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Terry J Gaymes,
Azim M Mohamedali,
Miranda Patterson,
Nazia Matto,
Alexander Smith,
Austin Kulasekararaj,
Rajani Chelliah,
Nicola Curtin, Farzin Farzaneh,
Sydney Shall,
Ghulam Mufti
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ABSTRACT: Background Inactivation of the DNA mismatch repair pathway manifests as microsatellite instability, an accumulation of mutations that drives carcinogenesis. Here, we determined whether microsatellite instability in acute myeloid leukaemia and myelodysplastic syndrome correlated with chromosomal instability and PARP inhibitor sensitivity through disruption of DNA repair function. Design and Methods Acute myeloid leukaemia cell lines (n=12) and primary cell samples (n=18) and bone marrow mononuclear cells from high risk myelodysplastic syndrome (n=63) were profiled for microsatellite instability using fluorescent fragment PCR. PARP inhibitor sensitivity was performed using cell survival, annexin V staining and cell cycle analysis. Homologous recombination was studied using immunocytochemical analysis. SNP karyotyping was used to study chromosomal instability. RNA silencing, western blotting and gene expression analysis was used to study the functional consequences of mutations. Results Acute myeloid leukaemia cell lines (4/12, 33%) and primary samples (2/18, 11%) exhibited microsatellite instability with mono-allelic mutations in CtIP and MRE11. These changes were associated with reduced expression of mismatch repair pathway components, MSH2, MSH6 and MLH1. Both microsatellite instability positive primary acute myeloid leukaemia samples and cell lines demonstrated a down-regulation of homologous recombination DNA repair conferring marked sensitivity to PARP inhibitors. Similarly, bone marrow mononuclear cells from 11/56 (20%) patients with de novo high risk myelodysplastic syndrome exhibited microsatellite instability. Significantly, all 11 patients with microsatellite instability had cytogenetic abnormalities with 4/11 (36%) patients possessing a mono-allelic microsatellite mutation in CtIP. Furthermore, 50% reduction in CtIP expression by RNA silencing also down-regulated homologous recombination DNA repair responses conferring PARP inhibitor sensitivity, whilst CtIP differentially regulated the expression of homologous recombination modulating RecQ helicases, WRN and BLM. Conclusions Microsatellite instability dependent mutations in DNA repair genes, CtIP and MRE11 are detected in myeloid malignancies conferring hypersensitivity to PARP inhibitors. Microsatellite instability is significantly correlated with chromosomal instability in myeloid malignancies.
Haematologica 01/2013; · 6.42 Impact Factor
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ABSTRACT: Background. Expansion of regulatory T cells occurs in high-risk myelodysplastic syndrome and correlates with a poor prognosis. DNA methyltransferase inhibitors, particularly 5-azacytidine, have been shown to increase the survival of high-risk myelodysplastic syndrome patients. It is not entirely clear whether this improvement in patient survival is related to the effects of DNA methyltransferase inhibitors on the immune system and/or the direct effect of these drugs on the dysplastic clone. In this study we investigated the effect of 5-azacytidine on the function and proliferation capability of regulatory T cells and T helper cells. Design and methods. The number and function of CD4+ T cell subsets in 68 intermediate-2/high-risk myelodysplastic syndrome patients were serially assessed at diagnosis and following treatment. The in-vitro effects of 5-azacytidine on CD4+ T cell subsets isolated from both healthy donors and myelodysplastic syndrome patients were also investigated. Results. The number of peripheral blood regulatory T cells was significantly higher in myelodysplastic syndrome patients compared with healthy donors and responders to treatment (p=0.01). The absolute numbers of T-helper 1 and T-helper 2, but not T-helper 17, cells were significantly reduced following 12 months of treatment (p=0.03, p=0.03). The in vitro addition of 5-azacytidine to CD4+ T cells reduced the proliferative capacity of regulatory T cells (p=0.03). In addition, the 5-azacytidine-treated regulatory T cells had reduced suppressive function and produced larger amounts of IL-17. The FOXP3 expression in 5-azacytidine-treated T effectors was also increased. Interestingly, these FOXP3+/IL-17+ cells originated mainly from effector T cells rather than regulatory T cells. Conclusions. Our data suggest that 5-azacytidine has profound effects on CD4+ T cells, which correlate with disease status post treatment. Furthermore, despite the demethylation of the FOXP3 promoter and increased FOXP3 expression following 5-azacytidine treatment, these phenotypic 'regulatory T cells-like cells' lack the regulatory function and cytokine profile of regulatory T cells. These findings are important in correlating the clinically relevant immunomodulatory effects of 5-azacytidine.
Haematologica 12/2012; · 6.42 Impact Factor
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ABSTRACT: Targets for cancer therapy are conventionally selected by identification of molecules acting downstream of established tumour suppressors and oncoproteins, such as p53, c-Myc and Ras. However, the forward genetics approach provides an alternative, conceptually distinct, strategy for identifying target molecules de novo. This approach, which uses unbiased selection protocols relying directly on the effects of the genes themselves on cell fate, has the potential to identify novel cancer targets which have not been highlighted by conventional approaches. PLAC8, a small cysteine-rich protein with little homology to other proteins, has been identified by both these strategies. Here we confirm that PLAC8 overexpression protects some cancer cell lines from apoptosis, but we also demonstrate for the first time that, in other cell lines, the effect of PLAC8 overexpression is reversed, and, in this context, PLAC8 induces apoptosis. In both cases siRNA-mediated down-regulation of PLAC8 confirms that the activity of endogenously expressed PLAC8 is consistent with that shown by exogenous PLAC8. The striking reversal of the effects of PLAC8 in different cell types is not readily explained by the level of PLAC8 expressed within the cells, by the differential expression of PLAC8 splice variants observed, or by the p53 status of the host cells. This intriguing contrast in the effects of PLAC8 on cell fate in different cellular contexts presents attractive possibilities for the development of novel therapies for cancers, such as pancreatic cancers, where PLAC8 has been shown to be overexpressed.
Current cancer drug targets 08/2012; · 5.13 Impact Factor
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ABSTRACT: BackgroundGlypican 3 (GPC-3) is an oncofoetal protein that is expressed in most hepatocellular carcinomas (HCC). Since it is a potential
target for T cell immunotherapy, we investigated the generation of functional, GPC-3 specific T cells from peripheral blood
mononuclear cells (PBMC).
MethodsDendritic cells (DC) were derived from adherent PBMC cultured at 37°C for 7 days in X-Vivo, 1% autologous plasma, and 800
u/ml GM-CSF plus 500 u/ml IL-4. Immature DC were transfected with 20 μg of in vitro synthesised GPC-3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 μF and 4 ms pulse time),
or pulsed with peptide, and subsequently matured with lipopolysaccharide (LPS). Six predicted GPC-3 peptide epitopes were
synthesized using standard f-moc technology and tested for their binding affinity to HLA-A2.1 molecules using the cell line
T2.
ResultsDC transfected with GPC-3 mRNA but not control DC demonstrated strong intracellular staining for GPC-3 and in vitro generated interferon-gamma expressing T cells from autologous PBMC harvested from normal subjects. One peptide, GPC-3522-530 FLAELAYDL, fulfilled our criteria as a naturally processed, HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope: i) it
showed high affinity binding to HLA-A2, in T2 cell binding assay; ii) it was generated by the MHC class I processing pathway
in DC transfected with GPC-3 mRNA, and iii) HLA-A2 positive DC loaded with the peptide stimulated proliferation in autologous
T cells and generated CTL that lysed HLA-A2 and GPC-3 positive target cells.
ConclusionsThese findings demonstrate that electroporation of GPC-3 mRNA is an efficient method to load human monocyte-derived DC with
antigen because in vitro they generated GPC-3-reactive T cells that were functional, as shown by interferon-gamma production. Furthermore, this study
identified a novel naturally processed, HLA-A2-restricted CTL epitope, GPC-3522-530 FLAELAYDL, which can be used to monitor HLA-A2-restricted CTL responses in patients with HCC. Further studies are required
to investigate whether anti-GPC-3 immunotherapy has a role in the treatment of GPC-3 dependent tumours, such as HCC.
Journal of Experimental & Clinical Cancer Research 04/2012; 29(1):1-11. · 2.15 Impact Factor
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Joop Gäken,
Azim M Mohamedali,
Jie Jiang,
Farooq Malik,
Doris Stangl,
Alexander E Smith,
Constantinos Chronis,
Austin G Kulasekararaj,
N Shaun B Thomas, Farzin Farzaneh,
Mahvash Tavassoli,
Ghulam J Mufti
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ABSTRACT: MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.
Nucleic Acids Research 02/2012; 40(10):e75. · 8.03 Impact Factor
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ABSTRACT: Small nucleolar RNAs (snoRNAs) have long been considered important but unglamorous elements in the production of the protein synthesis machinery of the cell. Recently, however, several independent lines of evidence have indicated that these non-coding RNAs might have crucial roles in controlling cell behaviour, and snoRNA dysfunction could consequently contribute to oncogenesis in previously unsuspected ways.
Nature Reviews Cancer 02/2012; 12(2):84-8. · 29.54 Impact Factor
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Shahram Kordasti,
Judith Marsh,
Sufyan Al-Khan,
Jie Jiang,
Alexander Smith,
Azim Mohamedali,
Pilar Perez Abellan,
Caroline Veen,
Benedetta Costantini,
Austin G Kulasekararaj,
Nana Benson-Quarm,
Thomas Seidl,
Syed A Mian, Farzin Farzaneh,
Ghulam J Mufti
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ABSTRACT: The role of CD4(+) T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4(+) T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.
Blood 12/2011; 119(9):2033-43. · 9.90 Impact Factor
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ABSTRACT: Non-coding RNA GAS5 (growth arrest-specific transcript 5) is a 5'-TOP (5'-terminal oligopyrimidine tract) RNA, whose translation, and consequently also stability, is controlled by the mTOR (mammalian target of rapamycin) pathway. GAS5 was identified by functional expression cloning and is necessary and sufficient for normal growth arrest in both leukaemic and untransformed human T-lymphocytes. GAS5 is also required for the inhibitory effects of rapamycin and its analogues on T-cells. The striking functional effects of GAS5 may be mediated through the snoRNAs (small nucleolar RNAs) encoded in its introns and/or through the unusual folding of the mRNA itself, which sequesters, and therefore inhibits, the glucocorticoid receptor.
Biochemical Society Transactions 04/2011; 39(2):482-6. · 3.71 Impact Factor
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ABSTRACT: This chapter describes some of the techniques in use in our laboratories for the investigation of PARP inhibitors in clinical medicine. More specifically, we are involved in investigating the utility of PARP inhibitors in the treatment of hematopoietic malignancies. We are also actively investigating the properties of the PARP systems in cell biology. We begin the chapter with a very brief history of the invention and use of PARP inhibitors. We then explain the underlying logic of the use of PARP inhibitors either in combination with chemo- or radiotherapy or as single agents used alone. We then provide in full detail the protocols that we use to study PARP inhibitors in cell biology to identify patients that should be susceptible to PARP inhibitor treatment and to manage and investigate these patients throughout their treatment.
Methods in molecular biology (Clifton, N.J.) 01/2011; 780:239-66.
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ABSTRACT: Leaves of Murraya exotica L. (Rutaceae) are used for the treatment of various disorders such as cough, fever, and infectious wounds, as well as alleviating pains in folk medicine in southern China.
The objectives of this study were to investigate the in vivo antinociceptive and anti-inflammatory activities of ethanol (70%) extracts and isolated compounds obtained from the dried leaves of M. exotica.
The antinociceptive activities were evaluated with the methods of acetic acid-induced writhing response and hot-plate latent pain response test. Carrageenan induced hind paw edema, xylene induced ear edema, and a rat knee osteoarthritis model were employed to measure the anti-inflammatory activities. The compounds were isolated using column chromatography and thin-layer chromatography, and the structures identified by ¹H NMR, ¹³C NMR, MS, and IR.
The ethanol (70%) extracts significantly decreased in the acetic acid-induced writhing response; increased in hot-plate latency; suppressed xylene induced ear swelling and the carrageenan-induced paw edema effectively. In the rat knee osteoarthritis model, the treatment of the ethanol (70%) extracts resulted in a significant increase in the activity of superoxide dismutase, an inhibition on inducible nitric oxide synthase activity, and a decrease in the contents of interleukin-1β and tumor necrosis factor-α of the rat serum. Following this, we explored the components of the ethanol (70%) extracts and isolated six known coumarins, including murracarpin, which exhibited the most potential in antinociceptive and anti-inflammatory activities.
M. exotica displayed remarkable antinociceptive and anti-inflammatory activities.
Pharmaceutical Biology 12/2010; 48(12):1344-53. · 0.88 Impact Factor
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ABSTRACT: The chicken anemia virus-derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However, the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein-apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines K562, HL60, U937, KG1, and NB4. In contrast, normal CD34(+) cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases, protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded through the upregulation of PKCβ, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downregulation of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma.
Cancer Research 09/2010; 70(18):7242-52. · 7.86 Impact Factor
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ABSTRACT: Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 07/2010; 878(22):1939-45. · 2.78 Impact Factor
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ABSTRACT: The central importance of the serine/threonine protein kinase mTOR (mammalian Target of Rapamycin) in the control of cell growth and proliferation is well established. However, our knowledge both of the upstream pathways controlling mTOR activity and of the downstream events mediating these effects is still seriously incomplete. We report a previously unsuspected role for the nonprotein-coding RNA GAS5 in the inhibition of T-cell proliferation produced by mTOR antagonists such as rapamycin. GAS5 transcripts are up-regulated during growth arrest and after rapamycin treatment, and GAS5 has recently been shown to be necessary and sufficient for normal T-cell growth arrest. Down-regulation of GAS5 using RNA interference protects both leukemic and primary human T cells from the inhibition of proliferation produced by mTOR antagonists. The GAS5 transcript is a member of the 5' terminal oligopyrimidine class of RNAs, which is specifically controlled at the level of translation by the mTOR pathway, and the effects of GAS5 on the cell cycle provide a novel and important link to the control of proliferation. These observations point to a significant advance in our understanding of the mechanism of action of mTOR inhibitors, which is likely to lead to improvements in immunosuppressive and cancer therapy.
Molecular pharmacology 07/2010; 78(1):19-28. · 4.53 Impact Factor
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ABSTRACT: Abstract
Background
Glypican 3 (GPC-3) is an oncofoetal protein that is expressed in most hepatocellular carcinomas (HCC). Since it is a potential target for T cell immunotherapy, we investigated the generation of functional, GPC-3 specific T cells from peripheral blood mononuclear cells (PBMC).
Methods
Dendritic cells (DC) were derived from adherent PBMC cultured at 37°C for 7 days in X-Vivo, 1% autologous plasma, and 800 u/ml GM-CSF plus 500 u/ml IL-4. Immature DC were transfected with 20 μg of in vitro synthesised GPC-3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 μF and 4 ms pulse time), or pulsed with peptide, and subsequently matured with lipopolysaccharide (LPS). Six predicted GPC-3 peptide epitopes were synthesized using standard f-moc technology and tested for their binding affinity to HLA-A2.1 molecules using the cell line T2.
Results
DC transfected with GPC-3 mRNA but not control DC demonstrated strong intracellular staining for GPC-3 and in vitro generated interferon-gamma expressing T cells from autologous PBMC harvested from normal subjects. One peptide, GPC-3<sub>522-530 </sub>FLAELAYDL, fulfilled our criteria as a naturally processed, HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope: i) it showed high affinity binding to HLA-A2, in T2 cell binding assay; ii) it was generated by the MHC class I processing pathway in DC transfected with GPC-3 mRNA, and iii) HLA-A2 positive DC loaded with the peptide stimulated proliferation in autologous T cells and generated CTL that lysed HLA-A2 and GPC-3 positive target cells.
Conclusions
These findings demonstrate that electroporation of GPC-3 mRNA is an efficient method to load human monocyte-derived DC with antigen because in vitro they generated GPC-3-reactive T cells that were functional, as shown by interferon-gamma production. Furthermore, this study identified a novel naturally processed, HLA-A2-restricted CTL epitope, GPC-3<sub>522-530 </sub>FLAELAYDL, which can be used to monitor HLA-A2-restricted CTL responses in patients with HCC. Further studies are required to investigate whether anti-GPC-3 immunotherapy has a role in the treatment of GPC-3 dependent tumours, such as HCC.
Journal of Experimental & Clinical Cancer Research. 01/2010;
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Advanced Materials 10/2009; 21(38-39):3910-3914. · 13.88 Impact Factor
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ABSTRACT: Despite being of the myeloid lineage, acute myeloid leukaemia (AML) blasts are of low immunogenicity, probably because they lack the costimulatory molecule CD80 and secrete immunosuppressive factors. We have previously shown that in vitro stimulation of autologous peripheral blood mononuclear cells (PBMCs) with primary AML cells modified to express CD80 and IL-2 promotes proliferation, secretion of Th1 cytokines and expansion of activated CD8(+) T cells. In this study, we show that allogeneic effector cells (from a healthy donor or AML patients) when stimulated with IL-2/CD80 modified AML blasts were able to induce the lysis of unmodified AML blasts. Effector cells stimulated with IL-2/CD80AML blasts had higher lytic activity than cells stimulated with AML cells expressing CD80 or IL-2 alone. Similarly, AML patient PBMCs primed with autologous IL-2/CD80 AML cells had a higher frequency of IFN-gamma secreting cells and show cytotoxicity against autologous, unmodified blasts. Crucially, the response appears to be leukaemia specific, since stimulated patient PBMCs show higher frequencies of IFN-gamma secreting effector cells in response to AML blasts than to remission bone marrow cells from the same patients. Although studied in a small number of heterogeneous patient samples, the data are encouraging and support the continuing development of vaccination for poor prognosis AML patients with autologous cells genetically modified to express IL-2/CD80.
Cancer Immunology and Immunotherapy 09/2009; 59(3):379-88. · 3.70 Impact Factor
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ABSTRACT: Coumarins are of many different structures. They constitute an important class of pharmacological agents possessing a range of different physiological activities including anti-cancer, anti-oxidant, anti- inflammation, anti-HIV, anti-coagulant, anti-bacterial, analgesic and comparative immune-modulation. Recently, coumarins have attracted intense research interest. Of great interest is the possibility that this class of molecules could be a source of drugs for the therapy of several diseases. These include recent insights into inhibiting cell proliferation by interfering with mitotic spindle microtubule function, decrease Matrix Metalloproteinase (MMP) activity, block the cell cycle in the S or G2/M phases to interfere with processes of cell division, suppress O2(-) generation in leukocytes, inhibit different protein kinases, modulate the signalings, induce carcinogen-detoxifying enzymes glutathione S-transferases (GSTs) and/or NAD(P)H quinine oxidoreductase (NQO1), suppress the phosphorylation of Akt/PKB as a mechanism inhibiting inflammation, progress in structure modification to increase in anti-fungal action, to broaden against bacteria spectrum, to enhance inhibiting activities of nitric oxide synthase (NOS) and cyclooxygenase (COX), to strengthen anti-oxidant activity and to exhibite a much higher cytotoxicity against human umbilical vein endothelial cell (HUVEC). With fewer non-hemorrhagic side effects than the indanedione derivatives, they can be applied as an oral anticoagulant commonly for preventing venous thromboembolism following orthopedic surgery, recurrent myocardial infarction and the treatment of systemic embolism in atrial fibrillation, together with the significant advances in the basis of drug action. It is therefore useful to build up some correlations with the data available in order to better explore the molecular and cellular mechanism of coumarin action in the treatment of diseases. This review will focus on recent advances in molecular and cellular mechanisms of coumarin action involved with the relationship between structure and activity.
Current Medicinal Chemistry 09/2009; 16(32):4236-60. · 4.86 Impact Factor
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ABSTRACT: Background Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors. DESIGN AND METHODS: Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5' aza-2'-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents. RESULTS: PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5' aza-2'-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes. Conclusions On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.
Haematologica 06/2009; 94(5):638-46. · 6.42 Impact Factor
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ABSTRACT: Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti-leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)-transduced AML cells stimulate in-vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2-expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up-regulation of the cytolytic marker CD107a was detected following autologous culture with LV-CD80/IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46.6% to 90.4%) and allogeneic AML cells (from 11.8% to 20.1%) following in-vitro stimulation by CD80/IL2-expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40-fold (from 0.4% to 22.5%). These studies demonstrated that vaccination of patients with CD80/IL2-transduced AML cells could provide a powerful strategy for T/NK cell-mediated stimulation of anti-leukaemic immunological responses.
British Journal of Haematology 05/2009; 145(6):749-60. · 4.94 Impact Factor
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ABSTRACT: Hepatitis C virus core (HCVcore) protein was expressed in myeloid dendritic cells (DC) from C57/B6 mice (H-2K(b)) by electroporation of HCVcore mRNA to investigate its effect on the ability of DC to prime CD8+ T cells displaying a T cell receptor specific for OVA(257-264) peptide (SIINFEKL)/H-2K(b) complex. Expression of full length HCVcore(191), which is directed to the endoplasmic reticulum (ER) membrane by a C-terminal signal sequence, but not a truncated variant HCVcore(152), which has a wider subcellular localization including the nucleus, significantly reduced surface levels of the H-2K(b)/SIINFEKL complex and impaired the ability of DC to prime naïve CD8+ T cells when they had to process endogenous antigen but not when MHC class I molecules were loaded directly with SIINFEKL peptide. Exploitation of the MHC class I antigen-processing pathway by HCVcore(191) impairs the ability of DC to stimulate CD8+ T cells and may contribute to the persistence of HCV infection.
Virology 05/2009; 389(1-2):1-7. · 3.35 Impact Factor
