Research Items (70)
The tumour microenvironment predominantly consists of macrophages with phenotypes ranging from pro-inflammatory (M1-like) to anti-inflammatory (M2-like). Trehalose-6,6′-dibehenate (TDB) displays moderate anti-tumour activity and stimulates M1-like macrophages via the macrophage inducible C-type lectin (Mincle) resulting in IL-1β production. In this study, we examined if monosodium urate (MSU), a known vaccine adjuvant, can boost IL-1β production by TDB-stimulated macrophages. We investigated the effect of MSU/TDB co-treatment on IL-1β production by GM-CSF (M1-like) and M-CSF/IL-4 (M2-like) differentiated mouse bone marrow macrophages (BMMs) and found that MSU/TDB co-treatment of GM-CSF BMMs significantly enhanced IL-1β production in a Mincle-dependent manner. Western blot analysis showed that increased IL-1β production by GM-CSF BMMs was associated with the induction of pro-IL-1β expression by TDB rather than MSU. Flow cytometry analysis showed that MSU/TDB co-stimulation of GM-CSF BMMs led to greater expansion of CD86high/MHC IIhigh and CD86low/MHC IIlow subpopulations; however, only the latter showed increased production of IL-1β. Together, these findings provide evidence of the potential to use MSU/TDB co-treatment to boost IL-1β-mediated anti-tumour activity in M1-like tumour-associated macrophages.
Objectives: To assess associations between occupation and motor neuron disease (MND). Methods: We conducted a population-based case-control study with cases (n=321) recruited through the New Zealand Motor Neurone Disease Association and hospital discharge data. Controls (n=605) were recruited from the Electoral Roll. Information on personal and demographic details, lifestyle factors and a full occupational history was collected using questionnaires and interviews. Associations with ever/never employed and employment duration were estimated using logistic regression stratified by sex and adjusted for age, ethnicity, socioeconomic deprivation, education and smoking. Results: Elevated risks were observed for field crop and vegetable growers (OR 2.93, 95% CI 1.10 to 7.77); fruit growers (OR 2.03, 95% CI 1.09 to 3.78); gardeners and nursery growers (OR 1.96, 95% CI 1.01 to 3.82); crop and livestock producers (OR 3.61, 95% CI 1.44 to 9.02); fishery workers, hunters and trappers (OR 5.62, 95% CI 1.27 to 24.97); builders (OR 2.90, 95% CI 1.41 to 5.96); electricians (OR 3.61, 95% CI 1.34 to 9.74); caregivers (OR 2.65, 95% CI 1.04 to 6.79); forecourt attendants (OR 8.31, 95% CI 1.79 to 38.54); plant and machine operators and assemblers (OR 1.42, 95% CI 1.01 to 2.01); telecommunications technicians (OR 4.2, 95% CI 1.20 to 14.64); and draughting technicians (OR 3.02, 95% CI 1.07 to 8.53). Industries with increased risks were agriculture (particularly horticulture and fruit growing), construction, non-residential care services, motor vehicle retailing, and sport and recreation. Positive associations between employment duration and MND were shown for the occupations fruit growers, gardeners and nursery growers, and crop and livestock producers, and for the horticulture and fruit growing industry. Conclusions: This study suggests associations between MND and occupations in agriculture and several other occupations.
The transfer of mitochondria between mammalian cells is a physiologically relevant phenomenon, however the signals driving this process are not yet clear. We hypothesise that therapeutics which impact mitochondrial function may enhance the rate of intercellular mitochondrial transfer, and this may serve as a component of resistance to therapy in many cancers. Using genetic methodologies, we have overcome serious limitations on techniques for study in this field, and may now accurately quantify the effect of therapies on the rate of mitochondrial transfer in vitro. In order to both detect and quantify intercellular mitochondrial transfer, a novel strategy for in situ molecular genotyping has been targeted toward single nucleotide polymorphisms (SNP) within the mitochondrial genome (mtDNA). This strategy exploits a highly selective ligase, that when combined with padlock oligonucleotides, generate a template for target-primed rolling circle amplification. This molecular technique allows amplification of individual mtDNA molecules carrying a target SNP in situ. Exogenous mtDNA which has transferred into 'recipient' cells is then detected by high throughput confocal imaging. These Images are analysed via an integrated machine-learning pipeline focused on reduction of false discovery rate, and allows us to accurately trace and quantify the transfer of mitochondrial genomes between cells in vitro. Using these tools, a robust, quantitative method to support microscopic observation of this phenomenon has been developed, which has enabled insights into the effect of cancer therapeutics on mitochondrial transfer.
Question - How do I best FACS sort GFP+ neural precursor cells?
Hi Anne - what are the physical parameters on your sorter? We have seen similar problems sorting GFP+ neural cells, and have modified as follows: 1. increase the nozzle size to get a larger droplet
2. reduce the pressure the sorter runs at
3. sort cells directly into optimal growth media
4. don't take the highest GFP+ expressing cells (these die the fastest in our hands, a lot of GFP is not compatible with good health post fluidics-esposure!)
Talk to the person running your sorter and see if they can reduce the physical stresses on your cells. This will depend on what sort of sorter it is.
Hope that helps
Question - A question for the DNA methylation community - is there any reason why bisulfite conversion would not work on a plasmid?
Thanks! We have tried the conversion again on linearised plasmid and fully expect to have the right answer shortly.
- Feb 2018
We have bisulfite-converted and sequenced a plasmid and find there are no cytosines that have converted to thymine ... we're puzzled because we don't believe that every cytosine is methylated. The conversion of genomic DNA works well, so the reagents are fine. Just wondering if plasmids need linearising or something first??? All thoughts appreciated!
Interest in the recently discovered phenomenon of mitochondrial transfer between mammalian cells has gained momentum since it was first described in cell culture systems more than a decade ago. Mitochondria-targeting fluorescent dyes have been repurposed and are now widely used in these studies and in acute disease models, sometimes without due consideration of their limitations, while vectors containing mitochondrially-imported fluorescent proteins have complemented the use of mitochondria-targeting dyes. Genetic approaches that use mitochondrial DNA polymorphisms have also been used in some in vitro studies and in tumor models and are particularly useful where mtDNA is damaged or deleted. These approaches can also be used to study the long-term consequences of mitochondrial transfer such as in bone marrow and organ transplantation and in tumour biology where inherent mitochondrial damage is often a key feature. As research on intercellular mitochondrial transfer moves from cell culture into animal models and human diseases it will be important to understand the limitations of the various techniques in order to apply appropriate methodologies to address physiological and pathophysiological conditions.
Question - Cell cycle and DNA analysis in symbiodinium algae?
Thanks for your answer - all indications are that they should have been actively replicating, but you're right, it is an important consideration and something we are checking more carefully. The EdU was left in for ~6 hours. The membrane is very tough, as you say - we might need to increase the permeabilization steps further. There is a suggestion in the literature that EdU may incorporate differently to BrdU in prokaryotes, perhaps this also occurs in other species? We're not sure.
- Oct 2017
Hello - we are working on analysis of cell cycle in symbiodinium algae, and struggling to get EdU-click-it labelling of the algal DNA. We have tried different fixation and strategies, detergents - 4% PFA and ethanol fixation, Triton, various Tweens, other permeabilisation strategies, and we have also titrated the concentration and timing of the EdU. Does anyone have expertise in this area? Thanks! Melanie, Victoria University of Wellington, New Zealand
The transfer of mitochondria between mammalian cells is a physiologically relevant phenomenon, however the signals driving this process are not yet clear. Cellular stress during disease or therapy may alter the rate of mitochondrial transfer between cells and surrounding tissue. Literature published in the field of intercellular mitochondrial transfer relies heavily on confocal microscopy to understand this process. Limitations of mitochondrial-specific fluorescent markers is of serious concern, thus ambiguous data on intercellular mitochondrial transfer have been presented throughout literature. In order to accurately detect and quantify intercellular mitochondrial transfer, a novel strategy for in situ molecular genotyping has been targeted toward single nucleotide polymorphisms within the mitochondrial genome (mtDNA). This strategy exploits the enhanced fidelity of an engineered, thermostable Taq ligase, that when combined with padlock oligonucleotides, may serve as template for a target-primed rolling circle amplification of a single SNP-containing mtDNA molecule. This enables detection of exogenous mtDNA within a 'recipient cell' in the context of mitochondrial transfer. In multiplexed reactions, detection of two (or more) variant mtDNAs present within a co-culture is possible, allowing bidirectional study of intercellular transfer of mitochondrial genomes. Application of this strategy allows us to trace and quantify the transfer of mitochondrial genomes, and provides robust, quantitative data to support microscopic observation of this phenomenon.
Background Tumour hypoxia limits the effectiveness of radiation therapy. Delivering normobaric or hyperbaric oxygen therapy elevates pO2 in both tumour and normal brain tissue. However, pO2 levels return to baseline within 15 minutes of stopping therapy. Aim To investigate the effect of perfluorocarbon (PFC) emulsions on hypoxia in subcutaneous and intracranial mouse gliomas and their radiosensitising effect in orthotopic gliomas in mice breathing carbogen (95%O2 and 5%CO2). Results PFC emulsions completely abrogated hypoxia in both subcutaneous and intracranial GL261 models and conferred a significant survival advantage orthotopically (Mantel Cox: p = 0.048) in carbogen breathing mice injected intravenously (IV) with PFC emulsions before radiation versus mice receiving radiation alone. Carbogen alone decreased hypoxia levels substantially and conferred a smaller but not statistically significant survival advantage over and above radiation alone. Conclusion IV injections of PFC emulsions followed by 1h carbogen breathing, radiosensitises GL261 intracranial tumors.
We have previously shown that exposure to high dose ascorbate causes double stranded breaks (DSBs) and a build-up in S-phase in glioblastoma (GBM) cell lines. Here we investigated whether or not this was due to genotoxic stress as well as metabolic stress generated by exposure to high dose ascorbate, radiation, ascorbate plus radiation and H₂O₂ in established and primary GBM cell lines. Genotoxic stress was measured as phosphorylation of the variant histone protein, H2AX, 8-oxo-7,8-dihydroguanine (8OH-dG) positive cells and cells with comet tails. Metabolic stress was measured as a decrease in NADH flux, mitochondrial membrane potential (by CMXRos), ATP levels (by ATP luminescence) and mitochondrial superoxide production (by mitoSOX). High dose ascorbate, ascorbate plus radiation, and H₂O₂ treatments induced both genotoxic and metabolic stress. Exposure to high dose ascorbate blocked DNA synthesis in both DNA damaged and undamaged cell of ascorbate sensitive GBM cell lines. H₂O₂ treatment blocked DNA synthesis in all cell lines with and without DNA damage. DNA synthesis arrest in cells with damaged DNA is likely due to both genotoxic and metabolic stress. However, arrest in DNA synthesis in cells with undamaged DNA is likely due to oxidative damage to components of the mitochondrial energy metabolism pathway.
Introduction: Macrophages play a significant role in the progression of diseases, such as cancer, making them a target for immune-modulating agents. Trehalose dibehenate (TDB) is known to activate M1-like macrophages via Mincle, however, the effect of TDB on M2-like macrophages, which are found in the tumor microenvironment, has not been studied. Methods: qRT-PCR, flow cytometry, cytokine ELISA, and Western Blotting were used to study the effect of TDB on GM-CSF and M-CSF/IL-4 derived bone marrow macrophages (BMMs) from C57BL/6 and Mincle(-/-) mice. Results: TDB treatment up-regulated M1 markers over M2 markers by GM-CSF BMMs, whereas M-CSF/IL-4 BMMs down-regulated marker gene expression overall. TDB treatment resulted in Mincle-independent down-regulation of CD11b, CD115, and CD206 expression by GM-CSF macrophages and CD115 in M-CSF/IL-4 macrophages. GM-CSF BMMs produced of significant levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α), which was Mincle-dependent and further enhanced by LPS priming. M-CSF BMMs produced little or no cytokines in response to TDB regardless of LPS priming. Western blot analysis confirmed that the absence of cytokine production was associated with a lack of activation of the Syk kinase pathway. Conclusion: This study illustrates that TDB has the potential to differentially regulate M1- and M2-like macrophages in the tumor environment.
Supporting Information SI1. TDB induces the production of cytokines in M‐CSF differentiated bone marrow macrophages. BMMs from WT bone marrow were differentiated over 8 days using M‐CSF followed by stimulation with 40 μg/mL TDB, or 100 ng/mL LPS as positive control. Levels of IL‐1β, IL‐6, and TNFα were measured by the ELISA from supernatant at 48 h. Mean ± SEM of triplicate samples from one experiment are shown. *P ≤ 0.05; ****P ≤ 0.001 (1‐way ANOVA).
- Jul 2017
Glioblastoma (GBM) is the most common and most deadly brain tumor to occur in adults. Initially patients respond to radiation and chemotherapy, which primarily work by causing large amounts of DNA damage, causing apoptosis of the cells. However, this process does not happen effectively in GBM and understanding how these cells resist cell death in response to therapy is key to improving the efficacy of treatment. BCL6 is a transcription factor that stops cell death in response to DNA damage. Recent work in our lab has shown BCL6 to be present in untreated GBM tumors and up-regulated in treated GBM cells. This evidence indicates that BCL6 may be used as a mechanism of therapy resistance by GBM cells. In this study, the objective was to determine the mechanism of action of BCL6 in GBM cells using luciferase reporter assays, quantitative chromatin immunoprecipitation (qChIP) and RNA sequencing. We observed that BCL6 was transcriptionally active in GBM as shown by a reduction in luciferase activity when BCL6 was present. qChIP experiments revealed that BCL6 binding changed over time and was different with different types of DNA-damaging treatment. Preliminary analysis of our RNA sequencing data has identified a unique subset of genes which are upregulated when BCL6 is inhibited and downregulated in response to chemotherapy. These changes indicate that these genes may be regulated by BCL6 in chemotherapy treated cells. All of these results illustrate that BCL6 appears to have an active and relevant function in GBM cells, which demonstrates that BCL6 is an attractive therapeutic target in GBM. Citation Format: Nicole M. Jones, Marie-Sophie Fabre, Dinindu Sachindra Senanayake, Katerina Hatzi, Ari M. Melnick, Melanie J. McConnell. The mechanism of action of BCL6 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1523. doi:10.1158/1538-7445.AM2017-1523
The dendritic cell signals required for the in vivo priming of IL-4–producing T cells are unknown. We used RNA sequencing to characterize DCs from skin LN of mice exposed to two different Th2 stimuli: the helminth parasite Nippostrongylus brasiliensis ( Nb ) and the contact sensitizer dibutyl phthalate (DBP)-FITC. Both Nb and DBP-FITC induced extensive transcriptional changes that involved multiple DC subsets. Surprisingly, these transcriptional changes were highly distinct in the two models, with only a small number of genes being similarly regulated in both conditions. Pathway analysis of expressed genes identified no shared pathways between Nb and DBP-FITC, but revealed a type-I IFN (IFN-I) signature unique to DCs from Nb -primed mice. Blocking the IFN-I receptor at the time of Nb treatment had little effect on DC migration and antigen transport to the LN, but inhibited the up-regulation of IFN-I–induced markers on DCs and effectively blunted Th2 development. In contrast, the response to DBP-FITC was not affected by IFN-I receptor blockade, a finding consistent with the known dependence of this response on the innate cytokine TSLP. Thus, the priming of Th2 responses is associated with distinct transcriptional signatures in DCs in vivo, reflecting the diverse environments in which Th2 immune responses are initiated.
Background: The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. Methods: We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. Results: We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. Conclusion: We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.
Background Apart from increasing age and a few specific genetic polymorphisms, the aetiology of Motor Neurone Disease is largely unknown. Only 5–10% of cases are familial with the vast majority being of the sporadic form. A role for environmental exposures is suggested by the lack of evidence for a genetic component, differences in incidence by geographical region, increases in incidence observed in some countries (including New Zealand) over a relatively short period, associations observed in epidemiological studies, and the higher proportion of males affected. We conducted a New Zealand population-based case-control study to investigate associations between occupational exposures and MND. Methods We recruited both prevalent and incident cases from a voluntary register, supplemented by notifications from neurologists. General population controls were selected from the Electoral Roll. A standardised questionnaire was used to obtain information on personal and demographic details, lifestyle factors and a full occupational history. We estimated odds ratios by occupation and industry, with analyses adjusted for age, gender, ethnicity, SES and smoking using logistic regression. Results We interviewed 265 cases and 532 controls. Two thirds of cases were male, with 70% aged over 60. Significantly elevated risks were observed for Telecommunications Technicians OR = 4.2 (95% CI: 1.2–14.0), Forecourt Attendants OR = 6.2 (1.3–30.2), Agriculture and Fishery Workers OR = 1.5 (1.1–2.2), Market Farmers and Crop Growers OR = 1.9 (1.1–3.3), Fruit Grower/Worker OR = 2.7 (1.2–6.3), Building Trades Workers OR = 1.8 (1.1–3.0), Builders Labourers OR = 4.3 (1.1–16.7) and Agricultural, Earthmoving and Other Materials-Handling Equipment Operators OR = 2.6 (1.1–6.1). There was also a non-significant elevation in Electricians (OR = 3.1). Analyses by industry showed significantly elevated risks in agricultural (particularly horticulture and fruit growing), construction, automotive fuel retailing and computer services. Discussion These preliminary results are consistent with those reported in the literature, and suggest that occupational exposures such as pesticides and ELF-MF may play a role in MND in New Zealand.
Background Although there are no established environmental or occupational risk factors for Motor Neurone Disease, an association with work in “electrical occupations” has been observed in numerous studies. However, the results of investigations using job-exposure matrices for extremely low frequency EMF and for electric shocks have been equivocal. In a population-based case-control study conducted in New Zealand we examined the effect of both electric shocks and ELF-MF. Methods We recruited cases from a voluntary register supplemented by notifications by neurologists. General population controls were selected from the Electoral Roll. A standardised questionnaire was used to obtain demographic and personal data, information on lifestyle factors plus a lifetime occupational history. Odds ratios were estimated for occupation, adjusting for age, gender, ethnicity, SES and smoking using logistic regression. The occupational histories of all participants were linked to job-exposure matrices on ELF-MF exposure and on electric shocks. Results We included 259 cases and 474 controls. There was no association between ELF-MF exposure and MND, with an OR = 1.1 (95% CI: 0.6–2.1) for the high exposure group. For electric shock the risk was elevated (but not statistically significantly) for both the medium OR = 1.3 (0.9–1.9) and high risk OR = 1.3 (0.0–1.9) groups when compared with the reference low risk group. Conclusions We found no strong evidence of elevated risk of MND associated with either ELF-MF or electric shock.
The transfer of mitochondria between mammalian cells is a physiologically relevant phenomenon, however the signals driving this process are not yet clear. Cellular stress during disease may alter the rate of mitochondrial transfer between cells and surrounding tissues. We hypothesise this form of intercellular communication to be fundamental to cell survival in disease, given the critical role of mitochondria within cell biology. Literature published in the field of intercellular mitochondrial transfer relies heavily on confocal microscopy to understand this process, however it is challenging to evaluate the frequency of transfer events by microscopy alone. In order to understand how cellular injury may alter the rate of mitochondrial transfer, two genetic tools have been developed. Application of these tools to trace and quantify the transfer of mitochondrial genomes among in vitro co-cultures provides quantitative data to support microscopic observations. These tools are highly sensitive, cost-effective and provide the throughput necessary to accurately evaluate how cellular injury alters mitochondrial transfer within a population of cells.
Intercellular mitochondrial transfer has been shown in tumor models, following lung injury and in xenotransplants of leukemic cells, but trafficking between cells in the brain remains unexplored. A suggestion that mitochondria move from astrocytes to neurons in a model of ischemia in a recent article in Nature by Hayakawa et al. (2016) should be interpreted with caution.
Current dogma holds that genes are the property of individual mammalian cells and partition between daughter cells during cell division. However, and rather unexpectedly, recent research has demonstrated horizontal cell-to-cell transfer of mitochondria and mitochondrial DNA in several mammalian cell culture systems. Furthermore, unequivocal evidence that mitochondrial DNA transfer occurs in vivo has now been published. While these studies show horizontal transfer of mitochondrial DNA in pathological settings, it is also possible that intercellular mitochondrial transfer is a fundamental physiological process with a role in development and tissue homeostasis.
- May 2016
Cancer stem cells (CSC) exhibit therapy resistance and drive self-renewal of the tumour, making cancer stem cells an important target for therapy. The PI3K signalling pathway has been the focus of considerable research effort, including in glioblastoma (GBM), a cancer that is notoriously resistant to conventional therapy. Different isoforms of the catalytic sub-unit have been associated with proliferation, migration and differentiation in stem cells and cancer stem cells. Blocking these processes in CSC would improve patient outcome. We examined the effect of isoform specific PI3K inhibitors in two models of GBM CSC, an established GBM stem cell line 08/04 and a neurosphere formation model. We identified the dominant catalytic PI3K isoform for each model, and inhibition of the dominant isoform blocked AKT phosphorylation, as did pan-PI3K/mTOR inhibition. Analysis of SOX2, OCT4 and MSI1 expression revealed that inhibition of the dominant p110 subunit increased expression of cancer stem cell genes, while pan-PI3K/mTOR inhibition caused a similar, though not identical, increase in cancer stem cell gene expression. This suggested that PI3K inhibition enhanced, rather than blocked, CSC activity. Careful analysis of the response to specific isoform inhibition will be necessary before specific subunit inhibitors can be successfully deployed against GBM CSC.
- Apr 2016
A series of N,N-bis(glycityl)amines with promising anti-cancer activity were prepared via the reductive amination of pentoses and hexoses, and subsequently screened for their ability to selectively inhibit the growth of cancerous versus non-cancerous cells. For the first time, we show that this class of compounds possesses anti-proliferative activity, and, while the selective killing of brain cancer (LN18) cells versus matched (SVG-P12) cells was modest, several of the amines, including d-arabinitylamine 1a and d-fucitylamine 1g, exhibited low micromolar IC50 values for HL60 cells. Moreover, these two amines showed good selectivity towards HL60 cells when compared to non-cancerous HEK-293 cells. The compounds also showed low micromolar inhibition of the leukaemic cell line, THP-1. The modes of action of amines 1a and 1g were then determined using yeast chemical genetics, whereby it was established that both compounds affect similar but distinct sets of biochemical pathways. Notably purine nucleoside monophosphate biosynthesis was identified as an enriched mechanism. The rapid synthesis of the amines and their unique mode of action thus make them attractive targets for further development as anti-cancer drugs.
Background: The transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) is critical for the regulation of normal stem cells maintenance by establishing specific epigenetic landscape. We have previously shown that CBP/p300 acetyltransferase induces PLZF acetylation in order to increase its deoxynucleotidic acid (DNA) binding activity and to enhance its epigenetic function (repression of PLZF target genes). However, how PLZF is inactivated is not yet understood. Results: In this study, we demonstrate that PLZF is deacetylated by both histone deacetylase 3 and the NAD+ dependent deacetylase silent mating type information regulation 2 homolog 1 (SIRT1). Unlike other PLZF-interacting deacetylases, these two proteins interact with the zinc finger domain of PLZF, where the activating CBP/p300 acetylation site was previously described, inducing deacetylation of lysines 647/650/653. Overexpression of histone deacetylase 3 (HDAC3) and SIRT1 is associated with loss of PLZF DNA binding activity and decreases PLZF transcriptional repression. As a result, the chromatin status of the promoters of PLZF target genes, involved in oncogenesis, shift from a heterochromatin to an open euchromatin environment leading to gene expression even in the presence of PLZF. Conclusions: Consequently, SIRT1 and HDAC3 mediated-PLZF deacetylation provides for rapid control and fine-tuning of PLZF activity through post-transcriptional modification to regulate gene expression and cellular homeostasis.
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Despite the advances in surgery, radiotherapy and chemotherapy, patient survival averages only 14.6 months. In most GBM tumors, tyrosine kinases show increased activity and/or expression and actively contribute to the development, recurrence and onset of treatment resistance; making their inhibition an appealing therapeutic strategy. We compared the cytotoxicity of 12 tyrosine kinase inhibitors in vitro. A combination of crizotinib and dasatinib emerged as the most cytotoxic across established and primary human GBM cell lines. The combination treatment induced apoptotic cell death and polyploidy. Furthermore, the combination treatment led to the altered expression and localization of several tyrosine kinase receptors such as Met and EGFR and downstream effectors as such as SRC. Furthermore, the combination treatment reduced the migration and invasion of GBM cells and prevented endothelial cell tube formation in vitro. Overall, our study demonstrated the broad specificity of a combination of crizotinib and dasatinib across multiple GBM cell lines. These findings provide insight into the development of alternative therapy for the treatment of GBM.
- Aug 2015
- Queenstown Molecular Biology
BCL6 is a transcription factor which binds DNA and is best known to silence the expression of cell cycle checkpoint genes and prevent apoptosis. The identity of BCL6 binding genes has been well characterised in B-cells, T-follicular helper cells and breast cancer using chromatin immunoprecipitation and sequencing (ChIP seq). ChIP is useful in the investigation of a wide range of nuclear protein interactions with DNA, including histones, transcription factors and chromatin modifying enzymes. The data generated from ChIP experiments gives functional relevance to protein and gene expression data, which allows researchers to build a bigger picture of the pathways they are interested in. We are interested in ChIP seq as there are major differences in the DNA binding profile of BCL6 between B-cells, T-follicular helper cells and breast cancer cells, which indicates that the target genes of BCL6 is different in different cell types. The McConnell lab has shown that BCL6 is expressed in Glioblastoma Multiforme (GBM) at a low level pre-treatment and upregulated in response to therapy, which lead us to investigate the DNA binding of BCL6 in GBM. Preliminary experiments have validated controls and show early indications of BCL6 binding to cell cycle checkpoint gene CCND2 and the DNA damage mediator ATR. Future work will sequence BCL6 ChIP products in order to give a full map of BCL6 binding sites in GBM and identify likely target genes.
- May 2015
- AACR Advances in Brain Cancer Research
Therapy induced DNA damage should result in cell death via the activation of DNA damage sensors, which then activate cell cycle checkpoints and ultimately apoptosis. The success of current chemo-radiation for Glioblastoma Multiforme (GBM) relies on this process, but this does not happen effectively. Understanding the mechanism behind resistance to DNA damage is key to finding effective treatments for GBM. BCL6 is a factor that drives survival in a number of cancers, by preventing activation of cell cycle checkpoints in response to DNA breaks, thereby preventing apoptosis. DNA damage can induce BCL6 expression, so we hypothesized that therapy-induced damage would induce BCL6 expression in GBM, and contribute to intrinsic therapy resistance. The specific objectives of this study were to examine basal BCL6 expression in human GBM tumors and induction of BCL6 in response to therapy. BCL6 expression was examined in formalin-fixed paraffin-embedded tumor sections, a panel of human GBM cell lines in vitro, and the mouse glioma cell line GL261, both in vitro and as an orthotopic intracranial tumor. Cells were treated with doxorubicin, ionizing radiation and temozolomide in vitro, and tumor-bearing mice were irradiated to look at BCL6 expression in vivo. BCL6 was examined using immunohistochemistry, immunofluorescence, western blotting and flow cytometry. We observed basal BCL6 expression localized to discrete regions of a subset of GBM tumors, consistent with a recent report. BCL6 was not detected, or was at a very low level in untreated mouse and human cell lines. However, BCL6 was consistently up-regulated in response to therapy, both in vitro and in the GL261 mouse model after irradiation. This data indicates that BCL6 is present in GBM, and that induction in response to DNA damage in vivo may play a role in therapy resistance.
Pharmacological ascorbate is currently used as an anti-cancer treatment, potentially in combination with radiation therapy, by integrative medicine practitioners. In the acidic, metal-rich tumor environment, ascorbate acts as a pro-oxidant, with a mode of action similar to that of ionizing radiation; both treatments kill cells predominantly by free radical-mediated DNA damage. The brain tumor, glioblastoma multiforme (GBM), is very resistant to radiation; radiosensitizing GBM cells will improve survival of GBM patients. Here, we demonstrate that a single fraction (6 Gy) of radiation combined with a 1 h exposure to ascorbate (5 mM) sensitized murine glioma GL261 cells to radiation in survival and colony-forming assays in vitro. In addition, we report the effect of a single fraction (4.5 Gy) of whole brain radiation combined with daily intraperitoneal injections of ascorbate (1 mg/kg) in an intracranial GL261 glioma mouse model. Tumor-bearing C57BL/6 mice were divided into four groups: one group received a single dose of 4.5 Gy to the brain 8 days after tumor implantation, a second group received daily intraperitoneal injections of ascorbate (day 8–45) after implantation, a third group received both treatments and a fourth control group received no treatment. While radiation delayed tumor progression, intraperitoneal ascorbate alone had no effect on tumor progression. Tumor progression was faster in tumor-bearing mice treated with radiation and daily ascorbate than in those treated with radiation alone. Histological analysis showed less necrosis in tumors treated with both radiation and ascorbate, consistent with a radio-protective effect of ascorbate in vivo. Dis-crepancies between our in vitro and in vivo results may be explained by differences in the tumor microenvironment, which determines whether ascorbate remains outside the cell, acting as a pro-oxidant, or whether it enters the cells and acts as an anti-oxidant.
- Nov 2014
Background: Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an extremely short time to relapse following standard treatment. Since recurrent GBM is often resistant to subsequent radiotherapy and chemotherapy, immunotherapy has been proposed as an alternative treatment option. Although it is well established that GBM induces immune suppression, it is currently unclear what impact prior conventional therapy has on the ability of GBM cells to modulate the immune environment. In this study, we investigated the interaction between immune cells and glioma cells that had been exposed to chemotherapy or irradiation in vitro. We demonstrate that treated glioma cells are more immunosuppressive than untreated cells and form tumors at a faster rate in vivo in an animal model. Cultured supernatant from in vitro-treated primary human GBM cells were also shown to increase suppression, which was independent of accessory suppressor cells or T regulatory cell generation, and could act directly on CD4+ and CD8+ T cell proliferation. While a number of key immunosuppressive cytokines were overexpressed in the treated cells, including IL-10, IL-6 and GM-CSF, suppression could be alleviated in a number of treated GBM lines by inhibition of prostaglandin E2. These results reveal for the first time that conventional therapies can alter immunosuppressive pathways in GBM tumor cells, a finding with important implications for the combination of immunotherapy with standard treatment. © 2014 Wiley Periodicals, Inc.
High dose ascorbate is used as an anti-cancer treatment by complementary and alternative medicine. In the acidic, metal-rich tumour environment ascorbate acts as a pro-oxidant, generating free radicals and DNA damage, similar to ionising radiation. We showed that addition of high-dose ascorbate to radiation blocked repair of radiation-induced DNA damage in primary GBM cell lines, effectively radio-sensitising. We examined the effect of ascorbate and radiation on the murine glioma GL261 in vitro, and combined with intra-peritoneal ascorbate in an intra-cranial GL261 model. As previously seen, high dose ascorbate radio-sensitized GL261 cells in a clonogenicity assay. Tumour-bearing mice were treated with: 4.5Gy to the brain 8 days post-implantation; repeated high-dose ascorbate from day 8-45; both treatments; or no treatment. While radiation increased survival, intraperitoneal ascorbate alone had no effect. In contrast with in vitro data, tumour-bearing mice treated with radiation and daily ascorbate had poorer survival than those treated with radiation alone. Histological analysis of the tumours showed less necrosis and bleeding within tumours treated with both radiation and ascorbate, consistent with a radio-protective effect of ascorbate in vivo. While the mechanism of protection is not yet defined, this finding might have important clinical implications for combining high-dose ascorbate with radiation therapy.
- Oct 2014
- New Zealand Society of Oncology
Glioblastoma Multiforme (GBM) is a grade IV CNS tumour of glial cells, which are the support cells for neurons in the brain. It is the most common and most deadly brain tumour to occur in adults. While initially patients respond to treatment, the tumour will inevitably relapse and become resistant to these therapies. Understanding how these cells resist therapy is key to improving the efficacy of treatment, and therefore the prognosis for people with GBM. B cell lymphoma 6 (BCL6) is a transcription factor which represses expression of the DNA damage response pathway to prevent apoptosis. BCL6 has been well characterised as a driver of lymphoma, where it was originally discovered, as it similarly blocks normal cell death in cancer cells with DNA damage. Recent work in the McConnell lab has shown that BCL6 is up-regulated in a panel of GBM cells in response to both radiation and chemotherapy. In this study we used a luciferase reporter assay to determine if BCL6 is transcriptionally active in the same panel of GBM cell lines. Our data indicated that BCL6 is transcriptionally active, as shown by a reduction in luciferase activity where BCL6 is present. This is key evidence towards our hypothesis that BCL6 represses cell cycle checkpoints after DNA damage and prevents apoptosis in GBM cells, which is likely to be a cause of therapy resistance.
We previously showed that 5mM ascorbate radiosensitized early passage radioresistant glioblastoma multiforme (GBM) cells derived from one patient tumour. Here we investigate the sensitivity of a panel of cell lines to 5mM ascorbate and 6Gy ionising radiation, made up of three primary human GBM cells, three GBM cell lines, a human glial cell line and primary human vascular endothelial cells. The response of different cells lines to ascorbate and/or radiation was determined by measuring viability, colony-forming ability, generation and repair of double stranded DNA breaks (DSBs), cell cycle progression, antioxidant capacity and generation of reactive oxygen species. Individually, radiation and ascorbate both decreased viability and clonogenicity by inducing DNA damage, but had differential effects on cell cycle progression. Radiation led to G2/M arrest in most cells whereas ascorbate caused accumulation in S-phase, which was moderately associated with poor DSB repair. While high dose ascorbate radio-sensitized all cell lines in clonogenic assays, the sensitivity to radiation, high dose ascorbate and combined treatment varied between cell lines. Normal glial cells were similar to GBM cells with respect to free radical scavenging potential and effect of treatment on DNA damage and repair, viability and clonogenicity. Both GBM cells and normal cells coped equally poorly with oxidative stress caused by radiation and/or high dose ascorbate, dependent primarily on their antioxidant and DSB repair capacity.
The addition of high-dose ascorbate to existing anticancer treatment strategies can improve efficacy and decrease toxicity-but not in all patients or with all combination therapies (Ma et al., this issue).
- Feb 2013
Trans-Plasma Membrane Electron Transport (tPMET) is a ubiquinone-dependent cell survival pathway for maintaining intracellular redox homeostasis in rapidly-dividing cells. To target this pathway, fifteen ubiquinone-based compounds were designed and synthesized to position at the plasma membrane and disrupt tPMET. We established that quaternary ammonium salt moieties carrying highly-hindered, positive electronic charges located to the plasma membrane, and that a 10-carbon chain link was effective at positioning the redox-active ubiquinone-like function within the lipid bilayer to disrupt tPMET in human leukaemic cells (IC50 9 + 1µM). TPMET inhibition alone was not sufficient to induce significant cell death, but positively-charged compounds could also enter the cell and disrupt intracellular redox balance, distinct from their effects on mitochondrial electron transport. The synergistic effect of tPMET inhibition plus intracellular redox disruption gave strong anti-proliferative activity (IC50 2 + 0.2µM). Positively-charged ubiquinone-based compounds inhibit human leukaemic cell growth.
- Nov 2012
Purpose: The prognosis for patients with glioblastoma multiforme (GBM) remains extremely poor despite recent treatment advances. There is an urgent need to develop novel therapies for this disease. Experimental design: We used the implantable GL261 murine glioma model to investigate the therapeutic potential of a vaccine consisting of intravenous injection of irradiated whole tumor cells pulsed with the immuno-adjuvant α-galactosylceramide (α-GalCer). Results: Vaccine treatment alone was highly effective in a prophylactic setting. In a more stringent therapeutic setting, administration of one dose of vaccine combined with depletion of regulatory T cells (Treg) resulted in 43% long-term survival and the disappearance of mass lesions detected by MRI. Mechanistically, the α-GalCer component was shown to act by stimulating "invariant" natural killer-like T cells (iNKT cells) in a CD1d-restricted manner, which in turn supported the development of a CD4(+) T-cell-mediated adaptive immune response. Pulsing α-GalCer onto tumor cells avoided the profound iNKT cell anergy induced by free α-GalCer. To investigate the potential for clinical application of this vaccine, the number and function of iNKT cells was assessed in patients with GBM and shown to be similar to age-matched healthy volunteers. Furthermore, irradiated GBM tumor cells pulsed with α-GalCer were able to stimulate iNKT cells and augment a T-cell response in vitro. Conclusions: Injection of irradiated tumor cells loaded with α-GalCer is a simple procedure that could provide effective immunotherapy for patients with high-grade glioma.
- Sep 2012
Immunology and Cell Biology focuses on the general functioning of the immune system in its broadest sense, with a particular emphasis on its cell biology. Areas that are covered include but are not limited to: Cellular immunology, Innate and adaptive immunity, Immune responses to pathogens,Tumour immunology,Immunopathology, Immunotherapy, Immunogenetics, Immunological studies in humans and model organisms (including mouse, rat, Drosophila etc)
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Glioblastoma multiforme (GBM) has a very poor prognosis because of its chemo-and radiation therapy resis-tance. Here we investigated the ability of pharmacological concentrations of ascorbate to radiosensitize pri-mary cells isolated from six GBM patients, mouse astrocytoma cells, and mouse astrocytes. We measured cell viability by trypan blue exclusion, generation of double-stranded DNA breaks by H2AX phosphorylation using fluorescently labeled antibodies and FACS analysis, apoptosis by annexin V/propidium iodide staining, inhibition of autophagy by 3-methyladenine, and cell cycle progression by propidium iodide staining of per-meabilized cells. We showed that 5 mM ascorbate in combination with 6 Gy radiation killed more GBM pri-mary cells by generating significantly more double-stranded breaks than either treatment alone (p b 0.05). Combined treatment affected viability and double-stranded break generation in normal astrocytes to a much smaller extent. Radiation, but not 5 mM ascorbate, caused G2/M arrest in GBM cells and ascorbate pre-vented radiation-induced G2/M arrest in combined treatment. Cell death in response to 5 mM ascorbate or combination treatment was not mediated by apoptosis or autophagy. In conclusion, pharmacological concen-trations of ascorbate radiosensitize GBM primary cells to a much greater extent than astrocytes; this large therapeutic ratio may be of clinical significance in radiation-resistant cancers. Glioblastoma multiforme (GBM) is the most prevalent and aggres-sive primary brain cancer in adults. Median survival time of GBM patients, with debulking surgery and radiation therapy with concom-itant and adjuvant temozolomide  is only 19 months and the 5-year overall survival rate is 10% . The poor prognosis for GBM patients is due to the GBM resistance to chemotherapy and ionizing radiation . As radiation kills cells primarily by generating free rad-icals that produce double-stranded DNA breaks (DSBs) [3,4], radia-tion resistance depends on the ability to neutralize free radicals  and effectively repair DSBs . Enhancing free radical generation may therefore present a valid therapeutic approach for augmenting radiation therapy for tackling radiation resistance in GBM patients. The use of the antioxidant ascorbate (vitamin C) as an anticancer treatment has been the topic of heated debate since the 1970s. Early clinical studies showed that high-dose ascorbate, given intrave-nously or orally, conferred a significant survival benefit on terminally ill cancer patients [6–8]. High-dose intravenous ascorbate has also generated impressive survival data in selected case studies . How-ever, double-blinded randomized controlled trials in the 1980s using high-dose ascorbate delivered orally did not validate these findings [10,11]. Nevertheless, high-dose intravenous ascorbate is still widely used in the complementary and alternative oncology setting and is well tolerated in patients with terminal cancer [12–14]. More recent research has demonstrated that ascorbate specifically kills a wide range of cancer cells when plasma concentrations reach 1 mM or more [15–20]. However, such high concentrations of ascor-bate can be achieved only by intravenous administration, which may explain the differential outcomes in previous survival data based on oral versus intravenous administration [12,13,17,21]. Plas-ma ascorbate concentrations are normally tightly controlled, varying between 50 and 70 μM in healthy volunteers . At these physiolog-ical concentrations, ascorbate acts as an antioxidant, conferring protection against oxidative stress and possibly acting as a cancer-preventative agent (see  for an in-depth recent review). However, at higher, pharmacologically relevant doses (>1 mM), ascorbate has been shown to act as a pro-oxidant, generating extracellular ascorbyl radicals [16,23–25] that drive H 2 O 2 -mediated oxidative stress leading to intracellular free radical production and damage to macromole-cules [16,19,20,26]. Pharmacological concentrations of ascorbate have been shown to augment the anticancer activity of several anticancer drugs [23,27–32]. A recent study showed that these high concentrations of ascorbate increase radiation-induced apoptosis in human leukemia HL60 cells in vitro via caspase 8 activation and upregulation of Bax expression . Radiation therapy employs ionizing radiation, which produces clusters of ionization events (particularly in water Free Radical Biology & Medicine xxx (2012) xxx–xxx
- Mar 2012
Breast cancer stem cells (BCSCs) initiate and sustain breast cancers, and several putative markers have been proposed to prospectively isolate BCSC from the non-cancer stem cell population. The candidate BCSC marker Sca-1 is a GPI-linked membrane protein expressed on activated lymphocytes, hematopoietic stem cells and mammary stem cells. Sca-1+ cells were purified from the murine mammary tumour cell line 4T1. However, this did not enrich for a stem-like, tumour initiating or metastatic cell population in vitro or in vivo. Sphere formation, which induced high levels of Sca-1, reduced BCSC gene expression with near complete loss of spontaneous metastasis from sphere-derived tumours. This was associated with decreased expression of TGFB2 and reduced activation of the TGFβ signalling pathway in spheres. Both TGFB2 expression in vitro and spontaneous metastasis in vivo could be restored upon re-differentiation of sphere cells by exposure to serum, and this occurred with retention of the majority of Sca-1 expression. We conclude that while putative BCSC, including spheres, can have high Sca-1 expression, Sca-1 itself is not a marker of BCSC in established 4T1 tumours or the cell line.
Glioblastoma multiforme (GBM) has a very poor prognosis because of its chemo- and radiation therapy resistance. Here we investigated the ability of pharmacological concentrations of ascorbate to radiosensitize primary cells isolated from six GBM patients, mouse astrocytoma cells, and mouse astrocytes. We measured cell viability by trypan blue exclusion, generation of double-stranded DNA breaks by H2AX phosphorylation using fluorescently labeled antibodies and FACS analysis, apoptosis by annexin V/propidium iodide staining, inhibition of autophagy by 3-methyladenine, and cell cycle progression by propidium iodide staining of permeabilized cells. We showed that 5 mM ascorbate in combination with 6 Gy radiation killed more GBM primary cells by generating significantly more double-stranded breaks than either treatment alone (p<0.05). Combined treatment affected viability and double-stranded break generation in normal astrocytes to a much smaller extent. Radiation, but not 5 mM ascorbate, caused G2/M arrest in GBM cells and ascorbate prevented radiation-induced G2/M arrest in combined treatment. Cell death in response to 5 mM ascorbate or combination treatment was not mediated by apoptosis or autophagy. In conclusion, pharmacological concentrations of ascorbate radiosensitize GBM primary cells to a much greater extent than astrocytes; this large therapeutic ratio may be of clinical significance in radiation-resistant cancers.
- Jul 2011
The switch from oxidative phosphorylation to glycolytic metabolism results in cells that generate fewer reactive oxygen species (ROS) and are resistant to the intrinsic induction of apoptosis. As a consequence, glycolytic cancer cells are resistant to radiation and chemotherapeutic agents that rely on production of ROS or intrinsic apoptosis. Further, the level of glycolysis correlates with tumor invasion, making glycolytic cancer cells an important target for new therapy development. We have synthesized a novel redox-active quinone phloroglucinol derivative, PMT7. Toxicity of PMT7 was in part due to loss of mitochondrial membrane potential in treated cells with subsequent loss of mitochondrial metabolic activity. Mitochondrial gene knockout ρ0 cells, a model of highly glycolytic cancers, were only half as sensitive as the corresponding wild-type cells and metabolic pathways downstream of MET were unaffected in ρ0 cells. However, PMT7 toxicity was also due to a block in autophagy. Both wild-type and ρ0 cells were susceptible to autophagy blockade, and the resistance of ρ0 cells to PMT7 could be overcome by serum deprivation, a situation where autophagy becomes necessary for survival. The stress response class III deacetylase SIRT1 was not significantly involved in PMT7 toxicity, suggesting that unlike other chemotherapeutic drugs, SIRT1-mediated stress and survival responses were not induced by PMT7. The dependence on autophagy or other scavenging pathways makes glycolytic cancer cells vulnerable. This can be exploited by induction of energetic stress to specifically sensitize glycolytic cells to other stresses such as nutrient deprivation or potentially chemotherapy.
There is strong evidence for the existence of cancer stem cells (CSCs) in the aggressive brain tumor glioblastoma multiforme (GBM). These cells have stem-like self-renewal activity and increased tumor initiation capacity and are believed to be responsible for recurrence due to their resistance to therapy. Several techniques have been used to enrich for CSC, including growth in serum-free defined media to induce sphere formation, and isolation of a stem-like cell using exclusion of the fluorescent dye Hoechst 33342, the side population (SP). We show that sphere formation in GBM cell lines and primary GBM cells enriches for a CSC-like phenotype of increased self-renewal gene expression in vitro and increased tumor initiation in vivo. However, the SP was absent from all sphere cultures. Direct isolation of the SP from the GBM lines did not enrich for stem-like activity in vitro, and tumor-initiating activity was lower in sorted SP compared with non-SP and parental cells. Transient exposure to doxorubicin enhanced both CSC and SP frequency. However, doxorubicin treatment altered the cytometric profile and obscured the SP demonstrating the difficulty of identifying SP in cells under stress. Doxorubicin-exposed cells showed a transient increase in SP, but the doxorubicin-SP cells were still not enriched for a stem-like self-renewal phenotype. These data demonstrate that the GBM SP does not necessarily contribute to self-renewal or tumor initiation, key properties of a CSC, and we advise against using SP to enumerate or isolate CSC.
To understand the changes in gene expression in polycythemia vera (PV) progenitor cells and their relationship to JAK2V617F. Messenger RNA isolated from CD34(+) cells from nine PV patients and normal controls was profiled using Affymetrix arrays. Gene expression change mediated by JAK2V617F was determined by profiling CD34(+) cells transduced with the kinase and by analysis of leukemia cell lines harboring JAK2V617F, treated with an inhibitor. A PV expression signature was enriched for genes involved in hematopoietic development, inflammatory responses, and cell proliferation. By quantitative reverse transcription-PCR, 23 genes were consistently deregulated in all patient samples. Several of these genes such as WT1 and KLF4 were regulated by JAK2, whereas others such as NFIB and EVI1 seemed to be deregulated in PV by a JAK2-independent mechanism. Using cell line models and comparing gene expression profiles of cell lines and PV CD34(+) PV specimens, we have identified panels of 14 JAK2-dependent genes and 12 JAK2-independent genes. These two 14- and 12-gene sets could separate not only PV from normal CD34(+) specimens, but also other MPN such as essential thrombocytosis and primary myelofibrosis from their normal counterparts. A subset of the aberrant gene expression in PV progenitor cells can be attributed to the action of the mutant kinase, but there remain a significant number of genes characteristic of the disease but deregulated by as yet unknown mechanisms. Genes deregulated in PV as a result of the action of JAK2V617F or independent of the kinase may represent other targets for therapy.
- Jun 2010
The transmembrane glycoprotein CD133 is a marker commonly used for isolation and analysis of putative cancer stem-like cells. However, analysis of CD133 expression is potentially confounded by the fact that two of the commonly used anti-CD133 antibodies, AC133 and 293C, only recognize CD133 that has undergone glycosylation. Therefore, our aim was to thoroughly examine antibody recognition and mRNA expression of CD133 in glioblastoma multiforme. Glioblastoma cell lines and primary cultures obtained from resected tumor tissue were analyzed by real-time PCR, Western blot analysis, and flow cytometry for CD133, and immunofluorescence was used to determine cellular localization. The AC133 and 293C antibodies did not detect any CD133 on the surface of the glioblastoma cells despite the fact that a protein was detected using C24B9, an anti-CD133 antibody that recognizes an unglycosylated epitope. This CD133 variant was truncated ( approximately 16 kDa) and, unlike typical expression of full-length CD133 protein, was found throughout the cytoplasm instead of localized to the plasma membrane. Levels of mRNA and protein for the variant increased with stress, indicating potential for it to be a functional molecule. Because AC133 and 293C antibodies do not detect all CD133 variants in glioblastoma cells, alternate detection methods need to be utilized for complete analysis of CD133 expression and for accurately determining the relationship between CD133 and cancer stem-like cells.
The transmembrane glycoprotein CD133 is a marker commonly used for isolation and analysis of putative cancer stem-like cells. However, analysis of CD133 expression is potentially confounded by the fact that two of the commonly used anti-CD133 antibodies, AC133 and 293C, only recognize CD133 that has undergone glycosylation. Therefore, our aim was to thoroughly examine antibody recognition and mRNA expression of CD133 in glioblastoma multiforme. Glioblas-toma cell lines and primary cultures obtained from resected tumor tissue were analyzed by real-time PCR, Western blot analysis, and flow cytometry for CD133, and immuno-fluorescence was used to determine cellular localization. The AC133 and 293C antibodies did not detect any CD133 on the surface of the glioblastoma cells despite the fact that a protein was detected using C24B9, an anti-CD133 antibody that recognizes an unglycosylated epitope. This CD133 variant was truncated (~16 kDa) and, unlike typical expression of full-length CD133 protein, was found throughout the cyto-plasm instead of localized to the plasma membrane. Levels of mRNA and protein for the variant increased with stress, indicating potential for it to be a functional molecule. Because AC133 and 293C antibodies do not detect all CD133 variants in glioblastoma cells, alternate detection methods need to be utilized for complete analysis of CD133 expression and for accurately determining the relationship between CD133 and cancer stem-like cells.
- Oct 2009
The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RARalpha) and RARalpha-PLZF. Using a combination of chromatin immunoprecipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RARalpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RARalpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RARalpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RARalpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RARalpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RARalpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RARalpha.
Interferons (IFNs) direct innate and acquired immune responses and, accordingly, are used therapeutically to treat a number of diseases, yet the diverse effects they elicit are not fully understood. Here, we identified the promyelocytic leukemia zinc finger (PLZF) protein as a previously unrecognized component of the IFN response. IFN stimulated an association of PLZF with promyelocytic leukemia protein (PML) and histone deacetylase 1 (HDAC1) to induce a decisive subset of IFN-stimulated genes (ISGs). Consequently, PLZF-deficient mice had a specific ISG expression defect and as a result were more susceptible to viral infection. This susceptibility correlated with a marked decrease in the expression of the key antiviral mediators and an impaired IFN-mediated induction of natural killer cell function. These results provide new insights into the regulatory mechanisms of IFN signaling and the induction of innate antiviral immunity.
Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15(INK4B), CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34(+) population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage-associated variant histone gamma-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination "epigenetic" therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179.
- Nov 2008
- 50th Annual Meeting of the American-Society-of-Hematology
The immune response to allergens starts with stimulation of a naïve T helper (Th) cell and its differentiation into a Th2 cell, expressing the cytokines interleukin (IL)-4, IL-5 and IL-13 responsible for the allergic response. The initial pattern of cytokine expression is retained during restimulation and division of the Th2 cell to create a population of specific allergen-responsive memory Th2 cells. Both, the coordinate cytokine expression and the inherited cytokine memory are specified by epigenetic mechanisms. Th2-specific changes in chromatin configuration at the Th2 locus act locally to open DNA, allowing recruitment of transcriptional machinery and rapid induction of cytokine expression. Induction of the transcription factor GATA3 is critical to this process. Loss of DNA methylation at the Th2 locus during differentiation from a naïve Th cell correlates to increased histone acetylation, consistent with the expression of IL-4, IL-5 and IL-13. The silencing of the Th2 locus in Th1 cells was associated with repressive histone methylation. These data indicate the formation of a 'poised' chromatin configuration at the Th2 locus that in combination with specific transcription factors specifies the cytokine repertoire in daughter cells and allows the immediate, rapid induction of cytokines by those cells in response to allergen.
Besides being a intracellular pH (pHi) regulator, Na+/H+ exchanger (NHE)1 has recently been postulated as a membrane scaffold that assembles protein complexes and coordinates various signaling pathways. The aim of the present study was to uncover NHE1 interactive partners and study their functional implications. NHE1 interactive partners were screened in the mouse brain with a signal transduction AntibodyArray. Ten of 400 tested proteins appeared to be potentially associated with NHE1. These partners have been shown to be involved in either cell proliferative or apoptotic pathways. The interactions between NHE1 and Src homology 2 domain-containing protein tyrosine phosphatase (SHP-2), Bin1, and heat shock protein (HSP)70 were reciprocally confirmed by coimmunoprecipitation. Moreover, in vitro binding data have shown that NHE1 COOH terminus interacts directly with SHP-2. The functional significance of the association between NHE1 and SHP-2 was further investigated by measuring pHi, cell proliferation, and cell death with the fluorescent dye BCECF, [3H]thymidine incorporation, and medium lactate dehydrogenase activity, respectively. Our results revealed that cells with SHP-2 overexpression exhibited a higher steady-state pHi and a faster, NHE1-dependent pHi recovery rate from acid load in HEPES buffer. In addition, SHP-2 overexpression diminished the HOE-642-induced inhibition of cell proliferation and protected cells from hypoxic injury, especially in the presence of HOE-642. Together, our findings demonstrate that SHP-2 not only is physically associated with NHE1 but also modulates NHE1 functions such as pHi regulation, cell proliferation, and cell death under hypoxia.
- Feb 2007
The PLZF gene is one of five partners fused to the retinoic acid receptor alpha in acute promyelocytic leukemia. PLZF encodes a DNA-binding transcriptional repressor and the PLZF-RARalpha fusion protein like other RARalpha fusions can inhibit the genetic program mediated by the wild tpe retinoic acid receptor. However an increasing body of literature indicates an important role for the PLZF gene in growth control and development. This information suggests that loss of PLZF function might also contribute to leukemogenesis.
- Nov 2005
- 47th Annual Meeting of the American-Society-of-Hematology
Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.
Fms-like tyrosine kinase 3 (Flt3) is a type III receptor tyrosine kinase (RTK). Between 20% and 30% of acute myeloid leukemia (AML) patients have either an internal tandem duplication (ITD) of the juxtamembrane region or a point mutation of the Flt3 receptor leading to the constitutive activation of downstream signaling pathways and aberrant cell growth. The silencing mediator of retinoic and thyroid hormone receptors (SMRT) corepressor mediates transcriptional repression by interacting with transcription factors such as the promyelocytic leukemia zinc finger (PLZF) protein. Previous reports indicate that SMRT interaction with transcription factors can be disrupted by phosphorylation through activation of RTK pathways. We report here that the Flt3-ITD interferes with the transcriptional and biologic action of the PLZF transcriptional repressor. In the presence of Flt3-ITD, PLZF-SMRT interaction was reduced, transcriptional repression by PLZF was inhibited, and PLZF-mediated growth suppression of leukemia cells was partially blocked. Furthermore, overexpression of Flt3-ITD led to a partial relocalization of SMRT protein from the nucleus to the cytoplasm. Nuclear export was dependent on the SMRT receptor interaction domain (RID), and Flt3-ITD enhances the binding of nuclear-cytoplasm shuttling protein nuclear factor-kappaB-p65 (NFkappaB-p65) to this region. These data suggest that activating mutations of Flt3 may disrupt transcriptional repressor function resulting in aberrant gene regulation and abnormal leukemia cell growth.
The eukaryotic translation initiation factor 4E (eIF4E) acts as both a key translation factor and as a promoter of nucleocytoplasmic transport of specific transcripts. Traditionally, its transformation capacity in vivo is attributed to its role in translation initiation in the cytoplasm. Here, we demonstrate that elevated eIF4E impedes granulocytic and monocytic differentiation. Our subsequent mutagenesis studies indicate that this block is a result of dysregulated eIF4E-dependent mRNA transport. These studies indicate that the RNA transport function of eIF4E could contribute to leukemogenesis. We extended our studies to provide the first evidence that the nuclear transport function of eIF4E contributes to human malignancy, specifically in a subset of acute and chronic myelogenous leukemia patients. We observe an increase in eIF4E-dependent cyclin D1 mRNA transport and a concomitant increase in cyclin D1 protein levels. The aberrant nuclear function of eIF4E is due to abnormally large eIF4E bodies and the loss of regulation by the proline-rich homeodomain PRH. We developed a novel tool to modulate this transport activity. The introduction of IkappaB, the repressor of NF-kappaB, leads to suppression of eIF4E, elevation of PRH, reorganization of eIF4E nuclear bodies, and subsequent downregulation of eIF4E-dependent mRNA transport. Thus, our findings indicate that this nuclear function of eIF4E can contribute to leukemogenesis by promoting growth and by impeding differentiation.
The transcriptional repressor PLZF was identified by its translocation with retinoic acid receptor alpha in t(11;17) acute promyelocytic leukemia (APL). Ectopic expression of PLZF leads to cell cycle arrest and growth suppression, while disruption of normal PLZF function is implicated in the development of APL. To clarify the function of PLZF in cell growth and survival, we used an inducible PLZF cell line in a microarray analysis to identify the target genes repressed by PLZF. One prominent gene identified was c-myc. The array analysis demonstrated that repression of c-myc by PLZF led to a reduction in c-myc-activated transcripts and an increase in c-myc-repressed transcripts. Regulation of c-myc by PLZF was shown to be both direct and reversible. An interaction between PLZF and the c-myc promoter could be detected both in vitro and in vivo. PLZF repressed the wild-type c-myc promoter in a reporter assay, dependent on the integrity of the binding site identified in vitro. PLZF binding in vivo was coincident with a decrease in RNA polymerase occupation of the c-myc promoter, indicating that repression occurred via a reduction in the initiation of transcription. Finally, expression of c-myc reversed the cell cycle arrest induced by PLZF. These data suggest that PLZF expression maintains a cell in a quiescent state by repressing c-myc expression and preventing cell cycle progression. Loss of this repression through the translocation that occurs in t(11;17) would have serious consequences for cell growth control.
FAZF, a member of the BTB/POZ family of transcriptional repressor proteins, has been shown to bind to FANCC, the protein defective in patients with the bone marrow failure syndrome Fanconi anemia complementation group C. Because bone marrow failure in Fanconi anemia has been attributed to a failure of the hematopoietic stem cell population to produce sufficient progeny, we documented the expression of FAZF in human CD34(+) hematopoietic progenitor cells. FAZF was expressed at high levels in early stages of differentiation but declined during subsequent differentiation into erythroid and myeloid lineages. Consistent with its presumed role as a transcriptional repressor, FAZF was found in the nuclear compartment, where it resides in distinct nuclear speckles at or near sites of DNA replication. Using a FAZF-inducible myeloid cell line, we found that enforced expression of FAZF was accompanied by accumulation in the G(1) phase of the cell cycle followed later by apoptosis. These results suggest an essential role for FAZF during the proliferative stages of primitive hematopoietic progenitors, possibly acting in concert with (a subset of) the Fanconi anemia proteins.
- Jan 2002
Monocyte differentiation induced by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is interrupted during the course of acute promyelocytic leukemia (APL). One form of APL is associated with the translocation t(11;17), which joins the promyelocytic leukemia zinc finger (PLZF) and retinoic acid receptor alpha (RARalpha) genes. Because PLZF is coexpressed in the myeloid lineage with the vitamin D(3) receptor (VDR), the interplay between PLZF and VDR was examined. It was found that PLZF interacts directly with VDR. This occurred at least partly through contacts in the DNA-binding domain of VDR and the broad complex, tram-trak, bric-a-brac/pox virus zinc finger (BTB/POZ) domain of PLZF. Moreover, PLZF altered the mobility of VDR derived from nuclear extracts when bound to its cognate binding site, forming a slowly migrating DNA-protein complex. Overexpression of PLZF in a monocytic cell line abrogated 1,25(OH)(2)D(3) activation from both a minimal VDR responsive reporter and the promoter of p21(WAF1/CIP1), a target gene of VDR. Deletion of the BTB/POZ domain significantly relieved PLZF-mediated repression of 1,25(OH)(2)D(3)-dependent activation. In addition, stable, inducible expression of PLZF in U937 cells inhibited the ability of 1,25(OH)(2)D(3) to induce surface expression of the monocytic marker CD14 and morphologic changes associated with differentiation. These results suggest that PLZF may play an important role in regulating the process by which 1,25(OH)(2)D(3) induces monocytic differentiation in hematopoietic cells.
The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RAR alpha fusion protein and, in a similar manner, inhibits RAR alpha target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RAR alpha and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia. (Blood. 2000;96:3939-3947)
PAX2 is a member of the paired box family of genes with an important role in kidney, genital tract and eye development. Another gene essential for kidney and genital tract development is the Wilms tumour gene, WT1. PAX2 and WT1 encode transcription factors expressed during fetal kidney development in patterns that overlap both spatially and temporally. The overlap of PAX2 and WT1 expression in fetal kidney prompted us to determine whether PAX2 regulates the WT1 gene. To investigate this possibility, the WT1 promoter and a series of WT1 promoter deletion fragments were cloned into a luciferase reporter vector, and used in co-transfection experiments with PAX2 expression constructs. PAX2 transactivated the WT1 promoter up to 35-fold in CHO-K1 cells, and from four- to sevenfold in 293 cells. Two regions of the WT1 promoter were required in the same promoter construct for efficient transactivation by PAX2 in CHO-K1 cells, and purified recombinant PAX2 protein was found to bind to two sites in the WT1 promoter, at -205/-230 and +377/+402. Removal of WT1 promoter sequences containing the -205/-230, or +377/+402 binding sites abolished transactivation of the WT1 promoter by PAX2 in CHO-K1 cells, and had a differential effect on transactivation of the WT1 promoter in 293 cells, depending on the PAX2 isoform used. A fragment containing the -205/-230 site alone could be transactivated by PAX2. These findings suggest that PAX2 is a tissue-specific modulator of WT1 expression, and is involved in cell growth control via WT1.