Extended silencing of the insulin-like growth factor-I receptor gene in MCF-7 cells by serial transfection with siRNA.
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Breast Neoplasms↗
Biomedical subjects
Stephen A Bustin
Publications and source records attributed to Stephen A Bustin.
Vitamin D metabolism in peripheral blood mononuclear cells is influenced by chewing "betel nut" (Areca catechu) and vitamin D status.
CONTEXT: Vitamin D deficiency, common in South Asians, is a risk factor for metabolic syndrome, type 2 diabetes, and ischemic heart disease. Vitamin D receptor (VDR) activation depends on activated vitamin D [1,25-dihydroxyvitamin D (1,25(OH)(2)D)] concentration, reflecting opposing actions of 25-hydroxyvitamin D-1alpha-hydroxylase [1-alpha(OH)ase] for formation and 25(OH)D-24-hydroxylase [24(OH)ase] for catabolism. We previously reported that circulating 1,25(OH)(2)D contributed to determination of VDR-protein levels and VDR genotype was a determinant of both VDR mRNA and VDR-protein in South Asians. OBJECTIVE: We hypothesized that chewing betel nut, an addictive habit common throughout South Asian communities, contributes to hypovitaminosis-D by modulating the enzymes regulating circulating 1,25(OH)(2)D concentration. DESIGN: Peripheral blood mononuclear cell (PBMC) 1-alpha(OH)ase and 24(OH)ase mRNA concentrations were measured and examined in relation to cross-sectional data on the vitamin-D axis, diet, smoking, and betel usage, including PBMC VDR-RNA and VDR-protein content in a pilot study of 33 healthy British Bangladeshis. RESULTS: PBMC 24(OH)ase mRNA correlated positively and serum 1,25(OH)(2)D negatively with betel quids per day (r = 0.49, P = 0.006 and r = -0.486, P = 0.006, respectively). Independent determinants for 24(OH)ase included betel quids per day (P < 0.0001) and serum 25-OHD (P = 0.024). Independent determinants for serum 1,25(OH)(2)D were gender, smoking, and betel quids per day. PBMC 1-alpha(OH)ase mRNA correlated inversely with VDR mRNA (r = -0.44; P = 0.013); its independent determinants were serum 1,25(OH)(2)D and VDR TaqI and BsmI polymorphisms (P = 0.03-0.0001). CONCLUSIONS: Betel chewing is a more powerful independent determinant of increased 24(OH)ase expression and of decreased serum calcitriol than serum 25-OHD, supporting the hypothesis that this habit could aggravate the effects of vitamin-D deficiency.
25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations.
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DNA↗
Real-time reverse transcription PCR and the detection of occult disease in colorectal cancer.
Molecular diagnostics offers the promise of accurately matching patient with treatment, and a resultant significant effect on improved disease outcome. More specifically, the real-time reverse transcription polymerase chain reaction (qRT-PCR), with its combination of conceptual simplicity and technical utility, has the potential to become a valuable analytical tool for the detection of mRNA targets from tissue biopsies and body fluids. Its potential is particularly promising in cancer patients, both as a prognostic assay and for monitoring response to therapy. Colorectal cancer provides an instructive paradigm for this potential as well as the problems associated with its use as a clinical assay. Currently, histopathological staging, which provides a static description of the anatomical extent of tumour spread within a surgical specimen, defines patient prognosis. The detection of lymph node (LN) metastasis constitutes the most important prognostic factor in colorectal cancer and as the primary indicator of systemic disease spread, LN status determines the choice of postoperative adjuvant chemotherapy. However, its limitations are emphasised by the considerable prognostic heterogeneity of patients within a given tumour stage: not all patients with LN-negative cancers are cured and not all patients with LN-positive tumours die from their disease. This has resulted in a search for more accurate staging protocols and has seen the introduction of the concept of "molecular staging", the incorporation of molecular parameters into clinical tumour staging. Quantification of disease-associated mRNA is one such parameter that utilises the qRT-PCR assay's potential for generating quantitative results. These are not only more informative than qualitative data, but contribute to assay standardisation and quality management. This review provides an assessment of the practical value to the clinician of RT-PCR-based molecular diagnostics. It points out reasons for the many contradictory results encountered in the literature and concludes that there is an urgent need for standardisation at every level, starting with pre-assay sample acquisition and template preparation, assay protocols and post-assay analysis.
Biomarkers, Tumor↗
Altered monocyte cyclooxygenase response to lipopolysaccharide in type 1 diabetes.
Type 1 diabetes is caused by adaptive immune responses, but innate immunity is important because monocytes infiltrate islets. Activated monocytes express cyclooxygenase (COX)-2, promoting prostaglandin-E(2) (PGE(2)) secretion, whereas COX-1 expression is constitutive. We aimed to define monocyte COX expression in type 1 diabetes basally and after lipopolysaccharide (LPS) stimulation. Isolated CD14(+) monocytes were analyzed for COX mRNA and protein expression from identical twins (discordant for type 1 diabetes) and control subjects. Basal monocyte COX mRNA, protein expression, and PGE(2) secretion were normal in type 1 diabetic subjects. After LPS, twins and control subjects showed a COX mRNA isoform switch with decreased COX-1 mRNA (P < 0.01), increased COX-2 mRNA (P < 0.01), and increased COX-2 protein expression (P < 0.01). Compared with control subjects, both diabetic and nondiabetic twins showed greater LPS-induced downregulation of monocyte COX-1 mRNA (P = 0.02), reduced upregulation of COX-2 mRNA and protein (P < 0.03), and greater inhibition by the COX-2 inhibitor di-isopropylfluorophosphate (DFP) of monocyte PGE(2) (P < 0.007). We demonstrate an alteration in monocyte COX mRNA expression as well as monocyte COX-2 and PGE(2) production after LPS in type 1 diabetic patients and their nondiabetic twins. Because COX-2 response to LPS is proinflammatory, an inherited reduced response would predispose to chronic inflammatory diseases such as type 1 diabetes.
Animals↗
The immunogenicity of colorectal cancers with high-degree microsatellite instability.
BACKGROUND: High-degree microsatellite instability (MSI-H) is a feature of approximately 15% of sporadic colorectal cancers. Patients with MSI-H cancers have been reported to have a better prognosis than those with non-MSI-H cancers. The MSI-H subset is also characterised by a dense infiltrate of intra-epithelial lymphocytes and the hypothesis that the latter represents an efficacious immune response contributing to improved outcome is very attractive. METHODS: Data for this review were identified by searches of MEDLINE, PubMed, and cross references from relevant articles using the search terms 'microsatellite instability', 'colorectal cancer' and 'immunology', 'immune response' or 'immunogenicity'. RESULTS: A total of 38 articles were identified by the search criteria and a further 95 articles by cross-referencing. The relevance of the articles to be interviewed was established by hand searching. Out of a total of 133 articles identified, 47 articles were rejected due to lack of relevance. A total of 86 articles were included in the review, pertaining to microsatellite instability in colorectal cancer, and immune mechanisms in colorectal cancer. CONCLUSION: It is suggested that this distinct group of colorectal cancers may have inherent immunogenic properties and that further elucidation of these may be invaluable to the development of successful immunotherapy.
Journal Article↗
Immunogenic hsp-70 is overexpressed in colorectal cancers with high-degree microsatellite instability.
PURPOSE: Colorectal cancers that display high-degree mi-crosatellite instability are associated with an improved prognosis and evidence of an activated host immune response. Molecular analyses have suggested that heat shock proteins, a family of proteins that have key immunologic functions, are upregulated in these cancers. We aimed to explore the expression of heat shock proteins 70 and 110 and their relationship to microsatellite instability, survival, and other clinicopathologic parameters. METHODS: Twenty-six colorectal cancers that displayed microsatellite instability were matched by age, stage, and site in the colorectum to 26 microsatellite-stable cancers. Immunohistochemistry was used to detect expression of both markers. RESULTS: The microsatellite-unstable group showed significantly higher expression of heat shock protein 70 than the microsatellite-stable group (P = 0.006), and patients undergoing curative resections for unstable cancers had improved prognosis compared with their stable counterparts (P = 0.026). Significantly, in a multivariate survival analysis, low or absent heat shock protein 70 expression was independently associated with a poor outcome (P = 0.001). CONCLUSIONS: Heat shock protein 70 has known functions that promote antitumor immune responses. Its overexpression in colorectal cancers with microsatellite instability may be pivotal to explaining these tumors' enhanced immunogenicity and improved prognosis.
Age Factors↗
Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis.
qRT-PCR (real-time reverse transcription-PCR) has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in novel clinical diagnostic assays. Quantitative results obtained by this technology are not only more informative than qualitative data, but simplify assay standardization and quality management. qRT-PCR assays are most established for the detection of viral load and therapy monitoring, and the development of SARS (severe acute respiratory syndrome)-associated coronavirus qRT-PCR assays provide a textbook example of the value of this technology for clinical diagnostics. The widespread use of qRT-PCR assays for diagnosis and the detection of disease-specific prognostic markers in leukaemia patients provide further examples of their usefulness. Their value for the detection of disease-associated mRNA expressed by circulating tumour cells in patients with solid malignancies is far less apparent, and the clinical significance of results obtained from such tests remains unclear. This is because of conceptual reservations as well as technical limitations that can interfere with the diagnostic specificity of qRT-PCR assays. Therefore, although it is evident that qRT-PCR assay has become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations if it is to fulfil its potential.
DNA, Complementary↗
Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences.
Real-time fluorescence-based quantitative PCR has become established as the benchmark technology for the quantification of nucleic acids, offering an immense choice of protocols, chemistries and instruments. However, whilst there are comparatively few technical problems associated with DNA-targeted quantitative PCR, this is not the case for real-time reverse transcription PCR assays, and there is considerable uncertainty regarding biological or clinical relevance of many real-time reverse transcription PCR results. A survey of working practices of nearly 100 delegates carried out prior to the Third qPCR Symposium held in London, UK, April 25-26, 2005, reveals some of the reasons underlying the variability of reported real-time reverse transcription PCR results. Specifically, the survey reveals extensive interlaboratory variation in assay design, validation and analysis that, together with other dubious practices, are the likely cause for the publication of variable results. These results emphasize the urgent need for the establishment of best practice guidelines for this technology, particularly in the context of its mounting adaptation as a high-throughput clinical diagnostic assay.
DNA Primers↗
Colorectal cancers with microsatellite instability display mRNA expression signatures characteristic of increased immunogenicity.
BACKGROUND: Colorectal cancers displaying high-degree microsatellite instability (MSI-H) have an improved prognosis compared to microsatellite stable (MSS) cancers. The observation of pronounced lymphocytic infiltrates suggests that MSI-H cancers are inherently more immunogenic. We aimed to compare the gene expression profiles of MSI-H and MSS cancers to provide evidence for an activated immune response in the former. RESULTS: We analysed tissue from 133 colorectal cancer patients with full consent and Local Ethics Committee approval. Genomic DNA was analysed for microsatellite instability in BAT-26. High-quality RNA was used for microarray analysis on the Affymetrix HG-U133A chip. Data was analysed on GeneSpring software version 6.0. Confirmatory real-time RT-PCR was performed on 28 MSI-H and 26 MSS cancers. A comparison of 29 MSI-H and 104 MSS cancers identified 2070 genes that were differentially expressed between the two groups [P < 0.005]. Significantly, many key immunomodulatory genes were up-regulated in MSI-H cancers. These included antigen chaperone molecules (HSP-70, HSP-110, Calreticulin, gp96), pro-inflammatory cytokines (Interleukin (IL)-18, IL-15, IL-8, IL-24, IL-7) and cytotoxic mediators (Granulysin, Granzyme A). Quantitative RT-PCR confirmed up-regulation of HSP-70 [P = 0.016], HSP-110 [P = 0.002], IL-18 [P = 0.004], IL-8 [0.002] and Granulysin [P < 0.0001]. CONCLUSIONS: The upregulation of a large number of genes implicated in immune response supports the theory that MSI-H cancers are immunogenic. The novel observation of Heat Shock Protein up-regulation in MSI-H cancer is highly significant in light of the recognised roles of these proteins in innate and antigen-specific immunogenicity. Increased mRNA levels of pro-inflammatory cytokines and cytotoxic mediators also indicate an activated anti-tumour immune response.
Colorectal Neoplasms↗
Quantification of cytokeratin 20, carcinoembryonic antigen and guanylyl cyclase C mRNA levels in lymph nodes may not predict treatment failure in colorectal cancer patients.
Conventional histopathologic staging of primary colorectal cancers does not allow accurate prognostic stratification within a given tumour stage. Therefore, PCR-based assays are increasingly used to try to predict more accurately the likelihood of disease progression for the individual patient. Real-time reverse transcription PCR (RT-PCR) assays were used to detect and quantitate cytokeratin 20 (ck20), carcinoembryonic antigen (CEA) and guanylyl cyclase C (GCC) mRNA in 149 lymph nodes (LN) from 17 patients with benign disease and 302 LN from 42 patients with colorectal cancer who had curative (R0) resections. None of the markers were specific, with ck20, CEA and GCC mRNA detected in 47%, 89% and 13% of 149 LN, respectively, from patients with benign disease. The sensitivity of all 3 markers was very high, with mRNA detected in 93%, 100% and 97% of 30 histologically involved LN, respectively. There was significant overlap in the mRNA levels of all 3 markers between histologically involved and uninvolved LN. There was no association between mRNA levels and distant recurrence (median follow-up: 3.94 years, range 3.35-5.12). We conclude that the use of molecular techniques to detect occult disease in LN may suffer from the same limitations as conventional methods. Instead, accurate prognostic stratification requires careful assessment of the likely metastatic potential of the primary cancer.
Adult↗
Age and diet act through distinct isoforms of the class II transactivator gene in mouse intestinal epithelium.
BACKGROUND & AIMS: Normal weaning induces class II major histocompatibility complex (Ia) and invariant chain (Ii) expression in the mouse intestinal epithelium. Because the class II transactivator protein (CIITA) induces Ia and Ii in most cell types, we hypothesized that diet-induced expression of these genes was through CIITA. METHODS: Mouse litters were split and weaned onto an elemental diet or a normal (complex) chow diet. On days 24, 31, and 45, epithelial cells were isolated from small intestine with EDTA, and the RNA was extracted from both wild-type and interferon (IFN)-gamma receptor knockout mice. Messenger RNA (mRNA) was measured by Northern blotting, RNase protection assay, and real-time polymerase chain reaction and Ia localized by immunohistochemistry. RESULTS: By day 31, CIITA mRNA was induced in the intestinal epithelium of normally weaned wild-type mice, and this mirrored the expression of Ii chain mRNA. Mice weaned onto an elemental diet did not exhibit Ii mRNA or increased CIITA mRNA in the intestinal epithelium by day 31, but low levels of Ii mRNA were detectable by day 45. Of the 3 isoforms of CIITA, weaning onto a complex diet induced only CIITA IV by day 31. Mice deficient in the IFN-gamma receptor expressed Ia in the epithelium and they also accumulated Ii mRNA (at low levels) by day 45, irrespective of diet. CIITA III mRNA accumulation mirrored the dietary-independent changes of Ii mRNA. CONCLUSIONS: Two mechanisms regulate Ii in the mouse intestinal epithelium: a rapid one, which is diet-induced acting through CIITA IV; and a slower, dietary-independent pathway, acting through CIITA III.
Age Factors↗
Evidence for a link between IGF-I and cancer.
Cancer risk is determined by a combination of environmental factors and genetic predisposition. Recent evidence suggests that dietary and related factors such as physical activity and body size may influence cancer risk through their effects on the serum concentration of IGF-I and its binding proteins. The growth hormone (GH)/IGF-I axis is involved in both human development as well as the maintenance of normal function and homeostasis in most cells of the body. In addition to their classical role as endocrine hormones, its members regulate a wide range of biological functions such as cell proliferation, differentiation and apoptosis through paracrine and autocrine mechanisms. During cancer development this complex network regulating tissue homeostasis breaks down, with inappropriate expression of the GH/IGF-I axis making an important contribution. The increased understanding of the molecular mechanisms and signalling pathways regulated by the GH/IGF-I axis has started to provide significant insights into the aetiology, prevention and therapy for a number of common cancers.
Human Growth Hormone↗
Gene expression profiling for molecular staging and prognosis prediction in colorectal cancer.
A key issue for patients undergoing surgery for colorectal cancer is the accurate prediction of treatment outcome. Currently, classification of a tumor by histopathologic stage is the most accurate prognostic factor for the risk assessment of treatment failure. However, despite improved histologic techniques and the application of novel immunohistochemical and molecular techniques, it is still not possible to delineate the underlying biochemical and genetic events that predict clinical outcome for individual cancer patients. One reason for this lack of progress is that the factors which determine the metastatic potential of a primary tumor are still unknown. This reality, coupled to dramatic technological developments in the field of expression profiling, has started a paradigm shift in the staging of colorectal cancers. It has raised expectations that genetic and/or transcriptome profiling of the primary tumor will result in the identification of prognostic determinants relevant to the individual patient. In turn, this may allow a clinically relevant definition of patient subgroups based on individual molecular parameters for rational decision making regarding choice of therapy.
Biomarkers, Tumor↗
Validation of housekeeping genes for normalizing RNA expression in real-time PCR.
Analysis of RNA expression using techniques like real-time PCR has traditionally used reference or housekeeping genes to control for error between samples. This practice is being questioned as it becomes increasingly clear that some housekeeping genes may vary considerably in certain biological samples. We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis. Housekeeping genes were selected from conventionally used ones and from genes reported to be invariant in human T cell culture. None of the commonly used housekeeping genes [e.g., glyceraldehyde-phosphate-dehydrogenase (GAPDH)] were found to be suitable as internal references, as they were highly variable (>30-fold maximal variability). Furthermore, genes previously found to be invariant in human T cell culture also showed large variation in RNA expression (>34-fold maximal variability). Genes that were invariant in blood were highly variable in peripheral blood mononuclear cell culture. Our data show that RNA specifying human acidic ribosomal protein was the most suitable housekeeping gene for normalizing mRNA levels in human pulmonary tuberculosis. Validations of housekeeping genes are highly specific for a particular experimental model and are a crucial component in assessing any new model.
Cells, Cultured↗
Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction.
Polymerase chain reaction (PCR)-based assays can target either DNA (the genome) or RNA (the transcriptome). Targeting the genome generates robust data that are informative and, most importantly, generally applicable. This is because the information contained within the genome is context-independent; i.e., generally, every normal cell contains the same DNA sequence--the same mutations and polymorphisms. The transcriptome, on the other hand, is context-dependent; i.e., the mRNA complement and level varies with physiology, pathology, or development. This makes the information contained within the transcriptome intrinsically flexible and variable. If this variability is combined with the technical limitations inherent in any reverse-transcription (RT)-PCR assay, it can be difficult to achieve not just a technically accurate but a biologically relevant result. Template quality, operator variability, the RT step itself, and subjectivity in data analysis and reporting are just a few technical aspects that make real-time RT-PCR appear to be a fragile assay that makes accurate data interpretation difficult. There can be little doubt that in the future, transcriptome-based analysis will become a routine technique. However, for the time being it remains a research tool, and it is important to recognize the considerable pitfalls associated with transcriptome analysis, with the successful application of RTPCR depending on careful experimental design, application, and validation.
Animals↗
Colorectal cancers with mononucleotide microsatellite instability can be identified using microfabricated chip technology.
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Colorectal Neoplasms, Hereditary Nonpolyposis↗
The GH-IGF-I axis and breast cancer.
The growth hormone-insulin-like growth factor-I (GH-IGF-I) axis plays a fundamental role in the development of the breast. The maintenance of breast tissue architecture is aided by its effect on proliferation, differentiation and apoptosis. There has been increasing recognition of its role as a major determinant of breast cancer and, more recently, its involvement in the development of resistance to both tamoxifen and an important novel therapy for advanced disease, trastuzumab (Herceptin). Here, we discuss the influence of the GH-IGF-I axis in normal mammary development and homeostasis, its putative role in breast tumorigenesis and its interactions with estrogen signalling.
Animals↗