PRISM :: Browsing by Author "Riabowol, Karl T."

Browsing by Author "Riabowol, Karl T."

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Open Access
A Role of ING1 Proteins in Cell Senescence
(2008) Soliman, Mohamed A.; Riabowol, Karl T.
Open Access
ARPC1B Gene: As a Potential Novel Prostate Cancer Driver
(2019-12) Zaaluk, Hend; Bismar, Tarek A.; Riabowol, Karl T.; Argiropoulos, Bob; Lees-Miller, Susan; Eszlinger, Markus
Prostate cancer is the most common malignancy in men and the second leading cause of cancer-related deaths in western countries. Currently, there is a lack of specific molecular markers that can predict cancer progression and prognosis. Characterization of prostate cancer driver genes is essential for investigating the cellular changes that influence the progression of cancer. This will provide a better understanding to prostate cancer carcinogenesis, elucidate novel biomarkers, and improve clinical outcomes. Previously our lab performed a bioinformatics screen using several public cohorts and identified a panel of genes that are deregulated on the mRNA and DNA levels. We hypothesize that these gene are potentially acting as oncogenes and tumor suppressors that could be related to the prognosis of prostate cancer. Actin-related protein -2/3 subunit B (ARPC1B) was found to be one of the most highly dysregulated genes. Dysregulation of ARPC1B expression has been detected in multiple human cancers and ARPC1B protein has been implicated in the control of actin polymerization. Moreover, ARPC1B is involved in many pathways such as cytoskeleton remodeling via actin; integrin mediated cell adhesion and movement of cell/subcellular compartments. The purpose of this research was to evaluate the expression levels of ARPC1B in different prostate cancer cell lines and investigate its potential role in disease progression. ARPC1B expression was analysed using western blot and qRT-PCR in multiple cell lines. We found ARPC1B protein and mRNA levels to be upregulated in the PC3 cell line compared to other cell lines. To validate the role of ARPC1B, siRNA was used to knockdown ARPC1B in PC3 cells which resulted in significant reduction of cell proliferation as measured using the MTS assay. Reduced cell growth and/or reduced migration in cells with ARPC1b knocked down was also seen using scratch assays. Tissue expression levels were also investigated on a progression tissue microarray and showed increased intensity with disease progression from benign to localized cancer and castrate resistant disease. These data suggest that ARPC1B could be a valid prostate cancer marker.
Open Access
ATRX as a Regulator of Telomerase Activity in Cancer Cells
(2020-03) Briggs, Sophie; Beattie, Tara L.; Cairncross, Gregory J.; Riabowol, Karl T.; Grewal, Savraj S.
Telomere maintenance is the central process governing cellular immortality. The two distinct pathways – 1) telomerase activity and 2) the alternative lengthening of telomeres (ALT) – result in telomere elongation and allow cells to evade normal cellular ageing mechanisms. Both telomere maintenance pathways can become activated through gene mutations, leading to genetically unstable cells capable of unlimited proliferation. Expression of the chromatin remodeling protein ATRX is lost in almost all ALT+ cancers, suggesting a role for ATRX in ALT repression, however its exact role in telomere maintenance remains unclear. This thesis provides novel evidence suggesting ATRX acts to resolve telomeric G-quadruplexes (G4), thereby facilitating telomerase activity and indirectly repressing ALT. Using RNA interference in human cancer cell lines and a novel DNA ELISA-based technique, the data presented here establish that loss of ATRX expression results in increased G4 at the telomere. This G4 enrichment correlates with a reduction in telomerase activity in vitro which is exacerbated following treatment with the G4 stabilizing agent, Pyridostatin. Further, loss of the full-length ATRX isoform alone does not directly correlate with the ALT phenotype in the selected cell panel. However, a truncated ATRX isoform, ATRXt, was found to be expressed in all selected telomerase+ cells and shown to maintain key functions of the full-length protein. Therefore, alternative ATRX isoforms may act to facilitate telomerase activity through G4 resolution, even in the absence of full-length protein. Taken together, these data provide novel evidence identifying ATRX as an important factor facilitating telomerase-mediated telomere elongation through G4 resolution at the telomere.
Open Access
Biochemical and functional characterization of the ING tumor suppressor family
(2006) Feng, Xiaolan; Riabowol, Karl T.
Open Access
Biochemical and molecular studies on the candidate tumor suppressor ING1 during apoptosis and senescence
(2003) Vieyra, Diego; Riabowol, Karl T.
Open Access
Characterising the Role of the Smc5/6 Complex at Telomeres
(2021-01-29) McLaren, William David John; Beattie, Tara L.; Zaremberg, Vanina; Riabowol, Karl T.
Smc5/6 is a member of the SMC family of proteins alongside cohesin and condensin. The complex has previously described roles in DNA repair and more recently a role in telomere binding and maintenance. Here, I demonstrate novel genetic interactions between the Smc5/6 complex and a nuclear pore complex protein Nup170. Both Nup170 and Esc1 have previously been shown to be necessary for telomere maintenance and these two proteins form a complex alongside Sir4, a well-defined regulator of heterochromatin at the telomeres. Loss of Nup170 when combined with a DNA binding-defective mutant of Nse3, a core component of the Smc5/6 complex, leads to increases in subtelomeric gene expression and loss of telomere clustering. It was observed that by combining a deletion mutant of Nup170 with an Nse3 mutant leads to a decrease in survival when exposed to the DNA damage inducing agent MMS as well as an increase in subtelomeric gene expression. Observing Rap1GFP clusters as a readout for telomere clustering shows no change between nse3-1 single mutant and the nup170Δ/nse3-1 double mutant. This indicates that both Smc5/6 and Nup170 operate together to maintain normal telomere function and that they operate in the same pathway to cluster telomeres. These findings help to further elucidate the role of the Smc5/6 complex in maintaining telomere clustering, suppressing expression of subtelomeric genes at telomeres and interactions with Nup170.
Open Access
Characterization and developmental functions of the c. elegans ing-3 gene product
(2007) Luo, Jingjing; Riabowol, Karl T.; Mains, Paul E.
Open Access
Characterization of the ING1 candidate tumor suppressor gene in breast cancer cells
(2001) Nelson, Rebecca; Riabowol, Karl T.
Open Access
Characterizing Inhibitor of Growth (ING) family evolution and ING1 structure and function
(2020-07-29) Bertschmann, Jessica; Riabowol, Karl T.; Cobb, Jennifer A.; De Koning, A. P. Jason
The INhibitior of Growth (ING) family of tumor suppressors have emerged as a versatile family of phospholipid effectors, histone mark sensors, and growth regulators. An updated phylogenetic analysis of this protein family using sequences from 42 eukaryotic species reveals that ING4 is likely most similar to the ancestral ING protein, not ING3 as previously reported. Previous studies have shown that the major ING1 isoforms, ING1a and ING1b serve distinct cellular functions by differentially regulating apoptosis and senescence in primary cells. The ING1a isoform encodes a sequence unique in the human proteome. To identify ING1a homologs in other species we searched all available databases and found that sequences corresponding to ING1a were only found in great apes and Old-World monkeys. However, only select primates had start codons capable of encoding full-length ING1a. Moreover, when we expressed ING1a with and without it’s unique N-terminal sequence, the unique sequence promoted localization to the mitochondria. Given the natural induction of this isoform as cells age in culture, expression of ING1a may serve to help limit the replicative lifespan of cells from long-lived primates, in part through its activity in the mitochondria.
Open Access
The Development and Validation of a Novel Non-Invasive Assay Based on Cell Free-DNA to Detect Acute Allograft Rejection After Heart Transplantation
(2019-08-22) Pattar, Sabrina Kaur; Greenway, Steven C.; Fine, Nowell M.; Riabowol, Karl T.
Immune-mediated injury (rejection) of a transplanted organ is a serious problem that can lead to allograft dysfunction and patient death. The gold standard for diagnosing acute cellular rejection (ACR) after heart transplantation (HT) is the endomyocardial biopsy (EMB), an invasive procedure with significant limitations. Dying cells release fragments of DNA into the circulation and increased levels of donor-derived cell-free DNA (dd-cfDNA) have been associated with ACR. Current methods to measure dd-cfDNA employ single nucleotide polymorphisms (SNPs) but an epigenetics-based assay could also accurately quantify dd-cfDNA in recipient blood. This thesis aimed to validate the use of ventricle-specific methylation patterns in human cfDNA as an alternative and novel biomarker for ACR following HT. We hypothesized that dd-cfDNA released due to ACR-mediated injury could be quantified in recipient plasma based on epigenetic differences and would correlate better with tissue apoptosis than EMB-based rejection. We identified increased cellular apoptosis within the myocardium as the severity of ACR increased, which provided a biological rationale for the use of cfDNA as a biomarker for rejection. We also initiated validation of an alternative sequencing platform and panel of highly polymorphic SNPs, which may improve a previously-established SNP-based assay. Finally, we established a bioinformatic pipeline for the identification of ventricle-specific differentially methylated regions (DMRs). These DMRs underwent retrospective validation using cfDNA samples from adult HT patients, which were associated with a known biopsy-proven rejection grade, to demonstrate the ability of these novel blood biomarkers to non-invasively detect acute rejection following HT. In conclusion, we successfully demonstrated the efficacy of cfDNA as a biomarker for immune-mediated tissue injury and introduced the potential use of two ventricle-specific DMRs for the identification and quantification of cfDNA released from a donated heart due to ACR.
Open Access
DNA Methylation Landscape of the Fibrinogen Gene Cluster in the Equine Embryo
(2018-09-26) Grant, Danielle Magann; Klein, C.; Riabowol, Karl T.; Demetrick, Douglas James
The initial weeks of equine pregnancy includes the unique phase of embryo mobility from day 9 until fixation on day 15. Fixation of the embryo to the uterine wall occurs before a microvillous attachment at ~day 40. Throughout the mobility period the equine embryo expresses and secretes fibrinogen, a protein best known for its involvement in the coagulation cascade and wound repair. The aim of this MSc project was to contribute to the characterization of conceptus-derived fibrinogen at the time of fixation. We confirmed early embryo expression in the absence of hepatic activity, as well as describe expression by the later fetal-placenta. We characterized DNA methylation across the fibrinogen gene cluster for equine liver, day 14 embryos, and endometrium. The methylation landscape of equine embryos is distinct from both liver and endometrium. However, in key regulatory regions the embryo and liver profiles were the same. The similarity to liver methylation in known regulatory regions supports fibrinogen expression by the embryo and suggests its involvement in gene regulation. In addition to our genetic characterization, we trialed various in vitro assays in an attempt to determine the possible role of fibrinogen at the embryo-maternal interface. Overall this study has contributed to our ongoing effort to provide context for the novel extra-hepatic expression of fibrinogen by the pre-implantation conceptus.
Open Access
Examination of the roles of ING1 in development
(2007) Quarrie, Jason Kenneth; Riabowol, Karl T.
Open Access
Genetic and epigenetic events regulating cellular senescence and aging
(2005) Berardi, Philip; Riabowol, Karl T.
Open Access
Identification of a novel conserved region in ing1 tumor suppressors as a lamin interacting domain
(2007) Han, Xijing; Riabowol, Karl T.
Open Access
In vitro investigation and characterization of ING1 protein interactions
(2005) Gong, Wei; Riabowol, Karl T.
Open Access
ING1 Impacts Ovarian Cancer by Altering EMT
(2019-08-30) Yang, Yang; Riabowol, Karl T.; Bonni, Shirin; Rancourt, Derrick E.; Muruve, Daniel A.
The INhibitor of Growth (ING) proteins are type II tumour suppressors that regulate epigenetic state and transcription by recruiting various chromatin-modifying complexes to chromatin. INGs are involved in multiple cellular processes such as DNA repair, apoptosis and cellular senescence. In this study, we investigated the potential role of ING1 in inhibiting the epithelial-mesenchymal transition (EMT) program that suppresses cancer metastasis and identifies the mechanism of how ING1 regulates gene transcription, as well as its clinical implication in epithelial ovarian cancer. Our analyses revealed that ING1 epigenetically regulates the transcription machinery of the EMT program through binding to the promoter region of Twist1, the critical gene encoding transcription factor (TF) that induce EMT. Subsequently, ING1 promoted the expression of epithelial cell markers, including E-cadherin, while suppressing the expression of mesenchymal markers such as N-cadherin. ING1 antagonized TGF-β or EGF induced EMT and inhibited cancer cell invasion and migration in a Twist1-dependent manner. Integration of ChIP-seq and RNA-seq data revealed the overall genomic binding characteristics of ING1 and its candidate target genes. Gene ontology (GO) and pathway analyses indicated that the genes targeted by ING1 were involved in diverse physiological processes and pathways, mostly associated with the EMT program. The bioinformatical analysis revealed the ING1 binding motif sequence, showing that ING1 recognized target genes through the TEAD family of transcription factors and their co-factor, YAP1, to further influence gene expression. Lastly, we found that high ING1 protein expression was associated with p16 and ARID1A levels and predicted better DSS in ovarian clear cell carcinoma (OCCC). Our data also suggested that high ING1/low N-cadherin expression predicts favourable disease-specific survival (DSS) in epithelial ovarian cancer. These data provide evidence that as a tumour suppressor, ING1 can impact ovarian cancer development through modulating the EMT process and play an essential role in regulating ovarian cancer chemoresistance. In summary, by downregulating the EMT-TF expression and hence and EMT program, ING1 suppresses tumour cell invasion and cancer metastasis. Global genomic profiling revealed new insights into ING1 biology and provided justification for further exploring ING1 functions. This study has also provided critical pre-clinical data that could help establish ING1 as a prognostic and therapeutic agent for ovarian cancer.
Open Access
Ing1 protein function in apoptosis and senescence
(2010) Bose, Pinaki; Riabowol, Karl T.
ING proteins act as readers and writers of the histone epigenetic code, affecting DNA repair, cellular senescence and apoptosis. My doctoral research focused upon understanding the molecular mechanisms underlying the involvement of ING1 in stress signaling. My work began with elucidating novel interacting partners of ING proteins using a cross-species (yeast, fly, and human) bioinformatics-based approach. We identified 381 proteins in a yeast interactome analysis that interacted with ING and also had human counterparts. I then biochemically confirmed the validity of the screen, showing that ING1 interacts with three proteins involved in stress signaling: p38MAPK, MEKK4 and RAD50. Our bioinformatics screen indicated that ING proteins in yeast interact with several mitochondrial proteins. Given that ING1 and p53 can functionally interact and that p53 has a transcription-independent role in apoptosis in the mitochondria, I asked if ING1 might also function outside the nucleus. We found that ING1 translocates to the mitochondria in primary fibroblasts and in established epithelial cell lines in response to apoptosis-inducing stimuli, independent of cellular p53 status. I also determined that endogenous ING1 specifically interacts with the pro-apoptotic BCL2 machinery in the mitochondria, suggesting a model in which the ING1-BAX interaction promotes mitochondrial membrane permeability, thereby promoting apoptosis. Since cellular aging is widely thought to represent a form of telomereinduced stress, I also investigated if ING1 interacted with the telomeric protein TRF2 that is involved in DNA damage and repair pathways. We found that, indeed, endogenous ING1 protein interacted with TRF2. Interestingly, TRF2 protein levels were also seen to decrease in senescent primary fibroblasts without a concomitant decrease in TRF2 mRNA levels. Further, our data provides evidence for the fact that telomere binding of TRF2 is required for its stability thereby indicating that a decrease in TRF2 levels might affect the induction of senescence caused by telomere attrition. Lastly, I characterized the DNA damage response in young versus senescent fibroblasts in which ING1 levels are altered, using 53BP1 focus formation as a surrogate marker for DNA damage. I found that decreased ability of senescent cells to process DNA damage foci correlates well with the loss of ING1 b during senescence, consistent with a role for ING1 bin DNA repair.
Open Access
Interspecies data mining to predict novel ING-protein interactions in human
(BioMed Central, 2008) Gordon, Paul; Soliman, Mohamed A; Bose, Pinaki; Sensen, Christoph W; Riabowol, Karl T.
Open Access
Investigating the biochemical and developmental role of the tumour suppressor ing-3 in caenorhabditis elegans
(2011) Shah, Sitar; Riabowol, Karl T.; Mains, Paul E.
Open Access
Lamin a regulates ing1b mrna stability
(2010) Smith, Heather Anne; Riabowol, Karl T.