lack of powerful prognostic marker and promising therapeutic agents. Accordingly, to identify novel biomarks to adjust therapeutic strategy is mandatory. The BCL6 proto-oncogene encodes a nuclear transcriptional repressor, it¡¦s inhibits DNA repair pathways and TP53. Perturbation of both these pathways may contribute to normal cell function by repressing DNA damage responses and permitting somatic hypermutation but , in the context of malignancy, this could lead to mutations promoting aggressive tumor. Several studies have demonstrated that BCL6 play a role in different cancer types, however, the function of BCL6 in bladder cancer is understood. Therefore, in this study, we will analyze the endogenous BCL6 mRNA and total/activated BCL6 protein in various bladder cancer cell lines, including BFTC905, and J82. Then we will knockdown of the BCL6 gene by shRNA interference and analyze how it implicates various cellular processes essential to cancerous states. And then we will be analyzed the affection of cell survival, migration and invasion. Conversely, Overexpression of BCL6 in bladder cancer cell lines will be assessed cell proliferation, migration and invasion. Finally, studying it¡¦s affection in vivo. We demonstrate that BCL6 is correlated with bladder cancer.

No description available.

No description available.

Loss of heterozygosity (LOH) of 16q is an early event occurring in 36-60% of primary sporadic breast cancers. CBFA2T3 (MTG16) is a putative breast cancer tumour suppressor gene, localized at chromosome band 16q24.3. CBFA2T3 (MTG16) belongs to the CBFA2T protein family and shares a high homology with other two members, CBFA2T1 (MTG8) and CBFA2T2 (MTGR1). CBFA2T1 and CBFA2T3 proteins form transcriptional repressor complexes with the DNA binding zinc finger proteins like BCL6, PLZF, Gfi1 and ZNF652. CBFA2T3 protein exists as isoform “a” and “b” that arise from alternate start sites. These isoform differ in their N-terminal sequences. Previous studies determined that CBFA2T3a localized to the nucleolus, while CBFA2T3b has a putative role as tumour suppressor protein. The present study confirms that the database entries of CBFA2T3a are incomplete and an extended N-terminus region is present to CBFA2T3a (NCBI NM_005187) isoform by RTPCR and DNA sequencing. Two rabbit polyclonal anti CBFA2T3 antibodies were raised against the region unique to CBFA2T3. These antibodies specifically detect the endogenous CBFA2T3 proteins and not CBFA2T1 and CBFA2T2. Cell fractionation studies show that endogenous CBFA2T3a localized to the cytoplasm, while CBFA2T3b targeted to the nucleus. The N-terminus region specific to “a” isoform determined the cytoplasmic localization. The detailed studies show that CBFA2T3a localized to centrosome and this was confirmed by co–localization with known centrosomal proteins γ- tubulin. This was further confirmed by immunoprecipitation of γ-tubulin with N-terminus regions of CBFA2T3a protein. Further investigation showed that CBFA2T3a localizes to the centrosome through out the centrosomal duplication. Presence of CBFA2T3a on procentriole was further confirmed by co-localization with known proteins having a crucial role in centrosome duplication like HsSAS6 and polyglutamilated tubulin. Experiments were conducted to determined if the different subcellular localization of “a” and “b” isoforms resulted into functional differences between two isoforms. Immunoprecipitation experiments with known DNA binding proteins like BCL6 and PLZF showed that CBFA2T3b interacts with BCL6, while no interaction was found with PLZF. Consistent with the known transcriptional co-repressor function, real time RT-PCR showed that CBFA2T3b has an additive effect on BCL6 mediated repression of its target cyclin D2, while no effect was observed with CBFA2T3a. Real time RT-PCR data also showed that BCL6 not only recruits CBFA2T3b to repress its target but also have repressive effects on CBFA2T3 transcription. CBFA2T3b transcription regulation by BCL6 was found to be mediated through one or two BCL6 putative binding sites in CBFA2T3b promoter. Immuno histochemical studies were carried out to analyse CBFA2T3b function as a breast cancer tumour suppressor. CBFA2T3 proteins are highly expressed in epithelial cell lineage of normal breast ducts, while its expression is lost in some tumours. CBFA2T3 expression was further analysed in a cohort of commercially available breast tumour sections. Data from these studies showed the loss of CBFA2T3 nuclear expression in some tumours, which was significantly correlated with tumours positive for HER2 expression, molecular subtypes and histological staging of the tumours. CBFA2T3 cytoplasmic expression was also down regulated in tumour sections. A significant association of CBFA2T3 cytoplasmic expression was observed with the TNM grading for tumour invasion and centrosomal abnormalities in BR701 TMA. Knock down studies using shRNA were conducted to investigate the role of CBFA2T3a. Following CBFA2T3 knock down in cells with minimal CBFA2T3b expression, an increase in centrosomal abnormalities was observed. These abnormalities were associated with a significant increase in metaphase anomalies. Since the “a” isoform is localized to cytoplasm and particularly centrosome, it was considered that this isoform is determining centrosome integrity. This work has provided a new insight into the localization pattern of CBFA2T3 isoforms, as CBFA2T3a and b isoforms were localized to different cellular compartments and were involved in distinct functions. CBFA2T3b function as a transcriptional co repressor, CBFA2T3b expression was lost in a group of breast tumours sections. Given that CBFA2T3a has a critical centrosomal function, the expression of this isoform would be expected to be maintained, even in the absence of the CBFA2T3b isoform in tumours. CBFA2T3a specific knock down studies may give a full insight on direct targets of CBFA2T3a, having a controlling role in normal centrosome duplication cycle. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1474414

The Early Growth Response genes 2 and 3 (Egr2/3) are zinc finger transcription factors that play an important role in the immune system. These transcription factors have reported functions in T cell receptor signaling, differentiation of effector T cell subsets and the development of lupus-like autoimmune diseases. Using CD2-Egr2-/- Egr3-/- mouse model, I investigate the development of inflammation pathology, differentiation of T follicular helper (Tfh) cells and the formation of germinal centers (GC) following viral challenge within these mice. The onset of inflammation pathology in CD2-Egr2-/- Egr3-/- mice was discovered to correlate with high levels of pro-inflammatory cytokines in the serum and the development of autoimmune diseases as previously reported by Li et al, 2012. Most importantly, a novel role for the Egr2/3 genes in the differentiation of T follicular helper (Tfh) cells was identified. Tfh cells are responsible for T cell dependent antibody immune response in the GC. They support the differentiation of GC B cells into plasma cells producing long lived high-affinity isotype-switched antibodies and memory B cells. Tfh cell differentiation is regulated by Bcl6 however; the regulators of Bcl6 during Tfh differentiation remain largely unknown. We have now discovered that Egr2/3 genes are required for Bcl6 expression during Tfh cell differentiation. In the absence of the Egr2 and 3 genes, Tfh cell differentiation is severely impaired and GC formation and functions were defective in response to Vaccinia Virus Western Reserve strain (VVWR) infection. Further investigation revealed that Egr2 regulated Bcl6 expression in a Tfh-specific manner as adoptive transfer of WT CD4+ T cells into Egr2-/- Egr3-/- mice was able to rescue Bcl6 expression, Tfh differentiation and GC formation. When the molecular mechanism of how Egr2 regulated Bcl6 was investigated, it was uncovered that Egr2 directly bound to the promoter region of Bcl6 gene in CD4 T cells to regulated Bcl6 expression. Indeed constitutive expression of either Egr2 or Bcl6 in CD2-Egr2-/- Egr3-/- CD4+ T cells rescued Tfh cell differentiation and GC formation. Our results inferred that the Egr2/3 genes are essential for Tfh differentiation and GC formation by regulating Bcl6 expression in CD4 T cells under Tfh condition. Our studies thus suggest that the Egr2/3 genes are paramount for minimising immunopathology and are also critical for efficient antibody production by regulating Tfh cell differentiation.

The germinal centre (GC) response is critical for generating highly effective humoral immune responses and immunological memory that forms the basis of successful immunisation. Control of the output of the GC response requires Follicular regulatory T (Tfr) cells, a subset of Foxp3+ Treg cells located within germinal centres. Tfr cells were first characterised in detail in 2011 and because of this relatively little is known about the exact role of Tfr cells within the GC, and the mechanism/s through which they exert their suppressive function. At the outset of this work, the major barrier to understanding Tfr cell biology was the lack of appropriate tools to study Tfr cells specifically, without affecting Tfh cells or other Treg cell subsets. This thesis set out to develop a strain of mice that specifically lacks Tfr cells. A unique feature of Tfr cells is their CXCR5-dependent localisation within the GC. Therefore, genetic strategies that exclude Treg cells from entering the GC are a rational approach to generating a mouse model that lacks Tfr cells. To this end, I generated a strain of mice that lacks CXCR5 on Foxp3+ Treg cells. These animals show a ~50% reduction in GC localised Tfr cells, and a GC response that is comparable to control animals. These data indicated that redundant mechanisms are involved in Treg cell homing to the GC. I identified CXCR4 as a chemokine receptor that is also highly expressed on Tfr cells, and hypothesised that it may also be involved in Tfr cell localisation to the GC. Surprisingly, simultaneous deletion of both CXCR4 and CXCR5 in Treg cells resulted in a less marked reduction in Tfr cells compared to deletion of CXCR5 alone, suggesting that CXCR4 might be involved in negative regulation of Treg homing to the GC. These data identify both CXCR4 and CXCR5 as key regulators of Tfr cell biology. Bcl6 drives Tfr cell differentiation, but how this transcriptional repressor facilitates commitment to the Tfr cell subset is unknown. I hypothesised that Bcl6 drives Tfr cell differentiation by repressing Tbx21, the transcriptional regulator involved in the differentiation of Th1-like Treg cells. I tested this hypothesis in Bcl6fl/fl CD4cre/+ animals and unexpectedly found that loss of Bcl6 regulates Treg cell differentiation in the absence of immunisation or infection. I have demonstrated that thymic loss of Bcl6 results in an increase in activated effector Treg cells, which occurs very early in life. These data point to a novel role for Bcl6 in preventing early thymic Treg activation, indicating that Bcl6 has a global role in Treg development and differentiation that is not simply limited to Tfr cells.

During optic cup morphogenesis, the neuroectodermal layers of the optic vesicle (OV) invaginate ventrally, and fuse at the choroid fissure (CF) along the proximo-distal axis such that the retina and retinal pigment epithelium (RPE) are confined within the cup. Failure of CF closure results in colobomas, which are characterized by the persistence of a cleft or hole at the back of the eye. While CF closure is a critical aspect of ocular development, the molecular and cellular mechanisms underlying this process are poorly understood. My research examined CF closure and colobomas using zebrafish as a model system. In the first study, I determined that early cell fate changes within the eye field could cause colobomas using the zebrafish mutant blowout. Colobomas in blowout resulted from defects in optic stalk morphogenesis whereby the optic stalk extended into the retina and impeded the edges of the CF from meeting and fusing. Positional cloning of blowout identified a nonsense mutation in patched1, a negative regulator of the Hedgehog pathway. Up-regulation of Hedgehog pathway activity causes disruption in the patterning of the OV into proximal and distal territories, revealing that cell fate determination, mediated by Hedgehog signaling, is intimately involved in regulating CF closure. In the second study, I examined Bcl6 function and regulation during zebrafish eye development. bcl6 encodes a transcriptional repressor expressed in the ventral retina during zebrafish eye development. Loss of Bcl6 function leads to colobomas along with up-regulation of p53, a previously known Bcl6 target, and an increase in the number of apoptotic cells in the retina, demonstrating that Bcl6 plays a critical role in preventing apoptosis in the retina during early eye development. I also showed that Vax1 and Vax2 act upstream of bcl6 in the ventral retina. Furthermore, I identified functional interactions between Bcl6, Bcor and Hdac1 during eye development, demonstrating that Bcl6 functions along with Bcor and Hdac1 to mediate cell survival by regulating p53 expression. Together my studies expand the gene regulatory network involved in cell fate determination and cell survival during CF closure and ventral retina formation, and provide mechanistic insight into coloboma formation. / text

Organische-Anionen-Transporter (OATs) sind maßgeblich an der Ausscheidung von körpereigenen und körperfremden Substanzen über die Niere beteiligt. In Ratten, einem häufig verwendeten Tiermodell in präklinischen Studien, ist bekannt, dass die basolateral lokalisierten Organischen-Anionen-Transporter 1 (Oat1) und 3 (Oat3) in männlichen Tieren stärker und darüber hinaus Testosteron abhängig exprimiert werden. Beide Transporter sind an der Ausscheidung von organischen Anionen, einschließlich negativ geladener Medikamente wie zum Beispiel Adefovir, Furosemid oder Penicillin, beteiligt. In den menschlichen Nieren zählen der OAT1 und der OAT3 zu den klinisch relevanten Transportern, deren Funktionen im Laufe neuer Medikamentenentwicklung berücksichtigt werden sollten. Für das Antibiotikum Penicillin wurde bei Frauen ein vermehrtes Auftreten von Nebenwirkungen im Vergleich zu Männern gezeigt. Dieses erhöhte Risiko könnte möglicher Weise auf einer geschlechtsabhängigen Expression des OAT1 und OAT3 zurückzuführen sein. Ziel der vorliegenden Arbeit war es, den molekularen Mechanismus, der für die höhere Oat1 und Oat3 Expression in den männlichen Rattennieren verantwortlich ist, zu identifizieren. Mit Hilfe von Luciferase assays wurde die Aktivierung von Ratten und menschlichen Oat1/OAT1 und Oat3/OAT3 Promotoren untersucht. Hierzu wurden zunächst Oat1/OAT1 und Oat3/OAT3 Promotorkonstrukte generiert, welche unterschiedlich lange Promotorregionen enthielten, und diese anschließend transient in OK oder LLC-PK1 Zellen transfiziert. Mittels Co-Transfektion potentieller transkriptioneller Regulatoren konnte deren Einfluss auf die Promotoraktivität von Oat1/OAT1 und Oat3/OAT3 untersucht werden. Zur Identifikation geschlechtsabhängig exprimierter Gene in der proximalen Tubuluszelle der Rattennieren wurden von vier männlichen und vier weiblichen Tieren je eine Niere präpariert und deren RNA mit Hilfe eines microarrays und real-time PCR analysiert. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die bereits bekannte männlich dominierende Expression von Oat1 und Oat3 in Rattennieren nicht durch den klassischen Testosteron/Androgenrezeptor vermittelten, transkriptionellen Mechanismus reguliert wird. Vergleichbar zu den Ratten Oat1 und Oat3, zeigten auch die menschlichen OAT1 und OAT3 Promotoren keine Aktivierung durch den Testosteron/Androgenrezeptor-Komplex. Während der Suche nach geschlechtsabhängig exprimierter transkriptioneller Regulatoren in der Rattenniere, konnte die Expression des Transkriptionsfaktors B-cell CLL/ lymphoma 6 (BCL6) erstmalig als männlich dominierend identifiziert werden. Die bereits bekannten Aktivatoren der Oats/OATs Expression, hepatocyte nuclear factor 1α (HNF1α), HNF1β und HNF4α zeigten keine geschlechtsabhängige Expression. Zudem konnte gezeigt werden, dass BCL6 die Promotoren der Ratten und menschlichen Oat1/OAT1 und Oat3/OAT3 aktiviert. Die BCL6-vermittelte Aktivierung von Oat1/OAT1 und Oat3/OAT3 erfolgt nicht über die bislang vorhergesagten BCL6-Bindungsstellen, aber möglicher Weise über Protein-Protein Interaktionen mit den Transkriptionsfaktoren HNF1 oder cAMP response element binding protein (CREB). Zusammenfassend konnte gezeigt werden, dass der Transkriptionsfaktor BCL6 einen vielversprechenden Regulator der geschlechtsabhängigen Expression von Oat1 und Oat3 in Ratten darstellt. Es ist anzunehmen, dass BCL6 ebenso die humane OAT1 und OAT3 Expression reguliert.

No description available.

Non-small cell lung cancer (NSCLC) makes up the majority of lung cancers, which remains the leading cause of cancer mortality worldwide. NSCLC with mutant epidermal growth factor receptor (EGFR) is currently treated with tyrosine kinase inhibitors (TKIs). TKIs have proven effective in improving survival until resistance is conferred, mostly by way of the exon 20, threonine 790 to methionine (T790M) point mutation in EGFR. The mechanism by which this point mutation arises is poorly understood. Herein we report a possible pathway by which the C to T transition that leads to T790M comes about. We show that activation-induced cytidine deaminase (AID) mRNA expression is induced upon treatment with EGFR TKIs in mutant-EGFR human lung cancer cell lines but not in control cell lines. We also show that stable expression of AID is sufficient to produce resistance to one such TKI, erlotinib, and is sufficient to produce T790M itself. We also report that B-cell lymphoma 6 (BCL6) may precede AID in this pathway. Our results show that BCL6 is upregulated in these cell lines treated with EGFR TKIs but not in normal bronchial cells. We then treated human lung cancer cell lines with EGFR TKIs in combination with BCL6 inhibitors. Our results show that AID is dependent upon BCL6 expression. Finally, we report on results from a transient BCL6 overexpression which lead us to believe that AID mRNA receives input from at least one alternate pathway in addition to BCL6. We also performed these experiments on a family of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, that show they may be involved in this pathway downstream of AID. Taken together, our results suggest a potential pathway involving BCL6, AID, and APOBEC cytidine deaminases that lead to the C to T transition that produces T790M, thereby conferring resistance to EGFR TKIs in mutant-EGFR NSCLC. They also provide potential new targets for treatment should further study confirm our results. / 2021-10-07T00:00:00Z