464 research outputs found
Presence of WT1, the Wilm's tumor suppressor gene product, in nuclear poly(A)+ ribonucleoprotein
No full textThe tumor suppressor gene WT1 encodes a zinc finger protein, which consists of four C-terminal C2-H2 zinc fingers of the Kruppel type, and at the N terminus a Q/P-rich trans-regulatory domain, both characteristic of transcription factors. However, recent findings suggest that WT1 may also be involved in a post-transcriptional process. Specifically, WT1 isoforms containing the alternatively spliced exon 9 (+lysine-threonine-serine (KTS)) preferentially associate with nuclear speckles and coimmunoprecipitate splicing antigens (Larsson, S. H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V., and Hastie, N. D. (1995) Cell 81, 391-401); furthermore, WT1 has been shown to interact with the ubiquitous splicing factor U2AF65 (Davies, R. C., Calvo, C., Larsson, S. H., Lamond, A. I., and Hastie, N. D. (1998) Genes Dev. 12, 3217- 3225) and binds to RNA in vitro (Caricasole, A., Duarte, A., Larsson, S. H., Hastie, N. D., Little, M., Holmes, G., Todorov, I., and Ward, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7562-7566; Bardeesy, N., and Pelletier, J. (1998) Nucleic Acids Res. 26, 1784-1792). To extend these findings, we have fractionated nuclear extracts to see if particles containing WT1 have the properties of ribonucleoprotein (RNP). In summary, WT1 is enriched by oligo(dT) chromatography, as are U2AF65, the U5 small nuclear RNP-associated protein p116 and hnRNP A1. Gel filtration and sedimentation profiles suggest that WT1 is present in RNase-sensitive particles, >2 MDa in size, peaking at ~60 S, and ~1.27 g/cm3 on Nycodenz. Similar results were obtained from two cell lines expressing WT1, fetal kidneys (day E17), and transiently transfected cells, suggesting that the presence of WT1 protein in nuclear poly(A)+ RNP is a general aspect of WT1 function
Atonal homolog 1 is a tumor suppressor gene.
No full textColon cancer accounts for more than 10% of all cancer deaths annually. Our genetic evidence from Drosophila and previous in vitro studies of mammalian Atonal homolog 1 (Atoh1, also called Math1 or Hath1) suggest an anti-oncogenic function for the Atonal group of proneural basic helix-loop-helix transcription factors. We asked whether mouse Atoh1 and human ATOH1 act as tumor suppressor genes in vivo. Genetic knockouts in mouse and molecular analyses in the mouse and in human cancer cell lines support a tumor suppressor function for ATOH1. ATOH1 antagonizes tumor formation and growth by regulating proliferation and apoptosis, likely via activation of the Jun N-terminal kinase signaling pathway. Furthermore, colorectal cancer and Merkel cell carcinoma patients show genetic and epigenetic ATOH1 loss-of-function mutations. Our data indicate that ATOH1 may be an early target for oncogenic mutations in tissues where it instructs cellular differentiation.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe
Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide.
No full textAdvanced-stage peritoneal carcinomatosis is resistant to current chemotherapy treatment and, in the case of metastatic ovarian cancer, results in a devastating 15%-20% survival rate. Therapeutics that restore genes inactivated during oncogenesis are predicted to be more potent and specific than current therapies. Experiments with viral vectors have demonstrated the theoretical utility of expressing the p53 tumor suppressor gene in cancer cells. However, clinically useful alternative approaches for introducing p53 activity into cancer cells are clearly needed. It has been hypothesized that direct reactivation of endogenous p53 protein in cancer cells will be therapeutically beneficial, but few tests of this hypothesis have been carried out in vivo. We report that a transducible D-isomer RI-TATp53C' peptide activates the p53 protein in cancer cells, but not normal cells. RI-TATp53C' peptide treatment of preclinical terminal peritoneal carcinomatosis and peritoneal lymphoma models results in significant increases in lifespan (greater than 6-fold) and the generation of disease-free animals. These proof-of-concept observations show that specific activation of endogenous p53 activity by a macromolecular agent is therapeutically effective in preclinical models of terminal human malignancy. Our results suggest that TAT-mediated transduction may be a useful strategy for the therapeutic delivery of large tumor suppressor molecules to malignant cells in vivo
Developmental context determines latency of MYC-induced tumorigenesis.
No full textOne of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a c-MYC human transgene can be conditionally regulated in murine hepatocytes. MYC's ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis
WT1 in the adult kidney : podocyte maintenance and the epithelial-mesenchymal balance
No full textGlomerular diseases are the leading cause of end stage kidney disease worldwide. Podocyte
injury plays a key role in the initiation and development of such diseases, which follow a
progressive course due to the limited capacity of podocytes to regenerate. Podocytes are highly
specialised, terminally differentiated cells, which play a vital role in the glomerular filtration
barrier. They are also the main sites of expression of the Wilms Tumour Suppressor gene, WT1,
in the adult. WT1 is a complex gene, which plays an essential role in renal development by
controlling the process of mesenchymal to epithelial transition that forms the nephron. Adult
podocytes maintain both epithelial and mesenchymal features and continue to express high
levels of WT1. Little is known about the role of WT1 in adult podocytes as previous studies have
been limited due to the confounding developmental effects and embryonic lethality of existing
animal models.
This thesis sought to investigate the hypothesis that WT1 is an essential gene in adult kidney and
plays a fundamental role in the adult podocyte. Given its role in nephron development, WT1 loss
was hypothesised to result in dedifferentiation and an alteration of the epithelial-mesenchymal
balance in the podocyte, affecting its specialised function. Using an inducible, conditional
animal model of Wt1 loss, Wt1 was deleted from the adult, confirming its essential role in adult
kidney. Wt1 deletion resulted in severe podocyte injury and failure of the glomerular filtration
barrier, as well as loss of expression of key podocyte genes. Preliminary analysis suggests this
was not simply due to podocyte apoptosis and/or detachment, supporting a role for Wt1 in
podocyte differentiation. This was corroborated by in vitro studies that demonstrated a
requirement for Wt1 for podocyte differentiation. Significantly, Wt1 loss resulted in a marked
change in the expression of epithelial and mesenchymal markers in podocytes, with upregulation
of mesenchymal characteristics, in keeping with a transitional stage consistent with an earlier
developmental form. To investigate the mechanism behind these findings a conditionally
immortalised podocyte cell line was generated as a model of Wt1 loss in vitro. In order to
confirm and specifically analyse the podocyte phenotype, BAC recombineering was utilised to
produce a promoter-reporter transgene construct to attempt to generate a fluorescent-labelled,
podocyte specific animal model of Wt1 loss. The findings of this thesis establish that Wt1 is essential for adult podocyte function, and appears
to be a key upstream regulator of podocyte differentiation. Extension of this work may allow the
identification of potential targets to promote podocyte differentiation and/or regeneration in the
setting of acquired and progressive glomerular disease
Pharmacogenomics:Clinical applications
No full textIntroduction: There is now a large body of evidence indicating that genetic factors can influence treatment response in a variety of human diseases. Pharmacogenetics, defined as the study of the genetic basis for individuality in response to drugs (Vogel, 1959) or use of genetic analysis to predict drug response, efficacy and toxicity (Roses, 2004) has seen an almost exponential rise in peer-reviewed publications in the last 10 years (Figure 34.1). The closely related field of pharmacogenomics can be defined as the use of genetic approaches in drug discovery and use and encompasses the study of all genes which may influence drug response although, as illustrated in Figure 34.1, the terms pharmacogenetics and pharmacogenomics are often used interchangeably. Inter-individual differences in response to commonly prescribed drugs are increasingly recognized. Serious adverse drug reactions (ADRs) have been estimated to occur in more than 2 million patients/year in the USA and result in more than 100 000 fatalities; meta-analysis of a number of US-based prospective studies suggest that ADRs are the fourth leading cause of death, after heart disease, cancer and stroke (Lazarou et al., 1998). A recent UK-based prospective study reported that up to 6.5% of all hospital admissions in the UK are related to adverse drug reactions, with a projected annual cost to the National Health Service of £466 million (Pirmohamed et al., 2004). Individuality in drug response can be both genetically and environmentally determined.</p
Analysis of biomarkers for complex human diseases
No full textThe aims of this study were to analyse known and potential biomarkers of common and genetically complex human disorders and to identify genetic and environmental variation associated with plasma biomarker concentrations. Two groups of protein
biomarkers were analysed. First, plasma complement factor H (CFH) was selected
as a potential biomarker for age-related macular degeneration (AMD),
since common variants in the CFH gene show strong association with this disorder.
Secondly, two isoforms of amyloid-β (Aβ40 and Aβ42) were selected as biomarkers
for Alzheimer disease (AD) since Aβ deposits are major constituents of the amyloid
plaques characteristic of this disorder.
Physiological and anthropometric measurements and samples of human
and genomic DNA were collected from a population sample of 1,021
individuals from the Croatian island of Vis. Quantitative determination of plasma
Aβ40 and Aβ42 concentrations was performed using enzyme-linked immunosorbent assays. Heritabilities and significant covariate effects were estimated for each trait in the Croatian data set. Genome-wide linkage and
association analyses were conducted for the biomarker traits.
A novel finding was the genome-wide significant association between
a CFH and several polymorphisms close to and within the CFH gene. The strongest association was with an intronic SNP within CFH, which explained 28% of the total trait variance (P < 10-50). The association was also replicated in a Dutch
sample set. A SNP haplotype was identified which accounted for a higher proportion of the phenotypic variance. Conditional haplotype analysis showed that the effect of
this haplotype on plasma CFH concentration was independent of the CFH Y402H
variant, and significantly stronger than a deletion of the adjacent CFHR3/CFHR1
which was already known to affect AMD susceptibility. Genetic analysis of
382 AMD cases and 201 controls was consistent with the CFH Y402H variant being the strongest AMD susceptibility locus. Variation in plasma CFH concentration was
found to explain up to 1.8% of the variation in susceptibility to AMD with an odds
2.1 (95% C.I. 1.3-3.4, P = 0.003). SNPs that were strongly associated with
a CFH concentration also influenced AMD susceptibility (P < 0.05) independently of the CFH Y402H polymorphism. Functional analysis of genomic regions associated with plasma CFH is needed to identify the causal variants. Associations were observed between plasma Aβ40 concentration and several novel candidate loci, spanning regions of approximately 0.2 Mb, on
chromosomes 9 and X. Similarly, novel associations with plasma Aβ42 were found
in several regions, each spanning 0.2-0.4 Mb, on chromosomes 2, 5, 9, 15 and 20.
The proportion of the phenotypic variance in plasma Aβ42 explained by these
putative associations ranged between 1.8 and 2.8%. However, none of the
associated SNPs was significant after correction for multiple testing, therefore
replication is required. Finally, attempts were made to identify and quantitate new protein
biomarkers of disease in human plasma using mass spectrometry. Development
and optimisation of techniques was initially undertaken to deplete high-abundance plasma proteins and improve signal:noise ratio. This allowed the assessment of
downstream proteomic approaches including MALDI-TOF mass spectrometry (MS),
capillary electrophoresis (CE) and ion exchange chromatography (IEC), each with
the potential for large-scale quantitation of plasma proteins. Although the analysis of
single protein analytes, using CE and IEC proved promising, the results highlighted
the difficulty associated with MALDI-TOF and protein ionisation techniques in
analysing complex mixtures such as plasma
The Vulnerable Spectator
No full textAdapted from the Lissa Evans novel Their Finest Hour and a Half, Their Finest (Lone Scherfig, 2016) is a fictional film based loosely on historical figures and circumstances, as it tells the story of the production of a feature film by the UK Ministry of Information (MOI) in 1940. What, Their Finest quietly asks, is real? What is fake? And what does it matter, if you are at the movies? Joy is real. Tears are real. And other things, too: the tea I sip, the arm of my companion next to me, the chattering women in the row below, the sighing man who has come to the movies alone. The light is real. The darkness, too. Hastie thinks through the implications of a female author of the original monograph, the female director of the current film, and the fictional composite female character Catrin Cole, the screenwriter in the film. The whole of Catrin Cole did and didn't exist before Their Finest. “Catrin Cole” is not a historical figure, hidden or otherwise. She is a composite of fact and fiction, the pieces stitched together to make a whole person. As asserted by producer Stephen Woolley, who initiated the project, Their Finest drew upon the lives of many women writers for the Film Division of the MOI, particularly that of Diana Morgan, the one woman in the Ealing Studios writers’ room.</jats:p
BMP-SMAD1/4 upregulates HNF4α in a subset of heterogeneous mouse pancreatic cancer cells while under metabolic stress
No full textIt is not known whether pancreatic cancers evolve from a single or multiple cells, or
from a particular pancreatic lineage. However, in the Pdx1-Cre; LSL-KrasG12D; LSLTp53R172H
mouse model of pancreatic cancer, all pancreatic lineages are susceptible
to express mutant KRas and p53. Hence, such mouse model implies a scenario of
maximal heterogeneity of cancer cell origins. On this basis, I isolated seven subclones
of heterogeneous mouse pancreatic cancer cells from a single tumour; each of
them had a distinct morphology and gene expression profile. Notably, they
possessed different intrinsic phospho-SMADs downstream of the TGFβ receptor
(phospho-SMAD2/3) or the BMP receptor (Phospho-SMAD1/5/8). I discovered that
SMAD4, a co-SMAD which is frequently found to be lost in pancreatic caner tissues,
upregulated HNF4α via the classical BMP-SMAD1 pathway, when cells were
experiencing metabolic stress upon deprivation of serum, or in the presence of excess
thymidine. Under serum starvation at a hypoglycemic-like glucose concentration, the
HNF4α-expressing sub-clones appeared to be more able to sustain an unstressed
morphology than other non-HNF4α-expressing sub-clones. Immunohistochemical
staining on pancreatic cancer sections revealed nuclear co-localization of SMAD4
and HNF4α in human (half of the cases) and in mouse samples. As a secondary
project conducted during characterization of cells, I also found that three of the subclones
more robustly proliferated under anchorage independent conditions, and they
relied on the MEK-ERK pathway and the canonical Wnt pathway, to a different
degree. Both studies demonstrate for the first time in primary cell culture that
pancreatic cancer cells within a tumour could be highly heterogeneous in terms of
both morphology and signaling pathways
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