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1. Attributes of Oct4 in stem cell biology: perspectives on cancer stem cells of the ovary

Author

Samardzija Chantel, Quinn Michael, Findlay Jock K, and Ahmed Nuzhat

Subjects
Ovarian carcinoma, Cancer stem cell, Metastasis, Chemoresistance, Recurrence, Embryonic stem cells, Induced pluripotent stem cells, Gynecology and obstetrics, RG1-991
Abstract

Abstract Epithelial ovarian cancer (EOC) remains the most lethal of all the gynaecological malignancies with drug resistance and recurrence remaining the major therapeutic barrier in the management of the disease. Although several studies have been undertaken to understand the mechanisms responsible for chemoresistance and subsequent recurrence in EOC, the exact mechanisms associated with chemoresistance/recurrence continue to remain elusive. Recent studies have shown that the parallel characteristics commonly seen between embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) are also shared by a relatively rare population of cells within tumors that display stem cell-like features. These cells, termed ‘cancer initiating cells’ or ‘cancer stem cells (CSCs)’ have been shown not only to display increased self renewal and pluripotent abilities as seen in ESCs and iPSCs, but are also highly tumorigenic in in vivo mouse models. Additionally, these CSCs have been implicated in tumor recurrence and chemoresistance, and when isolated have consistently shown to express the master pluripotency and embryonic stem cell regulating gene Oct4. This article reviews the involvement of Oct4 in cancer progression and chemoresistance, with emphasis on ovarian cancer. Overall, we highlight why ovarian cancer patients, who initially respond to conventional chemotherapy subsequently relapse with recurrent chemoresistant disease that is essentially incurable.

Published
2012
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2. Neuronal transcription factor Brn-3a(l) is over expressed in high-grade ovarian carcinomas and tumor cells from ascites of patients with advanced-stage ovarian cancer

Author

Ahmed Nuzhat, Latifi Ardian, Riley Clyde B, Findlay Jock K, and Quinn Michael A

Subjects
Gynecology and obstetrics, RG1-991
Abstract

Abstract Objectives In view of the recent association of Brn-3 transcription factors with neuroblastomas, cervical, breast, and prostate cancers we examined the expression of Brn-3a(l) in normal ovaries and in different histological grades of ovarian tumors. The expression of Brn-3a(l) was also evaluated in normal ovarian and cancer cell lines and tumor cells isolated from the ascites of advanced-stage ovarian cancer patients. Methods Normal ovaries, benign, borderline, grades 1, 2 and 3 ovarian tumors were analyzed by immunohistochemistry for Brn-3a(l) expression. A total of 46 ovarian specimens were included in the study. Immunofluorescence was used to investigate the expression of Brn-3a in normal ovarian and cancer cell lines. Brn-3a(l) expression was also evaluated by Western blot in tumor cells isolated from ascites of advanced-stage ovarian cancer patients and also in ovarian cancer cell lines. Results Nearly 12% of normal and benign ovarian tissues and 57% of borderline ovarian tumors were positive for epithelial Brn-3a(l) expression. Stromal staining was higher and it constituted 40% of normal non-cancerous ovaries compared to 50 and 86% in benign and borderline tumors. On the other hand, 85-100% of grades 1, 2 & 3 ovarian tumors demonstrated nuclear and cytoplasmic Brn-3a(l) staining in the epithelium. Stromal staining in grades1, 2 and 3 tumors constituted 71-88% of total staining. Overall, immunoreactive Brn-3a was present in all grades of ovarian tumors. The extent of epithelial and stromal Brn-3a staining was significantly different between the normal and histological grades of tumors (epithelial-χ2 = 41.01, df = 20, P = 0.004, stromal-χ2 = 24.66. df = 15, P = 0.05). The extent of epithelial staining was significantly higher in grades 1 and 2 ovarian tumors compared to normal ovaries and benign ovarian tumors (p < 0.05). In parallel, stromal staining was significantly higher in grade 3 tumors compared to normal ovaries (p < 0.05). In addition, cytoplasmic and nuclear Brn-3a expression was evident in ovarian cancer cell lines while no such expression was observed in SV40 antigen immortalized normal ovarian cell lines. Conclusion These data suggest that like other cancers, Brn-3a(l) expression is enhanced in ovarian tumors and its expression is consistent with its known role in inhibiting apoptosis and enhancing tumorigenesis. Specific targeting of Brn-3a may provide a useful strategy for regulating multiple tumor related genes involved with ovarian carcinomas.

Published
2010
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3. HTRA3 expression in non-pregnant rhesus monkey ovary and endometrium, and at the maternal-fetal interface during early pregnancy

Author

Findlay Jock K, Liu Yi-Xun, Li Ying, Bowden Marissa A, Salamonsen Lois A, and Nie Guiying

Subjects
Gynecology and obstetrics, RG1-991, Reproduction, QH471-489
Abstract

Abstract Background HTRA3 is a recently identified member of the mammalian serine protease family HTRA (high temperature requirement factor A). In both the rodent and the human HTRA3 is transcribed into two mRNA species (long and short) through alternative splicing. We have previously shown that HTRA3 is expressed in the mature rat ovary and may be involved in folliculogenesis and luteinisation. HTRA3 is also upregulated during mouse and human placental development. The current study investigated whether HTRA3 is also localised in the primate ovary (rhesus monkey n = 7). In addition, we examined the non-pregnant rhesus monkey endometrium (n = 4) and maternal-fetal interface during early pregnancy (n = 5) to further investigate expression of HTRA3 in primate endometrium and placentation. Methods HTRA3 mRNA levels in several rhesus monkey tissues was determined by semiquantitative RT-PCR. Protein expression and localisation of HTRA3 was determined by immunohistochemistry. Results Long and short forms of HTRA3 mRNA were detected in the ovary, aorta, bladder, small intestine, skeletal muscle, heart and uterus but not the liver nor the kidney. HTRA3 protein was immunolocalised to the oocyte of all follicular stages in the rhesus monkey ovary. Protein expression in mural and cumulus granulosa cells of late secondary follicles increased significantly compared to granulosa cells of primordial, primary and secondary follicles. Mural and cumulus granulosa cells of antral follicles also showed a significant increase in expression. Staining intensity was higher in the granulosa-lutein cells compared to the theca-lutein cells of corpora lutea (n = 3). In the non-pregnant monkey endometrium, HTRA3 was detected in the glandular epithelium. The basalis endometrial glands showed higher staining intensity than functionalis endometrial glands. During early pregnancy, strong staining for HTRA3 protein was seen in both maternal decidual cells and glands. Conclusion We propose that HTRA3 may be involved in folliculogenesis and luteinisation in the primate ovary. Furthermore, similar to previous findings in the human, HTRA3 is possibly a factor involved in and potentially important for primate placentation.

Published
2008
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8. Additional file 1 of Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer

Author

Escalona, Ruth M., Chu, Simon, Kadife, Elif, Kelly, Jason K., Kannourakis, George, Findlay, Jock K., and Ahmed, Nuzhat

Abstract

Additional file 1: Figure S1. siRNA suppression of TIMP-2 in the OVCAR5 cell line. Diagram showing the location of single siRNA duplexes A, B, C in the TIMP-2 gene. Figure S2. CRISPR/Cas9 editing of the TIMP-2 gene in the OVCAR5 cell line. A Diagram showing the configuration of TIMP-2 CRISPR/Cas9 plasmid. The TIMP-2 linear donor plasmid is ~ 2.74 kb and incorporates the donor GFP and puromycin (under the EF1a promoter). The transfection involves integration of Cas9/gRNA and the linear donor plasmid (GFP and puromycin) genes into cells. The Cas9 targets the TIMP-2 gene in exon1, guided by two gRNA sequences [the gRNA1 (yellow) and gRNA2 (pink)]. The donor genes can be inserted in the cells by transcription in the forward or reverse directions. In both situations, interruption of TIMP-2 expression coinciding with puromycin resistance and GFP expression should occur. (Figure adapted from https://www.origene.com/catalog/gene-expression/knockout-kits-crispr/kn409796/timp2-human-gene-knockout-kit-crispr ). B Puromycin “death” curve in the OVCAR5 cell line. The OVCAR5 cell line was incubated with puromycin concentrations ranging from 0 to 320 µg/mL followed by an MTT assay of OVCAR5 cells after 48 h. The concentration of 3 µg/mL (indicated by red arrow) was used for puromycin selection. Values are mean ± SEM and graph is representative of three experiments done in triplicate. C CRISPR transfected OVCAR5 cells after puromycin selection and GFP sorting. OVCAR5 cells were transfected with CRISPR/Cas9 vectors plus the donor plasmid containing puromycin and GFP genes. After puromycin selection, cells were GFP sorted and visualized under a confocal microscope. After a second GFP sorting, GFP fluorescence was only seen in gRNA2 cells but not in gRNA1 cells. 20× magnification; scale bar 1000 µM. D MTT assay of OVCAR5 TIMP-2 CRISPR/Cas9 transfected and Control cells. After 48 h of incubation the concentration of puromycin that killed 50% of cells was determined (IC50 values). Values are mean ± SEM and the graph is representative of three experiments done in triplicate. Figure S3. Expression of cellular TIMP-2 and corresponding GAPDH by Western blot. Representative full image of a Western blot of TIMP-2 and GAPDH proteins on the cell lysates of parental, CRISPR/Cas9 treated control, gRNA1 and gRNA2 cell lines. Figure S4. Quantification of EdU stained OVCAR5 parental, CRISPR control, and TIMP-2 knocked down gRNA2 and gRNA1 cells. The cells were stained with EdU and propidium iodide (PI) as described in “Methods”. A Flow cytometer representation of percentage of EdU stained cells in S-phase of the cycle for CRISPR transfected cell lines. B Flow cytometer representation of negative controls used to calculate the percentage of EdU stained cells in S-phase of the cycle for the OVCAR5 parental cell line. Red rectangles indicate the areas analysed for EdU positive cells. Table S1. Primers used in the study.

Published
2023
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15. Cisplatin-induced primordial follicle oocyte killing and loss of fertility are not prevented by imatinib

Author

Kerr, Jeffrey B., Hutt, Karla J., Cook, Michele, Speed, Terence P., Strasser, Andreas, Findlay, Jock K., Scott, Clare L., Maiani, Emiliano, Bartolomeo, Claudia Di, Klinger, Francesca G., Cannata, Stefano M., Bernardini, Sergio, Chateauvieux, Sebastien, Mack, Fabienne, Mattei, Maurizio, Felict, Massimo De, Diederich, Marc, Cesareni, Gianni, and Gonfloni, Stefania

Subjects
Oocytes -- Properties, Cisplatin -- Dosage and administration -- Patient outcomes, Fertility, Human -- Research, Biological sciences, Health
Abstract

To the Editor: TAp63α is the predominant member of the Trp53 family (consisting of the tumor suppressors Trp53, Trp63 and Trp73), expressed in primordial follicle oocytes (1) and is essential [...]

Published
2012

42. Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling

Author

Samardzija, Chantel, Greening, David W., Escalona, Ruth, Chen, Maoshan, Bilandzic, Maree, Luwor, Rodney, Kannourakis, George, Findlay, Jock K., Ahmed, Nuzhat, Samardzija, Chantel, Greening, David W., Escalona, Ruth, Chen, Maoshan, Bilandzic, Maree, Luwor, Rodney, Kannourakis, George, Findlay, Jock K., and Ahmed, Nuzhat

Abstract

Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas.

Published
2017

45. Coalition of Oct4A and β1 integrins in facilitating metastasis in ovarian cancer

Author

Samardzija, Chantel, Luwor, Rodney B., Quinn, Michael A., Kannourakis, George, Findlay, Jock K., Ahmed, Nuzhat, Samardzija, Chantel, Luwor, Rodney B., Quinn, Michael A., Kannourakis, George, Findlay, Jock K., and Ahmed, Nuzhat

Abstract

Background: Ovarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis. Methods: The expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice. Results: We report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant

Published
2016

46. Unique proteome signature of post-chemotherapy ovarian cancer ascites-derived tumor cells

Author

Ahmed, Nuzhat, Greening, David, Samardzija, Chantel, Escalona, Ruth M., Chen, Maoshan, Findlay, Jock K., Kannourakis, George, Ahmed, Nuzhat, Greening, David, Samardzija, Chantel, Escalona, Ruth M., Chen, Maoshan, Findlay, Jock K., and Kannourakis, George

Abstract

Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission afterinitial surgery and chemotherapy. However, most patients die within <5 years due to episodesof recurrences resulting from the growth of residual chemoresistant cells. In an effort to identifymechanisms associated with chemoresistance and recurrence, we compared the expression of proteinsin ascites-derived tumor cells isolated from advanced-stage ovarian cancer patients obtained atdiagnosis (chemonaive, CN) and after chemotherapy treatments (chemoresistant/at recurrence, CR)by using in-depth, high-resolution label-free quantitative proteomic profiling. A total of 2,999 proteinswere identified. Using a stringent selection criterion to define only significantly differentially expressedproteins, we report identification of 353 proteins. There were significant differences in proteinsencoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-celladhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/argininesynthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichmentof metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells.In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cellsfrom CN and CR ovarian cancer patients.

Published
2016

49. BCL2-modifying factor promotes germ cell loss during murine oogenesis

Author

Vaithiyanathan, Kavitha, primary, Liew, Seng H, additional, Zerafa, Nadeen, additional, Gamage, Thilini, additional, Cook, Michele, additional, O’Reilly, Lorraine A, additional, Bouillet, Philippe, additional, Scott, Clare L, additional, Strasser, Andreas, additional, Findlay, Jock K, additional, and Hutt, Karla J, additional

Published
2016
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50. A critical role of Oct4A in mediating metastasis and disease-free survival in a mouse model of ovarian cancer

Author

Samardzija, Chantel, Luwor, Rodney B., Volchek, Mila, Quinn, Michael A., Findlay, Jock K., Ahmed, Nuzhat, Samardzija, Chantel, Luwor, Rodney B., Volchek, Mila, Quinn, Michael A., Findlay, Jock K., and Ahmed, Nuzhat

Abstract

Background High grade epithelial ovarian cancer (EOC) is commonly characterised by widespread peritoneal dissemination and ascites. Metastatic EOC tumour cells can attach directly to neighbouring organs or alternatively, maintain long term tumourigenicity and chemoresistance by forming cellular aggregates (spheroids). Cancer stem-like cells are proposed to facilitate this mechanism. This study aimed to investigate the role of Oct4A, an embryonic stem cell factor and known master regulator of pluripotency in EOC progression, metastasis and chemoresistance. Methods To investigate the expression of Oct4A in primary EOC tumours, IHC and qRT-PCR analyses were used. The expression of Oct4A in chemonaive and recurrent EOC patient ascites-derived tumour cells samples was investigated by qRT-PCR. The functional role of Oct4A in EOC was evaluated by generating stable knockdown Oct4A clones in the established EOC cell line HEY using shRNA-mediated silencing technology. Cellular proliferation, spheroid forming ability, migration and chemosensitivty following loss of Oct4A in HEY cells was measured by in vitro functional assays. These observations were further validated in an in vivo mouse model using intraperitoneal (IP) injection of established Oct4A KD clones into Balb/c nu/nu mice. Results We demonstrate that, compared to normal ovaries Oct4A expression significantly increases with tumour dedifferentiation. Oct4A expression was also significantly high in the ascites-derived tumour cells of recurrent EOC patients compared to chemonaive patients. Silencing of Oct4A in HEY cells resulted in decreased cellular proliferation, migration, spheroid formation and increased chemosensitivity to cisplatin in vitro. IP injection of Oct4A knockdown cells in vivo produced significantly reduced tumour burden, tumour size and invasiveness in mice, which overall resulted in significantly increased mouse survival rates compared to mice

Published
2015

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