Clifford Steer - Academia.edu (original) (raw)

Papers by Clifford Steer

Biochemical and Biophysical Research Communications, 2010

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from h... more Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 microm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

The Veterinary Journal, 2012

Porcine hepatocytes are potentially important in liver regeneration and in the treatment of human... more Porcine hepatocytes are potentially important in liver regeneration and in the treatment of humans with acute and chronic liver diseases. Induced pluripotent stem (iPS) cells are a valuable source of hepatocytes for these applications as they have unlimited potential to propagate in vitro. An efficient and robust differentiation of iPS cells generated from porcine fetal fibroblasts into functional hepatocyte-like cells in vitro is reported. The methodology followed a three-step differentiation protocol using several growth factors, namely, activin A, basic fibroblast growth factor, bone morphogenetic protein-4, and oncostatin M. Porcine iPS cell-derived hepatocyte-like (piPS-Hep) cells were characterized by morphological analysis and were tested for the expression of hepatocyte-specific genes using RT-PCR. Functional analyses for albumin production and glycogen storage were also carried out. These differentiated hepatocyte-like cells could represent a valuable source for studies of drug metabolism and for cell transplantation therapy for a variety of liver disorders.

Hepatology, 2008

Hepatocellular carcinoma (HCC) typically has poor prognosis, because it is often diagnosed at an ... more Hepatocellular carcinoma (HCC) typically has poor prognosis, because it is often diagnosed at an advanced stage. Heterogeneous phenotypic and genetic traits of affected individuals and a wide range of risk factors have classified it a complex disease. HCC is not amenable to standard chemotherapy and is resistant to radiotherapy. In most cases, surgical resection and liver transplantation remain the only curative treatment options. Therefore, development of novel, effective therapies is of prime importance. Extensive research over the past decade has identified a number of molecular biomarkers as well as cellular networks and signaling pathways affected in liver cancer. Recent studies using a combination of "omics" technologies, microRNA studies, combinatorial chemistry, and bioinformatics are providing new insights into the gene expression and protein profiles during various stages of the disease. In this review, we discuss the contribution of these newer approaches toward an understanding of molecular mechanisms of HCC and for the development of novel cancer therapeutics.

Cell Death and Disease, 2015

This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to a... more This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to ascertain the relevance of MEK5/ERK5 signalling in colon cancer. Expression of MEK5 and ERK5 was evaluated in 323 human colon cancer samples. To evaluate the role of MEK5/ERK5 signalling in colon cancer, we developed a stable cell line model with differential MEK5/ERK5 activation. Impact of differential MEK5/ERK5 signalling was evaluated on cell cycle progression by flow cytometry and cell migration was evaluated by wound healing and transwell migration assays. Finally, we used an orthotopic xenograft mouse model of colon cancer to assess tumour growth and progression. Our results demonstrated that MEK5 and ERK5 are overexpressed in human adenomas (Po0.01) and adenocarcinomas (Po0.05), where increased ERK5 expression correlated with the acquisition of more invasive and metastatic potential (Po0.05). Interestingly, we observed a significant correlation between ERK5 expression and NF-κB activation in human adenocarcinomas (Po0.001). We also showed that ERK5 overactivation significantly accelerated cell cycle progression (Po0.05) and increased cell migration (Po0.01). Furthermore, cells with overactivated ERK5 displayed increased NF-κB nuclear translocation and transcriptional activity (Po0.05), together with increased expression of the mesenchymal marker vimentin (Po0.05). We further demonstrated that increased NF-κB activation was associated with increased IκB phosphorylation and degradation (Po0.05). Finally, in the mouse model, lymph node metastasis was exclusively seen in orthotopically implanted tumours with overactivated MEK5/ERK5, and not in tumours with inhibited MEK5/ERK5. Our results suggested that MEK5/ERK5/NF-κB signalling pathway is important for tumour onset, progression and metastasis, possibly representing a novel relevant therapeutic target in colon cancer treatment.

BMC Genomics, 2013

Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample ... more Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol W and TRIzol W LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.

Current Medicinal Chemistry, 2009

Nuclear steroid receptors (NSR) are ligand-activated transcription factors that play a key role i... more Nuclear steroid receptors (NSR) are ligand-activated transcription factors that play a key role in a variety of vital physiological phenomena including developmental or endocrine signaling, reproduction, and homeostasis. In addition, they are implicated in other important biological processes, such as apoptosis. Modulation of apoptosis by NSR is mostly associated with control of pro-apoptotic versus anti-apoptotic gene expression, and includes both induction and prevention of apoptosis depending on cell type. However, it is unclear how NSR can affect opposing expression of the same gene in different cells. Of note, recently described nongenomic mechanisms of NSR, in particular glucocorticoid receptor translocation to mitochondria, were suggested to be crucial steps for triggering apoptosis. NSR often act solely as nuclear transporters of other regulatory molecules, thus indirectly regulating several apoptosis-related genes. Curiously, NSR are thought to cooperate with the anti-apoptotic endogenous bile acid, ursodeoxycholic acid (UDCA), to prevent programmed cell death. In fact, as cholesterol-derived molecules and due to their chemical and structural similarities to steroid hormones, bile acids also modulate NSR activation. Although the precise link between NSR and UDCA remains unclear, we have demonstrated that the bile acid requires NSR for translocation to the cell nucleus as part of a ligand-receptor complex, using a mechanism similar to that of steroid hormones. Interestingly, other studies revealed that UDCA interacts with the glucocorticoid receptor as a novel and selective NSR modifier. The huge diversity of natural ligands and xenobiotics that bind to NSR and regulate their function represents one of the most exciting drug targets for potential therapeutic intervention. The next decade will almost certainly unveil the remarkable role of NSR in modulating cell fate in human health and disease.

The dynamic and multiple functions of p53, together with its involvement in the most common non-i... more The dynamic and multiple functions of p53, together with its involvement in the most common non-infectious diseases, underscore the need to elucidate the complexity of the p53 regulatory networks. Pathological conditions such as cancer, neurodegeneration, ischemia, cholestasis, and atherosclerosis are all strongly associated with deregulated levels of apoptosis in which p53 dysfunction has a prominent role. We will highlight recent developments of p53-induced apoptosis in human diseases, with a focus on modulation of liver cell apoptosis. In addition, we will discuss controversies arising from widespread p53 activation as a therapeutic approach to cancer. Recent studies have provided relevant and unprecedented information about mechanistic antiapoptotic functions of the endogenous bile acid, ursodeoxycholic acid (UDCA), suggesting that the finely tuned, complex control of p53 by Mdm-2 (mouse double minute-2, an oncoprotein) is a key step in UDCA modulation of p53-triggered apoptosis. We will also review recent therapeutic strategies and clinical applications of targeted agents, their safety, and efficacy, with particular emphasis on potential benefits of UDCA.

Global Advances in Health and Medicine, 2015

Numerous interspecies disease transmission events, Ebola virus being a recent and cogent example,... more Numerous interspecies disease transmission events, Ebola virus being a recent and cogent example, highlight the complex interactions between human, animal, and environmental health and the importance of addressing medicine and health in a comprehensive scientific manner. The diversity of information gained from the natural, social, behavioral, and systems sciences is critical to developing and sustainably promoting integrated health approaches that can be implemented at the local, national, and international levels to meet grand challenges. The Concept of One Medicine One Science (COMOS) as outlined herein describes the interplay between scientific knowledge that underpins health and medicine and efforts toward stabilizing local systems using 2 linked case studies: the food system and emerging infectious disease. Forums such as the International Conference of One Medicine One Science (iCOMOS), where science and policy can be debated together, missing pieces identified, and science-based collaborations formed among industry, governmental, and nongovernmental policy makers and funders, is an essential step in addressing global health. The expertise of multiple disciplines and research foci to support policy development is critical to the implementation of one health and the successful achievement of global health security goals.

The metabolism of eukaryotic mRNA occurs in both the nucleus and cytoplasm and involves a coordin... more The metabolism of eukaryotic mRNA occurs in both the nucleus and cytoplasm and involves a coordinated balance between transcriptional and posttranscrip- tional events. Posttranscriptional regulation can occur at many stages in the processing of a transcript, in- cluding its demise. A major contribution to the post- transcriptional regulation of mammalian gene expression is at the level of mRNA stability. The

The Future of Aging, 2010

The last two decades have shown significant advances in our understanding of age- and disease-rel... more The last two decades have shown significant advances in our understanding of age- and disease-related alterations in the regulation of gene expression and the underlying endogenous gene repair pathways. As a result, there have been some important strides in the development of engineered technologies for repairing mutated or damaged DNA via the development, delivery, and integration of specific and selective

Gut, Jan 17, 2015

Non-alcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma (HC... more Non-alcoholic fatty liver disease (NAFLD) is a major risk factor for hepatocellular carcinoma (HCC). However, the mechanistic pathways that link both disorders are essentially unknown. Our study was designed to investigate the role of microRNA-21 in the pathogenesis of NAFLD and its potential involvement in HCC. Wildtype mice maintained on a high fat diet (HFD) received tail vein injections of microRNA-21-anti-sense oligonucleotide (ASO) or miR-21 mismatched ASO for 4 or 8 weeks. Livers were collected after that time period for lipid content and gene expression analysis. Human hepatoma HepG2 cells incubated with oleate were used to study the role of miR-21 in lipogenesis and analysed with Nile-Red staining. microRNA-21 function in carcinogenesis was determined by soft-agar colony formation, cell cycle analysis and xenograft tumour assay using HepG2 cells. The expression of microRNA-21 was increased in the livers of HFD-treated mice and human HepG2 cells incubated with fatty acid. Mi...

Anticancer research, 2015

Treatment of rectal cancer has improved significantly with the addition of neoadjuvant chemoradia... more Treatment of rectal cancer has improved significantly with the addition of neoadjuvant chemoradiation. Certain patients have experienced a complete pathological response to chemoradiation, as observed in surgically resected tissue samples, thus calling into question the necessity of radical surgery in this population of patients. Pharmacogenetic studies now implicate the role that genetic biomarkers, such as single nucleotide polymorphisms, play in an individual's response to chemoradiation. The aim of this review was to provide a comprehensive evaluation of a group of candidate single nucleotide polymorphisms associated with chemoradiotherapy response and an assessment of techniques that can be used to easily identify the presence of these single nucleotide polymorphisms in patient samples. Relevant primary research articles were identified in the Medline Database from January 1, 2006 to May 31, 2012. We included nine relevant articles addressing the correlation between six can...

Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, Jan 30, 2015

Liver plays a major role in many inherited and acquired genetic disorders. It is also the site fo... more Liver plays a major role in many inherited and acquired genetic disorders. It is also the site for the treatment of certain inborn errors of metabolism that do not directly cause injury to the liver. The advancement of nucleic acid-based therapies for liver maladies has been severely limited due to the myriad of untoward side effects and methodological limitations. To address these issues, research efforts in recent years have been intensified towards the development of targeted gene approaches using novel genetic tools, such as the zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs), as well as various non-viral vectors, such as Sleeping Beauty transposons, piggyBac transposons and PhiC31 integrase. While each of these methods utilizes a distinct mechanism of gene modification, all of them are dependent upon the efficient delivery of DNA and RNA molecules into the cell. This r...

World journal of gastroenterology : WJG, Jan 21, 2015

To identify the genes induced and regulated by the MYC protein in generating tumors from liver st... more To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells. In this study, we have used an immortal porcine liver stem cell line, PICM-19, to study the role of c-MYC in hepatocarcinogenesis. PICM-19 cells were converted into cancer cells (PICM-19-CSCs) by overexpressing human MYC. To identify MYC-driven differential gene expression, transcriptome sequencing was carried out by RNA sequencing, and genes identified by this method were validated using real-time PCR. In vivo tumorigenicity studies were then conducted by injecting PICM-19-CSCs into the flanks of immunodeficient mice. Our results showed that MYC-overexpressing PICM-19 stem cells formed tumors in immunodeficient mice demonstrating that a single oncogene was sufficient to convert them into cancer cells (PICM-19-CSCs). By using comparative bioinformatics analyses, we have determined that > 1000 genes were differentially expressed between PICM-19 and PICM-19-CSCs. Gene ontology...

Journal of inherited metabolic disease, 1999

... Later that day she developed increasing cycling movements of arms and legs with ickering eye... more ... Later that day she developed increasing cycling movements of arms and legs with ickering eye movements. EEG showed contin-uous multifocal seizure activity and MRI indicated moderate cerebral oedema, sug-gesting possible asphyxia. ...

Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1995

Protein expression of the retinoblastoma (Rb) tumor suppressor gene product was examined by immun... more Protein expression of the retinoblastoma (Rb) tumor suppressor gene product was examined by immunoblot analysis of nuclei isolated from regenerating rat liver after 70% partial hepatectomy (PH). Levels were almost undetectable in quiescent 0-h livers but increased 15- to 60-fold 3 to 24 h post-PH, 105-fold at 30 h, and 20- to 50-fold at 60 to 72 h post-PH. Expression returned to near baseline levels at 18, 42, and 48 h post-PH. A similar pattern of Rb protein expression in the regenerating liver was observed by indirect immunofluorescence microscopy, with peak nuclear expression at 30 h post-PH. Rb-related proteins with apparent molecular masses of 300, 156, and 74 kDa were detected in regenerating liver using mAbs to the Rb protein. Their expression increased 6- to 8-fold during regeneration, and only p156 returned to baseline levels at 60 h post-PH. Rb and its related proteins were detected in cultured primary hepatocytes, and although total protein levels did not change appreciab...

Proceedings of the National Academy of Sciences of the United States of America, 1984

Clathrin-coated vesicles isolated from rat liver exhibited an enzymatic profile distinct from tha... more Clathrin-coated vesicles isolated from rat liver exhibited an enzymatic profile distinct from that of rat liver plasma membranes, lysosomes, microsomes, and mitochondria. The coated vesicles catalyzed ATP-dependent proton transport that acidified the vesicle interior, as measured by the fluorescence quenching of acridine orange. H+ transport by coated vesicles was not inhibited by vanadate (0.1 mM) or ouabain (2 mM) and differed from H+ transport by rat liver submitochondrial particles in its greater resistance to inhibition by oligomycin (10 pM to 10 microM) and N,N'-dicyclohexylcarbodiimide (DCCD) (0.1-100 microM) and its sensitivity to N-ethylmaleimide (0.1-2 mM). H+ transport was stimulated by valinomycin in the presence of K+, exhibited no specific cation requirement, but was dependent upon the presence of a permeant anion, with Cl- and Br- being the most effective of the anions studied. Finally, H+ transport was poorly supported by GTP, UTP, or ADP and exhibited no consist...

Translational Research, 2014

Tunneling nanotubes (TnTs) represent a novel mechanism by which intercellular components such as ... more Tunneling nanotubes (TnTs) represent a novel mechanism by which intercellular components such as proteins, Golgi vesicles, and mitochondria can be transferred from cell to cell in the complex tumor microenvironment. Here, we report data showing that microRNAs (miRNAs) are transferred through TnTs in osteosarcoma (OS) and ovarian cancer as in vitro model systems. miRNA array analysis demonstrated significant upregulation of miR-19a in OS tumors resected from human patients, and differential expression of miR-199a in ovarian cancer cell lines resistant or sensitive to platinum chemotherapy. K7M2 murine OS cells were transfected with miR-19a and cultured with nontransfected K7M2 cells in low-serum, hyperglycemic medium for up to 72 hours to induce TnT formation. miRNA transfer via TnTs was detected by time-lapse microscopic imaging. miR-19a was also transported via TnTs connecting transfected K7M2 cells and nontransfected stromal MC3T3 murine osteoblast cells. Similar findings were observed in studies of TnT-mediated transport of miR-199a among SKOV3 ovarian cancer cells and nonmalignant immortalized ovarian epithelial cells. To quantify TnT-mediated transport of miRNAs, we used modified Boyden chambers to separate miR-19a-transfected K7M2 cells (top chamber) and DiI-labeled MC3T3 cells (bottom chamber) compared with open culture of these cells. Fluorescence-activated cell sorting analysis of cells collected after 48 hours of culture indicated that miR-19a-positive MC3T3 cells were 3-fold higher in open culture; this finding suggests that miR-19a transfer occurred via TnTs, exclusive of other forms of cell-cell communication. These studies demonstrate that TnTs mediate direct transfer of genetic material between tumor and stromal cells.

The Journal of Pathology, 2014

Genetic changes in colon cancer are known to parallel the tissue abnormalities associated with th... more Genetic changes in colon cancer are known to parallel the tissue abnormalities associated with the disease, namely adenoma and adenocarcinoma. The role of microRNA dysregulation in dysplastic progression, however, is not well understood. Here, we show that miR-182 and miR-503 undergo sequential up-regulation and drive the progression of colon adenoma to adenocarcinoma by cooperatively down-regulating the tumour suppressor FBXW7. We identified that increased expression of miR-182 is a feature of adenomas. A subsequent increase in miR-503 expression works cooperatively with miR-182 to induce transformation of an adenoma to adenocarcinoma. We show that introducing miR-503 into AAC1 cells, which are derived from a benign adenoma, confers tumourigenic potential. We also demonstrated that blocking both miR-182 and miR-503 in HCT116 colon cancer cells resulted in increased FBXW7 expression and significantly reduced tumour size in xenograft models. We confirmed relevance of these results in patients by examining the expression levels of miR-182 and miR-503 in over 200 colon cancer patients with 12 year survival outcome data. Decreased patient survival was correlated with elevated expression of both miRNAs, suggesting that elevated levels of both miR-182 and miR-503 define a novel prognostic biomarker for colon cancer patients. In conclusion, we show that a sequential expression of miR-182 and miR-503 in benign adenoma cooperatively regulates the tumour suppressor FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma and miR-182 and miR-503 may prove to be novel therapeutic targets. Array differential isoform expression in normal colon and adenomas versus carcinoma . Furthermore, VEGF is targeted in the clinical management of metastatic colon cancer and miR-503-induced D) Predicted miR-182 binding site in the FBXW7 3 ′ UTR; two-point mutations were introduced at binding sites 1 and/or 2; mutated nucleotides are shown in bold (upper panel). Luciferase reporter assay demonstrating miR-182 binding to the FBXW7 3 ′ UTR. Luciferase activity of the FBXW7 3 ′ UTR reporter was repressed by co-transfection of miR-182 mimics, whereas mutations in both potential binding sites were required to rescue the luciferase repression mediated by miR-182. (E) Luciferase reporter assay demonstrating cooperative down-regulation of FBXW7 by miR-503 and miR-182. miRNA mimics for miR-503 (10 nM) and miR-182 (10 nM) individually show 28-30% luciferase repression. Combination of miR-503 and miR-182 mimics at equimolar concentrations (5 nM each) significantly decreased FBXW7-3 ′ UTR reporter luciferase activity by 83%. (F) Western blot from HCT116 cell treated with miRzip182 and miRzip503, demonstrating restored FBXW7 expression. Data are shown as mean ± SD in (B-E);